Split (1)

train · ~9.11M rows (showing the first 93.5k)

identifier stringlengths 11 32	pdf_url stringlengths 17 4.62k ⌀	lang stringclasses 120 values	error stringclasses 1 value	title stringlengths 2 500 ⌀	source_name stringlengths 1 435 ⌀	publication_year float64 1.9k 2.02k	license stringclasses 3 values	word_count int64 0 1.64M	text stringlengths 1 9.75M
W4210582816.txt	https://link.springer.com/content/pdf/10.1007/s00347-021-01556-5.pdf	de		Einsatz von künstlicher Intelligenz im Screening auf diabetische Retinopathie an einer diabetologischen Schwerpunktklinik	Deleted Journal	2,022	cc-by	5,477	Die Ophthalmologie vormals Der Opthalmologe Originalien Ophthalmologe 2022 · 119:705–713 https://doi.org/10.1007/s00347-021-01556-5 Eingegangen: 4. Juni 2021 Überarbeitet: 27. November 2021 Angenommen: 1. Dezember 2021 Online publiziert: 26. Januar 2022 © Der/die Autor(en) 2022 Einsatz von künstlicher Intelligenz im Screening auf diabetische Retinopathie an einer diabetologischen Schwerpunktklinik Sebastian Paul1 · Allam Tayar1 · Ewa Morawiec-Kisiel1 · Beathe Bohl1 · Rico Großjohann1 · Elisabeth Hunfeld1 · Martin Busch1 · Johanna M. Pfeil1 · Merlin Dähmcke1 · Tara Brauckmann1 · Sonja Eilts1 · Marie-Christine Bründer1 · Milena Grundel1 · Bastian Grundel1 · Frank Tost1 · Jana Kuhn2 · Jörg Reindel2 · Wolfgang Kerner2 · Andreas Stahl1 1 2 Zusatzmaterial online Zusätzliche Informationen sind in der Online-Version dieses Artikels (https:// doi.org/10.1007/s00347-021-01556-5) enthalten. QR-Code scannen & Beitrag online lesen Klinik und Poliklinik für Augenheilkunde, Universitätsmedizin Greifswald, Greifswald, Deutschland Klinik für Diabetes und Stoﬀwechselerkrankungen Karlsburg, Klinikgruppe Dr. Guth GmbH & Co. KG, Karlsburg, Deutschland Weltweit wird mit einer weiteren Zunahme der Erkrankungszahlen für Diabetes mellitus gerechnet. Die Gründe hierfür sind multifaktoriell und basieren unter anderem auf unseren modernen Lebens- und Ernährungsgewohnheiten sowie der demograﬁschen Entwicklung mit zunehmender Alterung der Bevölkerung. Eine frühzeitige Diagnose des Diabetes mellitus ist grundlegend für eine suﬃziente Therapie und somit für die Vermeidung schwerwiegender Komplikationen. Es wird geschätzt, dass etwa ein Drittel der Diabetiker Stadien einer diabetischen Retinopathie aufzeigen [12]. In Deutschland liegt die Prävalenz der diabetischen Retinopathie bei Patienten mit Typ-2-Diabetes bei 9–16 % und beim Typ-1-Diabetes bei 24–27 % [3]. Leider sind nicht alle Patienten mit Diabetes mellitus in ausreichender ophthalmologischer Kontrolle, sodass davon auszugehen ist, dass es eine signiﬁkante Dunkelziﬀer von nicht erkannter oder nicht sufﬁzient nachkontrollierter diabetischer Retinopathie gibt. Es besteht die Hoﬀnung, dass durch die Einführung von Diagnoseprozessen mittels künstlicher Intelligenz (KI) die Screeningrate auf diabetische Retinopathie erhöht werden kann und Patienten mit dringendem Behandlungsbedarf schneller identiﬁziert werden können. Basierend auf einer populationsgestützten Zulassungsstudie, wurde durch die amerikanische Food and Drug Administration (FDA) 2018 das KI-basierte Screeningmodul IDx-DR (IDx Technologies Inc., Coralville, Iowa, USA) zur Beurteilung der diabetischen Retinopathie zugelassen. Die KI erreichte in den Zulassungsstudien für die Diagnose einer schweren DR eine Sensitivität von 87,2 % (95 % CI, 81,8–91,2 %) und eine Speziﬁtät von 90,7 % (95 % CI, 88,3–92,7 %) sowie eine Abbildungsrate (Quote an auswertbaren Aufnahmen) von 96,1 % (95 % CI, 94,6–97,3 %), womit die Fähigkeit der KI zur Diagnostik der diabetischen Retinopathie in der Grundversorgung demonstriert wurde. Es benötigten 76,4 % der Teilnehmer keine pharmakologische Pupillendilatation, während 23,6 % eine Pupillendilatation benötigten. Basierend auf diesen Ergebnissen, hat die FDA das System zur medizindiagnostischen Verwendung zugelassen. Das IDx-DR-System basiert größtenteils auf dem Konzept des Deep-Learning (DL), wobei die angewandten Algorithmen darauf trainiert wurden, bestimmte Muster der diabetischen Retinopathie auf Fundusfotos zu erkennen. Die Detektion der Mikroaneurysmen fußt auf einer Multiscale-featurebank-Detektion [1]. Darüber Der Ophthalmologe 7 · 2022 705 Originalien hinaus sind DL-basierte Verfahren grundsätzlich in der Lage, sich ständig selbst weiter zu verbessern. Das KI-System von IDxDR verfügt über 2 Kernalgorithmen, einen auf die Bildqualität ausgerichteten KI-Algorithmus und den eigentlichen Diagnosealgorithmus. Beurteilt werden die deﬁnitionsgemäßen Veränderungen, die bei einer diabetischen Retinopathie auftreten können, wie Mikroaneurysmen, Cottonwool-Herde, intraretinale Blutungen, perlschnurartige Konﬁguration der retinalen Venen, intraretinale mikrovaskuläre Anomalien (IRMA), Neovaskularisationen, lipoproteinhaltige Exsudate und Glaskörpersanguinationen, allerdings ohne dass diese Veränderungen speziﬁsch als die jeweilige Entität erkannt und benannt werden. Der KI-Algorithmus gibt nur bei Vorliegen verwertbarer Fundusaufnahmen beider Augen eine summarische Einschätzung des Schweregrads, eingeteilt in keine, milde, moderate oder schwere (visusbedrohende) diabetische Retinopathie ohne eine Seitendiﬀerenzierung, aus [1]. Im Februar 2020 begannen wir an der diabetologischen Schwerpunktklinik in Karlsburg das IDx-DR-System begleitend zum dortigen ophthalmologischen Screening einzusetzen. Mit der Erprobung des IDx-DR-Systems sollte untersucht werden, ob KI zum Screening auf eine diabetische Retinopathie und zur Priorisierung der augenärztlichen Vorstellung an einer diabetologischen Schwerpunktklinik geeignet ist und wo mögliche Stärken und Schwächen des Systems in der täglichen Praxis liegen. Methoden Die Patientenklientel an der diabetologischen Schwerpunktklinik Karlsburg setzt sich aus Patienten sämtlicher Altersgruppen und mit allen Formen des Diabetes mellitus zusammen. Die Ablehnung der IDx-DR-Aufnahme oder der Datenauswertung durch den Patienten war das einzige Ausschlusskriterium für die hier vorgestellten Ergebnisse. Die Studie wurde in Übereinstimmung mit der Deklaration von Helsinki und nach positivem Votum der Ethikkommission der Universitätsmedizin Greifswald durchgeführt (BB 025/20). Das ophthalmologische Screening am Klinikum Karlsburg erfolgt im Rahmen der 706 Der Ophthalmologe 7 · 2022 Zusammenfassung Hintergrund: Seit 2018 ist mit IDx-DR ein Verfahren auf dem Markt, welches den Grad der diabetischen Retinopathie (DR) mittels künstlicher Intelligenz (KI) bestimmt. Methoden: Wir haben IDx-DR in die Sprechstunde an einer diabetologischen Schwerpunktklinik integriert und berichten über die Übereinstimmung zwischen IDx-DR (IDx Technologies Inc., Coralville, IA, USA) und Funduskopie sowie IDx-DR und ophthalmologischer Bildbeurteilung sowie über den Einﬂuss unterschiedlicher Kamerasysteme. Ergebnisse: Mit der Topcon-Kamera (n = 456; NW400, Topcon Medical Systems, Oakland, NJ, USA) konnte im Vergleich zur Zeiss-Kamera (n = 47; Zeiss VISUCAM 500, Carl Zeiss Meditec AG, Jena, Deutschland) häuﬁger eine ausreichende Bildqualität in Miosis erreicht werden. Insgesamt war bei etwa 60 % der Patienten eine IDx-DRAnalyse in Miosis möglich. Alle Patienten, bei denen keine IDx-DR-Analyse in Miosis möglich war, konnten in Mydriasis funduskopiert werden. Innerhalb der Gruppe der auswertbaren Befunde zeigte sich eine Übereinstimmung zwischen IDx-DR und augenärztlicher Funduoskopie in ca. 55 %, ein Überschätzen des Schweregrads durch IDx-DR in ca. 40 % und ein Unterschätzen in ca. 4 %. Die Sensitivität (Speziﬁtät) für das Erkennen einer schweren, behandlungsbedürftigen Retinopathie lag bei 95,7 % (89,1 %) für Fälle mit auswertbaren Fundusaufnahmen und bei 65,2 % (66,7 %), wenn alle Fälle betrachtet werden (inklusive derjeniger ohne verwertbare Aufnahme in Miosis). Der Kappa-Koeﬃzient zeigt mit 0,334 (p < 0,001) eine ausreichende Übereinstimmung zwischen IDx-DR und ärztlicher Bildauswertung anhand des Fundusfotos unter Berücksichtigung aller Patienten mit auswertbarer IDx-DR-Analyse. Der Vergleich zwischen IDx-DR mit der ärztlichen Funduskopie ergibt unter denselben Voraussetzungen eine geringe Übereinstimmung mit einem Kappa-Wert von 0,168 (p < 0,001). Schlussfolgerung: Die vorliegende Studie zeigt Möglichkeiten und Grenzen des KIgestützten DR-Screenings auf. Eine wesentliche Einschränkung liegt in der Tatsache, dass bei ca. 40 % der Patienten keine ausreichenden Aufnahmen in Miosis gewonnen werden konnten. Wenn ausreichende Aufnahmen vorlagen, stimmten IDx-DR und augenärztliche Diagnose in über 50 % der Fälle überein. Ein Unterschätzen des Schweregrades durch IDx-DR kam selten vor. Für die Integration in augenärztlich unterstützten Sprechstunden erscheint uns das System grundsätzlich geeignet. Die hohe Rate an fehlenden Aufnahmen in Miosis stellt allerdings eine Limitation dar, die einen Einsatz ohne augenärztliche Kontrollmöglichkeit schwierig erscheinen lässt. Schlüsselwörter IDx-DR · Künstliche Intelligenz · Screening · Diabetische Retinopathie · Telemedizin dort stationär durchgeführten internistischen Diagnostik und Therapie wöchentlich durch einen ophthalmologischen Konsiliardienst der Universitätsaugenklinik Greifswald. Mit Einführung des IDxDR-Systems wurde ein neuer Ablaufplan für das Screeningprogramm erstellt. Ein bis 2 Tage vor dem Sprechstundentermin werden durch den nichtärztlichen Funktionsdienst des Klinikums Karlsburg nichtmydriatische Fundusaufnahmen mit der herstellerseitig empfohlenen TopconKamera (NW400, Topcon Medical Systems, Oakland, USA) erstellt. Da in Europa das IDx-DR-System unabhängig vom Kameratyp zugelassen ist, untersuchten wir im Projekt zusätzlich den Einﬂuss des verwendeten Kamerasystems auf die Ergebnisqualität und führten auch Aufnahmen mit einem alternativen Kamerasystem durch (Zeiss VISUCAM 500, Carl Zeiss Meditec AG, Jena, Deutschland). Allerdings ist der Vergleich eingeschränkt zu bewerten, da mittels Zeiss-Kamera (Zeiss VISUCAM 500, Carl Zeiss Meditec AG, Jena, Deutschland) 47 Patienten und mit der Topcon-Kamera (NW400, Topcon Medical Systems, Oakland, NJ, USA) 456 Patienten untersucht wurden (s. Ergebnisteil). Das IDx-DR-Systems arbeitet mit 4 Fundusaufnahmen (pro Auge je 1 Foto mit Fokus auf den N. opticus und auf die Makula). Die Aufnahmen werden Cloud-basiert analysiert und (ausreichende Bildqualität vorausgesetzt) in kurzer Zeit beurteilt [1, 5]. Es handelt sich um keinen telemedizinischen Dienst, eine ärztliche Überprüfung des Ergebnisses ﬁndet seitens IDx- moderat 8,5 % schwer 10,3 % unzureichende Bildqualität 27,8 % mild 17,8 % kein Ergebnis 37,7 % kein Bild 9,9 % keine DR 25,7 % a schwer 10,6 % unzureichende Bildqualität 31,9 % moderat 2,1 % mild 8,5 % b keine DR 6,5 % DR nicht statt. Im Gegensatz zur augenärztlichen Untersuchung gibt IDx-DR nur Ergebnisse auf Patientenebene aus, ohne Diﬀerenzierung zwischen beiden Augen. Die augenärztliche Untersuchung dagegen gibt Resultate auf Augenebene aus. Zum Vergleich mit den IDx-DR-Ergebnissen wurde jeweils das augenärztlich als schlechter eingestufte Auge (also dasjenige mit dem höheren Grad an diabetischer Retinopathie) verwendet. Eine funduskopisch sichtbare diabetische Makulopathie wurde dabei beispielsweise mit berücksichtigt. Im Rahmen der vorliegenden Evaluierung erfolgte für jeden Patienten, der mittels IDx-DR untersucht wurde, in derselben Woche eine reguläre fachärztliche Fundusuntersuchung (ohne Kenntnis des Ergebnisses der bereits vorhandenen IDx-DRAnalyse). Zusätzlich erfolgte zu einem späteren Zeitpunkt die fachärztliche Einschätzung des Schweregrads der diabetischen kein Ergebnis 72,3 % Retinopathie ausschließlich anhand der Bilder, die in die IDx-DR-Analyse eingingen. Hierbei wurde der gesamte Bildausschnitt (45°) beurteilt. Unschärfen oder Verschattungen führten teilweise dazu, dass einige Aufnahmen seitens des Untersuchers als nicht graduierbar eingestuft wurden. Somit lagen zu jedem Datensatz 3 Ergebnisse vor: IDx-DR-Diagnose, fachärztliche Diagnose anhand der Bilder und fachärztliche Diagnose anhand der vollständigen binokularen Fundusuntersuchung in Mydriasis. Die Fundusaufnahmen wurden durch eine von 3 verschiedenen medizinischen Fachangestellten angefertigt, die augenärztlichen Untersuchungen sowie die Bewertung der Bilder erfolgten durch eine(n) von 4 Fachärzten/Fachärztinnen. Die fachärztliche Beurteilung der Aufnahmen und die Funduskopie erfolgten dabei immer von derselben Person, allerdings zeitlich versetzt. Die diagnostische Einstufung der kein Bild 40,4 % Abb. 1 9 a Ergebnis der IDx-DR-Analyse mittels Topcon-Kamera. b Ergebnis der IDx-DR-Analyse mittels Zeiss-Kamera Fundusbilder erfolgt durch IDx-DR 4-stuﬁg in eine negative, milde, moderate oder visusbedrohende diabetische Retinopathie. Ein beispielhafter Ausdruck des IDx-DRAnalyseberichtes ist Suppl. Abb. 1 zu entnehmen. Die Bewertung „negativ“ entspricht dabei dem Level 10 des ETDRSGradings und zeigt keine Anomalitäten [4]. Die „milde“ diabetische Retinopathie wird dem Level 20 der ETDRS zugeordnet und ist gekennzeichnet durch Mikroaneurysmen, isolierte Blutungen oder Cotton-wool-Herde. Eine „moderate“ diabetische Retinopathie entspricht den ETDRSLevels 35, 43 und 47. Die Einstufung der „visusbedrohenden“ diabetischen Retinopathie kommt den ETDRS-Levels 53 A–E, sowie 61, 65, 71, 75, 81 und 85 gleich und entspricht somit der schweren nichtproliferativen diabetischen Retinopathie (gemäß der 4:2:1-Regel) bzw. der proliferativen diabetischen Retinopathie [2]. Der Ophthalmologe 7 · 2022 707 Originalien Abb. 2 8 Vergleich Untersuchungsergebnis „IDx-DR-Analyse“ mit „ärztlicher Untersuchung“ mittels Funduskopie Abb. 3 8 Vergleich der Diagnosen durch IDx-DR mit der ärztlichen Diagnose durch Funduskopie; nicht dargestellt sind die Patienten, bei denen keine Aufnahme möglich war bzw. das Fundusfoto nicht auswertbar war Im IDx-DR-Modul werden die schwere nichtproliferative diabetische Retinopathie und die proliferative diabetische Retinopathie als visusbedrohend deﬁniert und im Ergebnis nicht unterschieden. Exsudate im Bereich der Makula sowie ein diabetisches Makulaödem werden ebenfalls als visusbedrohend eingestuft [1]. Die statistische Auswertung dieser Studie erfolgte mittels SPSS (IBM SPSS Statistics V.27, IBM Corporation, Armonk, NY, USA). 708 Der Ophthalmologe 7 · 2022 Ergebnisse Für diese Studie wurden insgesamt 503 Patienten, die aus einem bundesweiten Einzugsgebiet zur intensivierten Diagnostik und Therapieeinstellung eingewiesen wurden, ausgewertet. Das Durchschnittsalter der Patienten in unserer Studie lag mit 54,2 Jahren relativ niedrig. Der jüngste Patient war 9 Jahre und der älteste Patient 92 Jahre alt. Die Altersverteilung ist in Suppl. Abb. 2a aufgeführt. Das Patientenkollektiv war zu 42 % weiblich und zu 58 % männlich (Suppl. Abb. 2b). Wir untersuchten 273 Typ-II-Diabetiker (54,3 %) und 230 Typ-I-Diabetiker (45,7 %; Suppl. Abb. 2c). Um den Einﬂuss verschiedener Kameratypen auf die Ergebnisqualität zu analysieren, untersuchten wir 456 Patienten mit dem von IDx-DR empfohlenen Topcon-Kamerasystem und 47 Patientenmiteinem alternativen Zeiss-Kamerasystem. Die Handhabung mit dem Zeiss-Kamerasystem wurde von den durchführenden Fachkräften dabei als deutlich zeitaufwendiger und komplexer empfunden. Insbesondere die Auslöseautomatik und die erfolgreichere Aufnahme nichtmydriatischer Fundusbilder wurden bei der Topcon-Kamera als vorteilhaft beschrieben. Im Ergebnis konnten bei 40,4 % der mit der Zeiss-Kamera untersuchten Patienten keine verwertbaren Aufnahmen in Miosis gewonnen werden (19 von 47 Patienten). Mit der Topcon-Kamera war bei 9,9 % der Patienten (45 von 456 Patienten) keine verwertbare Fundusfotoaufnahme in Miosis möglich. Hinzu kommt, dass trotz erfolgter Aufnahme die Bildqualität für eine erfolgreiche IDx-DR-Analyse nicht immer ausreichend war. Dies war für Zeiss in weiteren 31,9 % der Fälle gegeben, für Topcon in 27,8 %. In . Abb. 1a, b sind die Ergebnisse der Analyse zu den einzelnen Kamerasystemen dargestellt. Als Hauptursachen für das Fehlen der Aufnahme oder die für eine IDx-DRAnalyse unzureichende Aufnahmequalität identiﬁzierten wir eine ausgeprägte Miosis, Trübungen der optischen Medien (Hornhaut, Linse, Glaskörper) oder eine unzureichende Kooperation des Patienten (beispielsweise reduzierter Allgemeinzustand zum Zeitpunkt der Untersuchung). Eine Katarakt, die so ausgeprägt war, dass gar keine Fundusaufnahme erfolgen konnte, lag allerdings nur bei einem Patienten vor. In . Abb. 2 werden die Resultate des IDx-DR-Systems mit den Untersuchungsergebnissen der etablierten binokularen Funduskopie in Mydriasis verglichen. Von den insgesamt 503 Patienten wiesen (basierend auf der augenärztlichen Untersuchung in Mydriase) 211 Patienten (42 %) Zeichen einer diabetischen Retinopathie in unterschiedlichen Schweregraden auf. Der Anteil an Patienten mit einem Dia- Abb. 4 8 Vergleich der Diagnosen durch IDx-DR mit der ärztlichen Diagnose durch Funduskopie nach Altersgruppen; nicht dargestellt sind die Patienten, bei denen keine Aufnahme möglich war bzw. das Fundusfoto nicht auswertbar war betes mellitus ohne Fundusveränderungen ist bei der ärztlichen Untersuchung deutlich höher als bei der IDx-DR-Analyse. Gleichzeitig wird der nicht unerhebliche Anteil an Patienten mit unzureichender Bildqualität oder fehlendem Bild aufseiten der IDx-DR-Auswertung deutlich (40,9 %). Funduskopisch war bei allen untersuchten Patienten eine Stadieneinteilung der diabetischen Retinopathie möglich. Wesentlich informativer als eine summarische Gegenüberstellung aller Patienten ist es, die Übereinstimmung der Ergebnisse beider Verfahren zu individuellen Patienten im Scatter-Plot darzustellen, da hier für jeden einzelnen Patienten individuell nachvollzogen werden kann, ob die beiden Methoden im Ergebnis übereinstimmen oder voneinander abweichen (. Abb. 3). Eine Übereinstimmung beider Untersuchungen lag in 55,9 % der Fälle vor (grün schraﬃerte Kästen). Das IDx-DR-System überschätzte den Schweregrad der diabetischen Retinopathie in 40,1 % der durch das IDx-DR-System auswertbaren Fundusbilder (blau hinterlegte Kästen unterhalb). Eine kritischer einzustufende Unterschätzung des Schweregrads der diabetischen Retinopathie durch das IDx-DRSystem wurde nur in 4 % festgestellt (rot unterlegte Kästen oberhalb). In . Abb. 4 istder Vergleichder Untersuchungen durch IDx-DR mit der herkömmlichen Funduskopie nach Altersgruppen aufgeteilt. Auﬀällig ist hier, dass sich in den Altersgruppen unter 40 Jahren die Diagnosen weniger gut decken. In den Altersgruppen <20 Jahre und 20 bis 39 Jahre liegt nur bei 25,0 % bzw. 35,2 % eine exakte Übereinstimmung zwischen der auswertbaren IDx-DR-Diagnose und dem Funduskopieergebnis vor, während dies in den Altersgruppen 40 bis 59 und >59 Jahre in 69,3 % bzw. 59,6 % der Patienten der Fall war (jeweils grün hinterlegte Kästen in . Abb. 4). Im Vergleich der Diagnose durch IDxDR mit der ärztlichen Diagnose anhand des Fundusbildes zeigt sich, dass auch hier die IDx-DR-Analyse den Befund als tendenziell höhergradig einschätzt (. Abb. 5). Dem Arzt liegen bei diesem Vergleich jeweils nur die insgesamt 4 Fundusfotos eines Patienten zur Bewertung vor, die auch von IDxDR bewertet wurden. Eine Übereinstimmung war bei 49,8 % der Patienten der Fall, für die eine auswertbare IDx-DR-AnaDer Ophthalmologe 7 · 2022 709 Originalien Abb. 5 8 VergleichderDiagnosendurchIDx-DRmit derärztlichenDiagnose anhand der Fundusfotograﬁen lyse vorlag (grün hinterlegt in . Abb. 5). Bei 43,7 % der Patienten mit auswertbarer IDx-DR-Analyse überschätzt das System den Schweregrad der diabetischen Retinopathie (blau hinterlegt in . Abb. 5). Das IDx-DR-System unterschätzt den Grad der diabetischen Retinopathie bei 1,35 % der Patienten, für die eine auswertbare IDx-DR-Analyse vorlag (rot hinterlegt in . Abb. 5). Insgesamt 25 Patienten (4,97 % vom Gesamtkollektiv bzw. 8,25 % des Kollektivs mit vorliegendem Fundusfoto), deren Fundusfotograﬁen durch IDx-DR nicht auswertbar waren, konnten durch den Arzt beurteilt werden und zeigten meist keine, in 4 Fällen aber auch eine schwere diabetischeRetinopathie. Es gab indem Datensatz auch insgesamt 15 Patienten (2,98 % vom Gesamtkollektiv bzw. 5 % des Kollektivs mit vorliegendem Fundusfoto), bei denen der Arzt anhand der Fundusbilder keine Stadieneinteilung der diabetischen Retinopathie vornehmen konnte, der IDx-DRAlgorithmus aber einen Schweregrad angab. Die Ergebnisse der Funduskopie deckten sich bei 8 dieser Patienten mit der IDxDR-Analyse. In 7 Untersuchungen zeigte sich eine Abweichung in der Beurteilung durch den Algorithmus. Dabei stellten wir mittels Funduskopie immer einen milderen besseren Befund fest. Je einmal lag die Diﬀerenz der Diagnose bei 2 (mild zu schwer) und bei 3 Stufen (keine zu schwer). Wichtig ist es auch zu untersuchen, wie sich der Schweregrad der diabetischen Re- 710 Der Ophthalmologe 7 · 2022 Abb. 6 8 Vergleich der ärztlichen Diagnose durch Funduskopie bei Patienten, für die entweder keine Fundusaufnahme in Miosis gewonnen werden konnte oder diese für IDx-DR nicht auswertbar war tinopathie bei denjenigen Patienten darstellt, für die entweder kein Bild für die automatisierte Auswertung gewonnen werden konnte oder bei denen das Bild von unzureichender Qualität für eine IDx-DRAnalyse war, da bei einem rein automatisierten Screening diese Patienten keinerlei Diagnose bekommen könnten. In diesem Zusammenhang stellten wir fest, dass bei allen Patienten, bei denen kein Foto möglich war oder die Aufnahme von unzureichender Qualität war, eine binokulare Funduskopie durchführbar und eine Schweregradeinteilung der DR durch den Arzt möglich war. Die . Abb. 6 zeigt, dass der größte Anteil der Patienten (20,7 % vom Gesamtkollektiv, grün hinterlegt in . Abb. 6), für die kein (ausreichendes) Fundusfoto für die IDx-DR-Analyse möglich war, keine diabetischen Netzhautveränderungen aufweist, also die mangelnde Aufnahmequalität nicht durch diabetogene Augenveränderungen zu erklären ist. Allerdings zeigten sich bei immerhin 4,6 % der Patienten in dieser Gruppe auch schwere, visusbedrohende Stadien der diabetischen Retinopathie, die bei einer alleinigen Diagnostik mittels automatisierter Fundusuntersuchung mangels adäquaten Bildmaterials nicht diagnostiziert worden wären (rot hinterlegt in . Abb. 6). Der Anteil an funduskopisch festgestellten milden Veränderungen ist in . Abb. 6 gelb hinterlegt und beträgt 11,3 %. Moderate Fundusveränderungen sind bei 4,4 % der Patienten dokumentiert (orange hinterlegt in . Abb. 6). DieErgebnisseder Berechnung der Sensitivität und Speziﬁtät sind in . Tab. 1 dargestellt. Dabei unterteilten wir die Werte für die jeweiligen Diagnosegruppen (keine DR, mild, moderat, schwer). Die Ergebnisse sind dreistuﬁg dargestellt: (1) das Kollektiv mit auswertbarer IDx-DR-Analyse, (2) das Kollektiv einschließlich nichtauswertbarer IDx-DR-Analyse und (3) das gesamte Kollektiv inklusive der Patienten, bei denen keine Aufnahme erfolgen konnte. Die höchste Sensitivität (0,957) und Speziﬁtät (0,891) erreicht IDx-DR bei Patienten mit einer schweren DR. Die Bewertung der Übereinstimmung zwischen der IDx-DR-Diagnose und der Bildauswertung anhand des Fundusfotos sowie zwischen der IDx-DR-Diagnose und der Funduskopie erfolgte statistisch durch Ermittlung des Kappa-Koeﬃzienten nach Cohen. Die Interpretation nach Landis und Koch beschreibt bei Kappa ≤ 0 eine schlechte, Kappa = 0–0,2 eine geringe, bei Kappa = 0,21–0,40 eine ausreichende, bei Kappa = 0,41–0,60 eine moderate, bei Kappa = 0,61–0,80 eine beachtliche und bei Kappa = 0,81–1,00 eine (fast) vollkommene Übereinstimmung. Der KappaKoeﬃzient zeigt mit 0,334 (p < 0,001) eine ausreichende Übereinstimmung zwischen IDx-DR und ärztlicher Bildauswertung anhand des Fundusfotos unter Berücksichtigung aller Patienten mit auswertbarer Tab. 1 Statistische Auswertung von Sensitivität, Speziﬁtät und positivem/negativem prädiktivem Wert (PPW/NPW) Diagnose + Gruppierung Sensitivität Spezifität Schwere DR und IDx-DR auswertbar 0,957 0,891 Schwere DR und IDx-DR auswertbar/nicht auswertbar 0,756 0,697 Schwere DR und IDx-DR auswertbar/nicht auswertbar + kein Bild 0,652 0,667 Moderate DR und IDx-DR auswertbar 0,536 0,907 Moderate DR und IDx-DR auswertbar/nicht auswertbar 0,520 0,705 Moderate DR und IDx-DR auswertbar/nicht auswertbar + kein Bild 0,514 0,672 Milde DR und IDx-DR auswertbar 0,328 0,724 Milde DR und IDx-DR auswertbar/nicht auswertbar 0,426 0,619 Milde DR und IDx-DR auswertbar/nicht auswertbar + kein Bild 0,442 0,600 Keine DR und IDx-DR auswertbar 0,585 0,908 Keine DR und IDx-DR auswertbar/nicht auswertbar 0,545 0,696 Keine DR und IDx-DR auswertbar/nicht auswertbar + kein Bild 0,540 0,642 Tab. 2 Ausschlusskriterien und Kontraindikationen für eine IDx-DR-Analyse [9] IDx-DR-Ausschlusskriterien: Okuläre Symptome (Verschwommensehen ...) Schwangerschaft Vorbekannte schwere nichtproliferative DR, proliferative DR, Makulaödem, Strahlenretinopathie, retinale Venenverschlüsse Zustand nach IVOM Zustand nach Laserkoagulation Zustand nach Netzhautchirurgie IDx-DR-Kontraindikationen für Fundusfotografie: Lichtempﬁndlichkeit Vor Kurzem durchgeführte photodynamische Therapie Medikamenteneinnahme, die zu Photosensitivität führt IDx-DR-Analyse. Der Vergleich zwischen IDx-DR mit der ärztlichen Funduskopie ergibt unter denselben Voraussetzungen eine geringe Übereinstimmung mit einem Kappa-Wert von 0,168 (p < 0,001). Diskussion Das Beispiel der diabetologischen Schwerpunktklinik in Karlsburg zeigt, wie ein sinnvoller Einsatz für die Anwendung von künstlicher Intelligenz mittels IDx-DR in Ergänzung zur augenärztlichen Sprechstunde vor Ort aussehen könnte. In einem ersten Schritt könnten Patienten mithilfe der KI-unterstützten Bildanalyse durch das IDx-DR-System vorab ein Screening auf diabetische Retinopathie erhalten. Screeningbefunde, die Netzhautpathologien aufzeigen, müssen dann augenärztlich abgeklärt werden. Ebenso müssen alle Patienten, für die keine KI-gestützte Auswertung vorliegt, weil kein ausreichendes Bildmaterial in Miosis gewonnen werden kann, zusätzlich augenärztlich untersucht werden. Bei Patienten, für die das IDx-DRSystem ein auswertbares Bild erhielt und „keine diabetische Retinopathie“ angab, lag dagegen in keinem Fall laut funduskopischer augenärztlicher Diagnose eine schwere (behandlungsbedürftige) und nur in 1 Fall eine moderate diabetische Retinopathie vor. Es ist bekannt, dass eine nicht zu unterschätzende Anzahl an Diabetikern, die empfohlenen Screeninguntersuchungen nicht wahrnimmt. In einer amerikanischen Untersuchung wird angegeben, dass etwa 40 % der Diabetiker die von der American Academy of Ophthalmology empfohlenen Untersuchungsintervalle nicht einhalten [1, 6]. Durch die schnelle und einfache Durchführung der IDx-DR-Untersuchung kann möglicherweise die Bereitschaft dieser Patienten erhöht werden, sich zu einem Screening vorzustellen. Die nichtmydriatische Untersuchung gestaltet sich für den Patienten zeiteﬃzient und weniger unangenehm. Es ist gut vorstellbar, dass dadurch die Motivation für eine Screeninguntersuchung ansteigt. Sollte durch die IDx-DR-Analyse eine milde, moderate oder visusbedrohende diabetische Retinopathie erkannt werden, wäre auch bei symptomlosen Patienten möglicherweise die Bereitschaft zur fachärztlichen Untersuchung in Mydriasis erhöht. Auch ausbleibende Untersuchungsergebnisse nach erfolgter IDx-DR-Analyse könnten den Patienten zum Nachdenken bewe- PPW 0,423 0,158 0,174 0,375 0,141 0,150 0,224 0,253 0,261 0,917 0,739 0,656 NPW 0,996 0,974 0,947 0,949 0,940 0,924 0,816 0,781 0,770 0,559 0,492 0,525 gen und die Motivation für nachfolgende augenärztliche Kontrollen erhöhen. Die Arbeit von Verbraak et al. gab auf Basis ihrer Studienergebnisse eine sichere Verwendung des IDx-DR in der Grundversorgung an. Hier wurden die Ergebnisse der IDx-DR mit einem unabhängigen Referenzzentrum verglichen. Die Sensitivität lag bei 100 % und die Speziﬁtät bei 97,8 % für die visusbedrohende DR [7, 11]. Unsere Berechnungen für die Gruppe der schweren, visusbedrohenden DR ergaben eine Sensitivität von 65,2–95,7 % und eine Speziﬁtät von 66,7–89,1 %, je nachdem ob nur das Kollektiv mit verwertbaren Aufnahmen berücksichtigt wird (Sensitivität 95,7 %) oder ob auch Patienten in die Analyse einbezogen werden, für die keine Aufnahme vorlag oder diese nicht auswertbar war (Sensitivität 65,2 %). Aus unserer Sicht muss der Aspekt der in Miosis auswertbaren Fundusaufnahmen in die gesamte Beurteilung mit aufgenommen werden (s. . Tab. 1), um eine umfängliche Aussage zu ermöglichen. Hier decken sich unsere Ergebnisse im Übrigen auch mit den Zulassungsstudien, die für verwertbare Fundusaufnahmen in 23,6 % eine Pupillendilatation benötigten [1]. Grundsätzlich bestätigen unsere Daten sowie die Daten anderer Arbeitsgruppen, dass das IDx-DR-System in vielen Fällen eine valide Einschätzung über das Vorliegen einer diabetischen Retinopathie geben kann [7, 8]. In Fällen, in denen IDxDR den Schweregrad der diabetischen Retinopathie im Vergleich zum Augenarzt überschätzt, lag dieser Unterschied meist im Rahmen von nur einer Diagnosestufe. Der Ophthalmologe 7 · 2022 711 Originalien In unserer Studie wurde im Cohens KappaTest eine geringe Übereinstimmung zwischen IDx-DR-Analyse und ärztlicher Funduskopie gefunden. Wir begründen diese Beobachtung dadurch, dass die IDx-DRAnalyse in etwa 40 % der Fälle ein schwereres Stadium angibt als der Augenarzt und eine volle Übereinstimmung mit dem Augenarzt nur in etwa 50 % der Fälle vorlag. Eine als medizinisch kritisch zu wertende Unterschätzung des Schweregrads der diabetischen Retinopathie kam allerdings nur in ca. 4 % der Fälle vor. Nicht vergessen werden darf, dass IDxDR nur zur Erkennung von diabetogenen Veränderungen entwickelt wurde und andere Augenerkrankungen nicht erkennen kann. Das System kann daher sicherlich nicht die allgemeine augenärztliche Untersuchung ersetzen, jedoch, wie oben skizziert, ergänzen. Zu beachten ist außerdem, dass IDx-DR 4 Fundusfotograﬁen aus 2 Augen benötigt, um einen Analysebericht zu erstellen. Eine Auswertung von nur 1 Auge ist nicht möglich. Somit können einäugige Patienten oder Patienten mit einseitigen optischen Trübungen, bei denen nur auf 1 Auge suﬃziente Abbildungsqualitäten erreicht werden, nicht mittels IDx-DR untersucht werden. Zudem werden werkseitig von IDx Technologies eine Anzahl an weiteren Ausschlusskriterien und Kontraindikationen für eine IDxDR-Analyse angegeben (s. . Tab. 2; [9]). Diese Kriterien des Herstellers sollten sicherlich vor jedem IDx-DR-Screening zumindest berücksichtigt werden. Insgesamt betrachtet handelt es sich beim Diabetes mellitus um eine Erkrankung, die oft ältere Menschen betriﬀt. Mit zunehmendem Alter nimmt die Pupillenweite ab, und die Linsentrübungen nehmen zu. Es kann davon ausgegangen werden, dass bei einem älteren Patientenkollektiv daher eine ausreichende Aufnahmequalität evtl. schlechter zu erreichen ist als bei einem jüngeren. Allerdings zeigten unsere Untersuchungen auch, dass jugendliche Fundusreﬂexe häuﬁg zu falschen Interpretationen führen und zu einer geringeren Speziﬁtät in jüngeren Altersgruppen beitragen können (Suppl. Abb. 3). Diskutiert wurde bereits häuﬁger der mögliche Einsatzort für automatisierte KIunterstützte Screeninguntersuchungen ohne ärztliche Kontrolle. Dabei sollte si- 712 Der Ophthalmologe 7 · 2022 cherlich auch, aufbauend auf den hier gezeigten Ergebnissen, auf ein sinnvolles Anwenderspektrum geachtet werden, um Patienten mit „falsch positiven“ Befunden nicht zu verunsichern [10]. Ein unkontrollierter Einsatz in einem nicht medizinisch betreuten und kontrollierten Umfeld könnte zu falsch positiven Befunden oder einer falschen Interpretation des Untersuchungsergebnisses mit Verunsicherung oder auch einem falschen Sicherheitsgefühl der oder des Untersuchten führen. Ein solcher Einsatz von KIunterstützten Screeninguntersuchungen ohne ärztliche Indikationsstellung und Begleitung könnte auch das Vertrauen in solche Systeme langfristig schädigen. Sinnvoller erscheint dagegen ein Einsatz in Bereichen, in denen Risikopopulationen in enger Kooperation mit Augenärzten auf diabetische Retinopathie gescreent werden sollen. Dann könnten auch Faktoren wie der Visus, wahrgenommene Metamorphopsie und subjektive Beschwerden im Gesamtbild mitbeurteilt und der Patient ggf. vollständig augenärztlich abgeklärt werden. Denkbar wäre auch eine Kombination der künstlichen Intelligenz mit einer telemedizinischen Beurteilung der hochauﬂösenden Fundusfotograﬁen. Vor einem Screening-basierten Einsatz der Analysesoftware sollte geklärt sein, ob im Bedarfsfall die Ressourcen für eine zeitnahe augenärztliche Nachuntersuchung bestehen. Patienten mit auﬀälligen Befunden sollten im Rahmen der Terminvergabe priorisiert werden. Da auch Befunde ohne auswertbare Ergebnisse einer weiteren Abklärung bedürfen, muss eine suﬃziente und ggf. interdisziplinäre Kooperation mit den mitbehandelnden Praxen/Kliniken erfolgen. Im konkreten Beispiel unserer Kooperation mit der diabetologischen Schwerpunktklinik Karlsburg war eine solche enge Zusammenarbeit sehr gut realisierbar. In einem weiteren Schritt könnte bei Patienten, die nach dem Screening noch eine augenärztliche Untersuchung benötigen, der Anstieg der Zuweisungsrate untersucht werden. Neben falsch positiven Befunden zählen hier auch Patienten dazu, bei denen der Algorithmus, bedingt durch die Bildqualität, keine Diagnose stellen konnte. Andere Untersuchungen zeig- ten hier teilweise einen Anstieg der Zuweisungsrate durch KI von über 70 % [13]. Letztendlich muss die Frage gestellt werden, inwieweit der Deep-learning-Algorithmus dem Patienten und Untersucher weiterhilft. Hierzu sind insbesondere Studien nötig, die neben den ökonomischen Parametern valide Daten dazu liefern, ob auf längere Sicht bessere Visusverläufe in Patientengruppen erzielt werden können, die Zugang zu KI-gestützten Diagnosesystemen haben, im Vergleich zu Patientengruppen, die einen solchen Zugang nicht haben. Fazit für die Praxis 4 4 Unsere Ergebnisse zeigen, dass eine Unterdiagnostizierung diabetischer Fundusveränderungen durch IDx-DR eher nicht zu befürchten ist. Wesentlich häufiger sind Fehleinschätzungen, bei denen die KI einen Befund schwerwiegender als der untersuchende Augenarzt einordnet, sowie Fälle, in denen in Miosis kein ausreichendes Bildmaterial für eine KI-gestützte Diagnose erhoben werden konnte. Für die Einbindung in augenärztliche Screeningprozesse sowie zur Dringlichkeitseinstufung innerhalb einer Risikopopulation erscheint uns IDx-DR aufgrund der hier vorgestellten Daten geeignet. Es ist vorstellbar, dass die Motivation zum Screening bei denjenigen Risikopatienten erhöht wird, die ansonsten zu wenig oder gar nicht gescreent würden. Ein zu breit angelegtes KI-Screening von Patienten ohne relevantes Risiko für eine diabetische Retinopathie oder ein Screening ohne die Möglichkeit, fragliche Screeningbefunde zeitnah augenärztlich verifizieren oder falsifizieren zu können, ist dagegen nicht zu empfehlen. Korrespondenzadresse Andreas Stahl Klinik und Poliklinik für Augenheilkunde, Universitätsmedizin Greifswald Ferdinand Sauerbruch Str., 17475 Greifswald, Deutschland andreas.stahl@med.uni-greifswald.de Danksagung. Ein besonderer Dank gilt dem nichtärztlichen Funktionsteam der Klinik für Diabetes und Stoﬀwechselerkrankungen Karlsburg für die tatkräftige Unterstützung bei diesem Projekt. Förderung. Dieses Projekt wurde zum Teil unterstützt durch Mittel aus dem EYEnovative Förderpreis von Novartis (an B.G.). Abstract Funding. Open Access funding enabled and organized by Projekt DEAL. Use of artificial intelligence in screening for diabetic retinopathy at a tertiary diabetes center Einhaltung ethischer Richtlinien Background: In 2018, IDx-DR was approved as a method to determine the degree of diabetic retinopathy (DR) using artiﬁcial intelligence (AI) by the FDA. Methods: We integrated IDx-DR into the consultation at a diabetology focus clinic and report the agreement between IDx-DR and fundoscopy as well as IDx-DR and ophthalmological image assessment and the inﬂuence of diﬀerent camera systems. Results: Adequate image quality in miosis was achieved more frequently with the Topcon camera (n = 456; NW400, Topcon Medical Systems, Oakland, NJ, USA) compared with the Zeiss camera (n = 47; Zeiss VISUCAM 500, Carl Zeiss Meditec AG, Jena, Germany). Overall, IDx-DR analysis in miosis was possible in approximately 60% of the patients. All patients in whom IDx-DR analysis in miosis was not possible could be assessed by fundoscopy with dilated pupils. Within the group of images that could be evaluated, there was agreement between IDx-DR and ophthalmic fundoscopy in approximately 55%, overestimation of severity by IDx-DR in approximately 40% and underestimation in approximately 4%. The sensitivity (speciﬁcity) for detecting severe retinopathy requiring treatment was 95.7% (89.1%) for cases with fundus images that could be evaluated and 65.2% (66.7%) when all cases were considered (including those without images in miosis which could be evaluated). The kappa coeﬃcient of 0.334 (p < 0.001) shows suﬃcient agreement between IDxDR and physician’s image analysis based on the fundus photograph, considering all patients with IDx-DR analysis that could be evaluated. The comparison between IDxDR and the physician’s funduscopy under the same conditions shows a low agreement with a kappa value of 0.168 (p < 0.001). Conclusion: The present study shows the possibilities and limitations of AI-assisted DR screening. A major limitation is that suﬃcient images cannot be obtained in miosis in approximately 40% of patients. When suﬃcient images were available the IDx-DR and ophthalmological diagnosis matched in more than 50% of cases. Underestimation of severity by IDx-DR occurred only rarely. For integration into an ophthalmologist’s practice, this system seems suitable. Without access to an ophthalmologist the high rate of insuﬃcient images in miosis represents an important limitation. Interessenkonflikt. B. Grundel weist auf folgende Beziehungen hin: Relationship: Novartis, Speciﬁcations: EYEnovative Förderpreis. S. Paul, A. Tayar, E. Morawiec-Kisiel, B. Bohl, R. Großjohann, E. Hunfeld, M. Busch, J.M. Pfeil, M. Dähmcke, T. Brauckmann, S. Eilts, M.-C. Bründer, M. Grundel, F. Tost, J. Kuhn, J. Reindel, W. Kerner und A. Stahl geben an, dass kein Interessenkonﬂikt besteht. Die Studie wurde in Übereinstimmung mit der Deklaration von Helsinki und nach positivem Votum der Ethikkommission Greifswald durchgeführt. Open Access. Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenz veröﬀentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem 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. Keywords IDx-DR · Artiﬁcial intelligence · Screening · Diabetic retinopathy · Telemedicine Literatur 1. Abràmoﬀ MD, Lavin PT, Birch M et al (2018) Pivotal trial of an autonomous AI-based diagnostic system for detection of diabetic retinopathy in primary care oﬃces. NPJ Digit Med 1:39. https://doi.org/10. 1038/s41746-018-0040-6 2. ACCORD Study Group, ACCORD Eye Study Group, Chew EY et al (2010) Eﬀects of medical therapies on retinopathy progression in type 2 diabetes. N Engl J Med 363:233–244. https://doi.org/10. 1056/NEJMoa1001288 3. Bundesärztekammer (BÄK), Kassenärztliche Bundesvereinigung (KBV), Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften (AWMF) (2015) Nationale VersorgungsLeitlinie Prävention und Therapie von Netzhautkomplikationen bei Diabetes – Langfassung, 2. Auﬂage. Version 2. www. netzhautkomplikationen.versorgungsleitlinien. de. Zugegriﬀen: 26. Nov. 2021. https://doi.org/10. 6101/AZQ/000318 4. Early Treatment Diabetic Retinopathy Study Research Group (1991) Fundus photographic risk factors for progression of diabetic retinopathy. ETDRS report number 12. Ophthalmology 98:823–833 5. FDA (2018) FDA permits marketing of artiﬁcial intelligence-based device to detect certain diabetes-related eye problems. https://www. fda.gov/news-events/press-announcements/ fda-permits-marketing-artiﬁcial-intelligencebased-device-detect-certain-diabetes-relatedeye. Zugegriﬀen: 21. März 2021 6. Flaxel CJ, Adelman RA, Bailey ST et al (2020) Diabetic retinopathy preferred practice pattern. Ophthalmology 127:P66–P145. https://doi.org/ 10.1016/j.ophtha.2019.09.025 7. Goldhagen BE, Al-Khersan H (2020) Diving deep into deep learning: an update on artiﬁcial intelligence in retina. Curr Ophthalmol Rep 8:121–128. https://doi.org/10.1007/s40135-02000240-2 8. van der Heijden AA, Abramoﬀ MD, Verbraak F et al (2018) Validation of automated screening for referable diabetic retinopathy with the IDx-DR device in the Hoorn Diabetes Care System. Acta Ophthalmol (Copenh) 96:63–68. https://doi.org/ 10.1111/aos.13613 9. Indications for use. https://dxs.ai/products/idx-dr/ indications-for-use/. Zugegriﬀen: 21. März 2021 10. Treder M, Diener R, Eter N (2020) Artiﬁcial intelligence in management of macular edema: opportunities and challenges. Ophthalmol Z Dtsch Ophthalmol Ges 117:989–992. https://doi.org/10. 1007/s00347-020-01110-9 11. Verbraak FD, Abramoﬀ MD, Bausch GCF et al (2019) Diagnostic accuracy of a device for the automated detection of diabetic retinopathy in a primary care setting. Diabetes Care 42:651–656. https://doi. org/10.2337/dc18-0148 12. Williams LB, Prakalapakorn SG, Ansari Z, Goldhardt R (2020) Impact and trends in global ophthalmology. Curr Ophthalmol Rep. https://doi. org/10.1007/s40135-020-00245-x 13. Wintergerst MWM, Bejan V, Hartmann V, Schnorrenberg M et al (2021) Telemedical diabetic retinopathy screening in a primary care setting: quality of retinal photographs and accuracy of automated image analysis. Ophthalmic Epidemiol 20:1–10. https://doi.org/10.1080/09286586.2021. 1939886 Der Ophthalmologe 7 · 2022 713
https://openalex.org/W4293071441	http://ojs.uhnsugriwa.ac.id/index.php/IJHSRS/article/download/806/568	English	null	THE WORK CULTURE INTEGRATION OF HINDU RELIGIOUS EXTENSION AGENTS WITH PARISADA IN COMMUNITY DEVELOPMENT IN TABANAN	Vidyottama Sanatana international journal of Hindu science and religious studies	2,022	cc-by	9,915	Vol. 6 No. 1 May 2022 THE WORK CULTURE INTEGRATION OF HINDU RELIGIOUS EXTENSION AGENTS WITH PARISADA IN COMMUNITY DEVELOPMENT IN TABANAN By: I Wayan Sukabawa 1, A.A. Made Putra Arsana2 Universitas Hindu Negeri I Gusti Bagus Sugriwa Denpasar E-mail: sukabawa@gmail.com Received: March 23, 2022 Accepted: April 30, 2022 Published: May 30, 2022 Abstract This study aimed to uncover, understand, describe, and analyze facts about the work cultur integration of the Hindu Religious Extension Agent with Parisada in Community Development in Tabanan. The work culture established by the Hindu institution between the Hindu Religiou Extension Agents and Parisada intended to improve work ethics as a service to the people. This i important to do to improve a positive image as a religious institution for religious communitie under the Ministry of Religious Affairs in the community. The work culture has been agreed upon which are the right and appropriate values to improve the state apparatus performance, namely Hindu Religious Extension Agents to provide excellent service to the community. By: I Wayan Sukabawa 1, A.A. Made Putra Arsana2 Universitas Hindu Negeri I Gusti Bagus Sugriwa Denpasar E-mail: sukabawa@gmail.com Received: March 23, 2022 Accepted: April 30, 2022 Published: May 30, 2022 Abstract This study aimed to uncover, understand, describe, and analyze facts about the work culture integration of the Hindu Religious Extension Agent with Parisada in Community Development in Tabanan. The work culture established by the Hindu institution between the Hindu Religious Extension Agents and Parisada intended to improve work ethics as a service to the people. This is important to do to improve a positive image as a religious institution for religious communities under the Ministry of Religious Affairs in the community. The work culture has been agreed upon, which are the right and appropriate values to improve the state apparatus performance, namely Hindu Religious Extension Agents to provide excellent service to the community. Keywords: Work Culture Integration; Hindu Religious Extension Agents; Parisada; Community Development Vol. 6 No.1 May 2022 Keywords: Work Culture Integration; Hindu Religious Extension Agents; Parisada; Community Development I. INTRODUCTION I. INTRODUCTION The Tabanan Government Regency continues to strive to improve human resources in various fields, especially in the education field, mental, and moral strengthening. It because the advancement of an area is caused by advancing education and mental education based on religion. This is carried out based on national education objectives, namely developing abilities and shaping the character and civilization of a nation with dignity to educate the nation’s life. It also aims to develop students' potential to become human beings who believe and fears God Almighty, noble, capable, creative, independent, democratic, and responsible citizens, article 39, paragraph 1 (Sisdiknas No. 20 of 2003). The essence of national development is human development as a whole. Human development as a whole means helping to improve his personal qualities and creating a balanced climate so that the quality and human resources can increase. y p g An institution is a system consisting of components (subsystems) that are interrelated or dependent (interdependence) with one another in the work process of Hindu Religious Extension Agents with Parisada. The interdependent subsystems are goals and values (goals and values subsystem), technical (technical system), management (managerial system), and sub- structure (structural system). In the process of interaction between one and another subsystem, there is a guarantee that will always be a match between Hindu Religious Extension Agents and Parisada and society or the compatibility between individuals and their implementers. The existence of work culture in a developed organization in an institution will influence the behavior of the members of Hindu Religious Extension Agents. The process of achieving progress, increasing human resources as well as in the fields of development and education, all of these cannot be separated from the readiness of the Hindu Religious Extension Agents with Parisada as a leader who guides Hindu communities and institutions in providing services that can compete in the Industrial Revolution 4.0 era. In that era, Hindus must be able to fight and win the battle for the future. The quality of a good leader is the main basis for preparing human resources that can compete in the global world, certainly, it is required a work culture sovereignty between Hindu Religious Extension Agents and Parisada as Hindu leaders in a professional manner in carrying out the task of coaching Hindus in the Tabanan area. g After the 1998 reformation, the Ministry of Religious Affairs has indeed shown many improvements. Vol. 6 No.1 May 2022 1 and assistance in the form of materials to support the development of the interests of Hindus that are directed towards achieving a harmonious life. PHDI Dharma Duta Institute is a religious institution formed by the central Parisada Hindu Dharma Indonesia (PHDI) with the Decree of Central Parisada Hindu Dharma Indonesia Number: 34/SK/PARISADA PUSAT/II/2013, February 19, 2013. Its duties to help Parisada Hindu Dharma Indonesia by fostering Hindus throughout Indonesia through enlightenment activities or Hindu Dharma Wacana. This existence has been formed by the Institutions in each Province and Regency to assist the community development in the regions. An institution is a system consisting of components (subsystems) that are interrelated or dependent (interdependence) with one another in the work process of Hindu Religious Extension Agents with Parisada. The interdependent subsystems are goals and values (goals and values subsystem), technical (technical system), management (managerial system), and sub- structure (structural system). In the process of interaction between one and another subsystem, there is a guarantee that will always be a match between Hindu Religious Extension Agents and Parisada and society or the compatibility between individuals and their implementers. The existence of work culture in a developed organization in an institution will influence the behavior of the members of Hindu Religious Extension Agents. and assistance in the form of materials to support the development of the interests of Hindus that are directed towards achieving a harmonious life. PHDI Dharma Duta Institute is a religious institution formed by the central Parisada Hindu Dharma Indonesia (PHDI) with the Decree of Central Parisada Hindu Dharma Indonesia Number: 34/SK/PARISADA PUSAT/II/2013, February 19, 2013. Its duties to help Parisada Hindu Dharma Indonesia by fostering Hindus throughout Indonesia through enlightenment activities or Hindu Dharma Wacana. This existence has been formed by the Institutions in each Province and Regency to assist the community development in the regions. Vol. 6 No.1 May 2022 II. METHOD II. METHOD The type of research used in this research was qualitative research. Qualitative methods are often called naturalistic research methods because it is carried out in natural conditions (Sugiyono, 2007: 7-8). Furthermore, Suprayogo (2001: 9) states that qualitative research aims to understand (understanding) the meaning shown in the community’s behavior according to the community’s perspective. Because it was understanding, the research data was naturalistic. The method was inductive, while the reporting was descriptive. In essence, this research observes the integration of Hindu Religious Extension Agents with Parisada. In qualitative research, data collection can be done in natural settings (natural conditions), primary data sources, and data collection techniques more on participant observation, in- depth interviews, literature study, and documentation. According to Bogdan and Biklen (1982: 74) in Satori and Komariah (2010: 179- 180) explained that after the author does observations, interviews, or research, the researcher must rewrite what is found based on the data collected into a written form or on a computer, tells about what happened and was found at the research location, namely in Tabanan related to the integration of Hindu Religious Extension Agents with Parisada in community development, researchers describe people, objects, places, events, activities, and conversations. When carrying out activities, it can help researchers express ideas, strategies, and reflections in the form of notes. It can be concluded that field notes are written notes about what is heard, seen, experienced, and thought to collect data and reflect on data in qualitative research. The type of research used in this research was qualitative research. Qualitative methods are often called naturalistic research methods because it is carried out in natural conditions (Sugiyono, 2007: 7-8). Furthermore, Suprayogo (2001: 9) states that qualitative research aims to understand (understanding) the meaning shown in the community’s behavior according to the community’s perspective. Because it was understanding, the research data was naturalistic. The method was inductive, while the reporting was descriptive. In essence, this research observes the integration of Hindu Religious Extension Agents with Parisada. Work culture is inherent in the community institutions, either big or small organization, wherever or whenever, a work culture will be found, including in government bureaucratic organizations. I. INTRODUCTION This can be seen from their seriousness in improving the performance of the state apparatus as a community service, improving the system, the realization of good and clean governance. Last but not least, efforts to foster a work culture, to keep the flow of good change in the Ministry of Religious Affairs running on the right track, thus the agreed and determined work culture needs to be implemented consistently. The work culture management of Hindu Religious Extension Agents with Parisada in fostering Hindus in Tabanan to create work discipline, in the process of achieving progress, increasing human resources, it is necessary to have commitment and efforts from a leader, in this case, is Parisada. This effort can be made through a process of coaching in the form of lectures in increasing religious understanding, The behavior consistency of Hindu Religious Extension Agents at the Ministry of Religious Affairs Tabanan is part of the organizational culture in dealing with the environment. However, that consistency can encourage or Vol. 6 No.1 May 2022 2 Parisada as a representative of the Ministry of Religious Affairs in the related district to coaching Hindus in Tabanan. otherwise hinder organizations from responding to environmental changes. In other words, although consistency can improve organizational functions as an institution, not all consistency is beneficial for the organization (Evers & Lakomski, 1992). The reason is the performance of Hindu Religious Extension Agents with the performance of Parisada and how the sense of belonging to the bureaucracy cannot be properly understood, except by understanding the culture. II. METHOD In this research, the work culture of the religious extension agents with Parisada in coaching Hindus in Tabanan was the subject of research related to work culture in community development, as a foundation for building the morale of the Hindus younger generation. The research subject was chosen based on the consideration that there is a section head, each section head in the Ministry of Religious Affairs Tabanan has the main duty to provide coach and service to the community by showing a work culture related to fostering religious and religious education in the community. One of the goals is to serve and protect the interests and needs of community members in a region. Extension Agents with Parisada. In qualitative research, data collection can be done in natural settings (natural conditions), primary data sources, and data collection techniques more on participant observation, in- depth interviews, literature study, and documentation. According to Bogdan and Biklen (1982: 74) in Satori and Komariah (2010: 179- 180) explained that after the author does observations, interviews, or research, the researcher must rewrite what is found based on the data collected into a written form or on a computer, tells about what happened and was found at the research location, namely in Tabanan related to the integration of Hindu Religious Extension Agents with Parisada in community development, researchers describe people, objects, places, events, activities, and conversations. When carrying out activities, it can help researchers express ideas, strategies, and reflections in the form of notes. It can be concluded that field notes are written notes about what is heard, seen, experienced, and thought to collect data and reflect on data in qualitative research. g While the reality on the field nowadays shows that coaching the Hindu Religious Extension Agents of the Ministry of Religious Affairs Tabanan and Parisada for Hindus in coaching has not been running optimally. This causes several things; first, the process of coaching the community is still awaiting the interests of the community which means that Hindu Religious Extension Agents with Parisada as representatives in coaching Hindus are not regularly scheduled every month. Second, the work culture of Hindu Religious Extension Agents with Parisada as representatives in coaching has not been able to reach every Hindu in remote areas of Tabanan. This phenomenon was made researchers interested in researching in the Tabanan Regency, especially the Work Culture Integration of Hindu Religious Extension Agents with III. RESULTS AND DISCUSSION Work culture integration is functional integration that is expected can meet needs. Functional integration can be formed by the leader. By functioning Hindu Religious Extension Agents Vol. 6 No.1 May 2022 Vol. 6 No.1 May 2022 3 with several binding regulations into one unit. In the process of meeting the needs for progress, increasing human resources as well as in the fields of development and education, all of this is inseparable from the readiness of the Hindu Religious Extension Agents with Parisada as the guide for Hindus. The consistency of the behavior of Hindu Religious Extension Agents is part of the organizational culture in dealing with the work culture. positive impact on religious life. As an institution and shelter for Hindus, the Ministry of Religious Affairs and PHDI should not have different understandings. Different understanding should be solved to create a single and integrated formula before conducting a coach to the people. Different understandings between the Ministry of Religious Affairs and PHDI will only provide a negative dualism perspective in society. It was resolved by the unification of understanding in fostering the community by the Ministry of Religious Affairs and PHDI in Tabanan Regency. The broad understanding of culture is all manifestations and activities of human creativity, taste, and initiative. Culture is the realization of the greatness that is passed on to humans and manifests it through their way of life. Culture is also well important in education. A person will be respected if he masters both education and culture. Education without culture can be likened to a rice field without water supply, an electric cable without electricity supply, and a temple without Gods. Modern youth have lost their purpose without cultural values. Education is supposed to enhance culture. Not only through education, but also through the culture of one’s personality will be cleansed. The term for such cultural activities as ‘samskara’ which consists of; sadguna (good character), sadaachara (good practice), and sadbhaavana (good feeling). With these good qualities, humans will care about the welfare of everyone. g y The creation of integrated coaching principles is also firmly adhered to in the collaboration between Hindu Religious Extension Agents of the Ministry of Religious Affairs and PHDI Tabanan. The existence of equality and unity in counseling principles reinforce the position of the Ministry of Religious Affairs and PHDI Tabanan. III. RESULTS AND DISCUSSION Given, the similarities in these two principles also affect public trust in the Ministry of Religious Affairs and PHDI. The same principles strengthen the achievement of counseling targets in the field. This principle will be the direction in realizing the counseling targets that have been established by the Ministry of Religious Affairs and PHDI. A work culture that is positively formed by both institution of Hindu Religious Extension Agents and Parisada will be beneficial because every Hindu who is in an organization needs a positive scope in building society for the progress of Hindus in Tabanan. Implementing the work culture of Hindu Religious Extension Agents with Parisada has a deep meaning because the two leaders of community institutions will change the attitude and behavior of Hindu human resources. y The extension Agents and Parisada towards Hindus in coaching have not been running optimally. This causes several things; first, the coaching process in the community is still awaiting the interests of the community which means that Hindu Religious Extension Agents with Parisada as representatives in coaching Hindus are not regularly scheduled every month. Second, the work culture of Hindu Religious Extension Agents with Parisada as representatives in coaching has not been able to reach every Hindu in prone areas that friction by other religion. The community’s most dominant expectation towards coaching, Hindu Religious Extension Agents with Parisada is inseparable institutions and as facilitators for Hindus to guide so that Hindus receive religious enlightenment. Through this kind of training, religious values will have a Vol. 6 No.1 May 2022 The Coaching Process Carried Out Extension agents are a group of people who carry out these targeted activities. Extension agents need communication, which is a desire from some coaching to take part in achieving goals with other members. In this case, the role of a person in counseling are some members who must be informed or motivated, and others who have to make decisions. b. The Core Stage of Determining Coaching Implementation Strategy b. The Core Stage of Determining Coaching Implementation Strategy After analyzing the program, public relations problems, and people’s requests, the next step is to determine the strategy for implementing coaching. Hindu religious extension agents and Parisada can work together to equalize planning steps and strategies in conducting coaching. The counseling implementation strategy must be precise and structured, thus can ensure the conducive implementation of coaching that will be implemented. This strategy covers several things, such as determining the procedure for the implementation of counseling and other technical aspects in the counseling that will be carried out. It followed by determining the material presented in the coaching. Hindu religious extension agents with Parisada must compile the counseling or coaching material to be delivered. Coaching materials must be prepared based on the counseling program, counseling topics requested by the community, as well as current problems in Hindu life in Tabanan. Counseling materials are arranged together to be structured, systematic, easy to understand. Counseling materials avoid p gy After analyzing the program, public relations problems, and people’s requests, the next step is to determine the strategy for implementing coaching. Hindu religious extension agents and Parisada can work together to equalize planning steps and strategies in conducting coaching. The counseling implementation strategy must be precise and structured, thus can ensure the conducive implementation of coaching that will be implemented. This strategy covers several things, such as determining the procedure for the implementation of counseling and other technical aspects in the counseling that will be carried out. It followed by determining the material presented in the coaching. Extension concessions as groupings of people who are deliberately arranged to achieve certain goals. Such groups have characteristics, power, and accountability that being communicated. This division is not done randomly (random), however, it is deliberately planned to increase efforts to achieve certain goals. There are one or more centers of power that can be used to control counseling efforts that have been planned and which can be directed to achieve the objectives. This center of power should also be used to randomly reassess extension services, and refine structures deemed necessary to increase efficiency. There are efforts to replace extension agents, for example, someone whose work method is unsatisfactory can be transferred and replaced by another person. The Coaching Process Carried Out g A religious extension agent is an organization that carries out tasks in community development and has a common goal to be realized. With this goal, religious extension agent members will work together on efforts to achieve the goal. The targets to be achieved by religious extension agents are from the initial stage of the procedure, the program of the coaching implementation pattern, to the final result of the coaching Vol. 6 No.1 May 2022 Vol. 6 No.1 May 2022 4 process. To achieve the goal, facilities and infrastructure are required, such as; office, funding materials, human resources, and others. process. To achieve the goal, facilities and infrastructure are required, such as; office, funding materials, human resources, and others. promotion. a. Analysis of the Work Program Coaching carried out in collaboration must begin with analysis. The first analysis is to analyze the counseling work program in the Ministry of Religious Affairs with Parisada, Tabanan Regency. It is important to analyze each counseling program, thus it is compatible with current conditions in the community. Ura Hindu of the Ministry of Religious Affairs and Parisada of Tabanan Regency are obliged to pay close attention to their counseling work programs, thus can choose the counseling program most relevant to the current conditions of the community. This analysis is also carried out hence activities adjust to the budget that has been announced. The Hindu religious extension agents with Parisada in the sub-district have their work program that adapts to the conditions of their respective sub- districts. Work programs in each sub-district must also be analyzed first. Thus, work programs can be selected that are most relevant to the current conditions of the people in their respective regions. Each religious extension agent has a role in contributing to the success or failure of counseling. Therefore, religious extension agents are an essential resource that must be respected and maintained. Human Resources (HR) or religious extension agents must be developed and prepared to face situations and challenges in the future. This human resource development helps develop and maintain religious extension agents, thus they become reliable resources and ultimately useful in coaching the people. The extensions will also develop a sense of attachment to coaching as a result of human resource development activities. Cooperation in counseling is an activity that is achieved through a process of targeted awareness, deliberation, and coordination. Vol. 6 No.1 May 2022 c. The final stage of the coaching process c. The final stage of the coaching process When the material is arranged, Hindu religious extension agents and Parisada are obliged to determine the targets and facilities that will be obtained by the counseling participants. Determination of participants and its facilities is mandatory if counseling activities are both the Ministry and Religious Affairs and Parisada programs. Given that the counseling program from the Ministry and Religious Affairs and Parisada is linked to the budget amount. Thus the number of participants and its facilities is adjusted to the available budget. If the counseling is a request from the community, then the determination of the number of participants and its facilities is entirely up to the Customary Villages which organizes the counseling activities. Hindu religious extension agents and PHUDI were only present as resource persons and provided counseling material according to the topic raised by Customary Villages. Another obstacle during counseling is unpredictable natural events. Natural events can occur in the Tabanan region both in the lowlands and highlands. During the rainy season, for example, the access road to the counseling site is in the mountains and is filled with landslides. Limited shortcuts eventually forced the extension agents to wait for further action to handle these natural events. Although there are other shortcuts, the location is far away and takes a very long time. Thus, if forced to choose a long shortcut, in which the extension agents will be late arriving at the counseling site. On the other hand, many other natural events can occur at any time and can be obstacles for extension agents to the counseling site. Work Cultural Integrity Constraints Constraints or obstacles interpreted as experienced obstacles. The integration obstacle is also known as a disturbance (semantic or mechanical), which this disturbance is still a communication obstacle. One of the communication effectiveness will be influenced by several experienced some obstacles. Integration activities certainly will face various obstacles. Obstacles in the integration process will affect the effectiveness of the communication process. There are some obstacles experienced by the extension agents in realizing tolerance between religious communities in Tabanan, namely some obstacles that arise due to physical factors. Based on the results of field observations, there were three obstacles in implementing the extension agent integration strategy, namely physical obstacles, differences in mindset, and social environmental factors. Vol. 6 No.1 May 2022 b. The Core Stage of Determining Coaching Implementation Strategy In counseling, efforts can also be made to re- integrate extension activities by transfer or p g Hindu religious extension agents with Parisada must compile the counseling or coaching material to be delivered. Coaching materials must be prepared based on the counseling program, counseling topics requested by the community, as well as current problems in Hindu life in Tabanan. Counseling materials are arranged together to be structured, systematic, easy to understand. Counseling materials avoid Vol. 6 No.1 May 2022 5 things that can trigger negative thoughts from the community. Therefore, Hindu religious extension agents and Parisada are obliged to complement each other in compiling the counseling material to be delivered. a. Physical Obstacles Physical or organic obstacles are obstacles that occur due to geographic location. For example, because of the long-distance that difficult to reach by extension agents and transportation. Extension agents as communicators in this study experienced physical obstacles, namely regarding the distance between extension members. Physical factors become an obstacle because long distances that cause extension agents with PHDI less effective. Hindu religious extension agents cannot be separated from the human character of not having access to many roads, especially to reach mountainous areas. Sometimes there is only one access road (main road) to mountainous areas. This condition slows down the counseling time if there are obstacles on the main road. The lack of short-cut access availability causes extension agents to wait for a conducive main road situation. Besides the lack of road access, thus the access to the mountains tends to be damaged. Road damage is almost always found on several access roads to the mountainous region of Tabanan. Damaged roads cause delays in counseling time. Extension agents are required to drive very carefully, thus it takes a lot of time on the way to the extension location. c. The final stage of the coaching process Many extension agents have short Hindu religious backgrounds and are confused when they get questions or ask for solutions from the community. This shows that the depth of religious knowledge among Hindu extension agents with Hindu scholarly background is not optimal. This condition is also an obstacle in developing counseling programs. Given that the counseling program requires Hindu human resources who have maximum skills in Hindu religious knowledge because of the inadequate level of education. The education level of Hindu extension agents at backgrounds such as Bachelor of Arts and other non-Hindu graduates. Hindu religious extension agents who are not Hindu scholarly background, seem to be quite difficult in conducting counseling. Many questions about Hindu issues are not answered maximally by Hindu religious extension agents with non-Hindu scholar backgrounds. Thus, many communities asked questions to other extension agents who have a Hindu scholarly background. Lack of knowledge about Hinduism. There is a possibility if the extension agents who have Hindu religious scholarly cannot answers questions about Hindu problems from the community. When rainy and other weather anomalies, tends to cause extension agents do not dare to go to the counseling sites. Because the condition is seen as dangerous. However, the extension agents still attend even though they are delayed by waiting for possible weather conditions. On the other hand, weather anomalies also often discourage counseling participants from participating in the counseling activities. Unfavorable weather causes some people who are far from the counseling site to choose not to attend the counseling conducted by the Ministry of Religious Affairs and PHDI. The counseling is the process of changing other people’s behavior. A person can influence the attitudes, opinions, and behavior of other people if there is communicative communication from the religious extension. I Wayan Tontro explained that PHDI consists of elements as answers to questions, including communicators, messages, media, communicants, and effects. PHDI can communicate effectively if the message sent by PHDI can be received well by the people, besides that the message sent can also be understood the content of what is conveyed in the message. To overcome physical obstacles in the form of long distances between people, it is formed a division of tasks by having the task of sending messages or information provided by the Ministry of Religious Affairs to all members of the extension. c. The final stage of the coaching process With this division of tasks, information sent by the Ministry of Religious Affairs and PHDI can be well received by the people, thus messages or information can be understood properly. Many extension agents have short Hindu religious backgrounds and are confused when they get questions or ask for solutions from the community. This shows that the depth of religious knowledge among Hindu extension agents with Hindu scholarly background is not optimal. This condition is also an obstacle in developing counseling programs. Given that the counseling program requires Hindu human resources who have maximum skills in Hindu religious knowledge because of the inadequate level of education. The education level of Hindu extension agents at the Ministry of Religious Affairs in Tabanan Regency is still inadequate. Some of the extension agents are recruited from high school graduates. The Directorate General of Hindu Community Guidance Decree Number 93 of 2019 does allow the recruitment of extension agents with a high school or Utama Widya Pasraman education level if there is an area that does not have a Bachelor of Hindu background. Despite having general knowledge about Hinduism, this will be a kind of obstacle in attracting public trust in the Hindu religious extension agents. Critical communities sometimes ask about the educational background of the extension agents. When they know that there are Hindu extension agents who do not have a maximum education about Hinduism, the public’s trust in these extension agents diminishes. This condition is sufficient to cause obstacles in carrying out the counseling. Thus, this must receive attention from the government to improve the educational qualifications of Hindu religious extension agents who are still The education level of Hindu extension agents at the Ministry of Religious Affairs in Tabanan Regency is still inadequate. Some of the extension agents are recruited from high school graduates. The Directorate General of Hindu Community Guidance Decree Number 93 of 2019 does allow the recruitment of extension agents with a high school or Utama Widya Pasraman education level if there is an area that does not have a Bachelor of Hindu background. Vol. 6 No.1 May 2022 c. The final stage of the coaching process Weather anomalies disrupt the counseling process carried out by the Extension of the Ministry of Religious Affairs or PHDI Tabanan. Weather anomalies affect mobility to the extension site and the enthusiasm of participants in participating in the counseling activities. Vol. 6 No.1 May 2022 6 When rainy and other weather anomalies, tends to cause extension agents do not dare to go to the counseling sites. Because the condition is seen as dangerous. However, the extension agents still attend even though they are delayed by waiting for possible weather conditions. On the other hand, weather anomalies also often discourage counseling participants from participating in the counseling activities. Unfavorable weather causes some people who are far from the counseling site to choose not to attend the counseling conducted by the Ministry of Religious Affairs and PHDI. The counseling is the process of changing other people’s behavior. A person can influence the attitudes, opinions, and behavior of other people if there is communicative communication from the religious extension. I Wayan Tontro explained that PHDI consists of elements as answers to questions, including communicators, messages, media, communicants, and effects. PHDI can communicate effectively if the message sent by PHDI can be received well by the people, besides that the message sent can also be understood the content of what is conveyed in the message. To overcome physical obstacles in the form of long distances between people, it is formed a division of tasks by having the task of sending messages or information provided by the Ministry of Religious Affairs to all members of the extension. With this division of tasks, information sent by the Ministry of Religious Affairs and PHDI can be well received by the people, thus messages or information can be understood properly. backgrounds such as Bachelor of Arts and other non-Hindu graduates. Hindu religious extension agents who are not Hindu scholarly background, seem to be quite difficult in conducting counseling. Many questions about Hindu issues are not answered maximally by Hindu religious extension agents with non-Hindu scholar backgrounds. Thus, many communities asked questions to other extension agents who have a Hindu scholarly background. Lack of knowledge about Hinduism. There is a possibility if the extension agents who have Hindu religious scholarly cannot answers questions about Hindu problems from the community. b. Differences in Mindset The reality in the field shows that Hindu religious extension agents (especially non-Civil Servants extension agents) have a side job apart from being an extension agent. Other professions or jobs that are carried out by Hindu religious extension agents, such as staff in several village offices, teachers, entrepreneurs, and other jobs. This is due to the insufficient allowances of Hindu religious extension agents. This condition calls for non-civil servant Hindu extension agents to seek additional income through other professions. Sometimes the side profession outside of extension agents, demands a lot of time and good concentration. Thus, time and concentration on developing oneself as extension agents decrease hence perception is an interpretation of a certain thing. Perception comes from the interpretation of each Lack of innovative development of counseling materials. Hindu religious extension agents, both Civil Servants (PNS) and non-Civil Servants (PNS) are less committed to innovating, especially in terms of counseling materials. This can be observed through counseling activities during the COVID-19 pandemic, which at first glance shows a monotonous side in counseling materials. The Ministry of Religious Affairs in Tabanan Regency has a very good strategy in accommodating counseling activities during the Pandemic, which is addressed by conducting counseling on Hindu religion virtually. But slowly, it seems that there is less innovative material delivery. This can be seen through the high similarity of topics and themes of the counseling material delivered by each extension agent. Thus, this condition is sufficient to provide saturation for audiences or people who are actively listening to Hindu religious counseling through the mass media. g p g The courage to explore oneself in conducting counseling is still lacking. Many extension agents feel down when assigned or challenged to provide the counseling. It was proven that many young extension agents who were considered competent did not dare to show their Hindu religious abilities through counseling activities. Fear of a large audience still often arises when extension agents will provide the counseling. This condition is sometimes cultured and transmitted to other extension workers. Thus, young extension agents who still exist to provide counseling both on social media, TV, and Hindu counseling cooperation with the Central Ministry of Religious Affairs are only filled by extension agents who have deep knowledge and courage to provide counseling. g g Lack of opportunity (time) for self-development of the extension agents. b. Differences in Mindset b. Differences in Mindset Hindu religious extension agents within the Ministry of Religious Affairs of Tabanan Regency come from various scientific backgrounds. This condition indeed shows the diversity of knowledge in the Hindu religious extension work unit. Some of the extension agents have Hindu religious education background, either a Bachelor of Hindu Philosophy, a Bachelor of Hindu Theology, a Bachelor of Hindu Religious Education, and a Bachelor of Religion and Culture. On the other hand, some come from other scientific Vol. 6 No.1 May 2022 7 high school graduates. High school graduates and non-Hindu Bachelor graduates extension agents who have a lot of knowledge about Hinduism do not need to be dismissed or replaced. However, it should be funded or included in the education of qualification improvement program, by implementing cooperation with PTKH in the Bali region. Thus, extension agents who have a high school education or a non-Hindu scholar can have the knowledge and a degree in Hindu religious education, and are less able to sustain the effectiveness of the Hindu religious counseling program in Tabanan hence they lack the courage to provide counseling. anything that interferes with the communication process and hinders effective communication, which can make it difficult to send clear messages. messages. Lack of innovative development of counseling materials. Hindu religious extension agents, both Civil Servants (PNS) and non-Civil Servants (PNS) are less committed to innovating, especially in terms of counseling materials. This can be observed through counseling activities during the COVID-19 pandemic, which at first glance shows a monotonous side in counseling materials. The Ministry of Religious Affairs in Tabanan Regency has a very good strategy in accommodating counseling activities during the Pandemic, which is addressed by conducting counseling on Hindu religion virtually. But slowly, it seems that there is less innovative material delivery. This can be seen through the high similarity of topics and themes of the counseling material delivered by each extension agent. Thus, this condition is sufficient to provide saturation for audiences or people who are actively listening to Hindu religious counseling through the mass media. Lack of opportunity (time) for self-development of the extension agents. Hindu religious extension agents at the Ministry of Religious Affairs do not have time to focus on developing themselves as extension agents. This is caused by the concentration of the extension agents who are not only focused on their duties as an extension agent. Vol. 6 No.1 May 2022 b. Differences in Mindset Mindset is a way of assessing and making conclusions on something based on a certain point of view. Mindset differences are caused by a different number of points of view that are used as a basis or reason. The number of perspectives to think is influenced by emotion, knowledge, and experience. In overcoming some obstacles arising from differences in the mindset of Hindus, Pasraman, or other educational activities is carried out in implementing a coaching activity. It carried out so that society knows and understands the culture of each community. It hoped that the mindset of the culture and background of each community could be synchronized. Life as a human being is never free from challenges. Philosophers say that people who have no challenges in life are the same as dying. The higher and harder the challenge, the more meaningful it will be in life. In general, the most difficult challenges faced in human life today are poverty, ignorance, and backwardness. In religious life, the most basic and severe poverty in the challenge of humanity is the moral poverty of srada and bhakti. It is easy to overcome material poverty, but the most severe is moral poverty. For many people who are well established economically, their social status will be destroyed by moral poverty. This can be proven that the correctional institution is filled with people whose economic status and social status are already good. There are three challenges faced in religious life today, namely: a) anarchists, always impose their will to destroy and destroy the established order, such as violent demonstrations, b) a sadistic attitude, which is beyond the primal attitude of humanity, c) the decline of the moral community. These three challenges initially developed in the big cities of Metropolitan, but the reality, it has spread to all corners of the village. This is evident from the fact that there are many anarchic and brutal attitudes of the community in facing their problems in the Tabanan area. The conflict case in Customary Villages has proven these three elements, which have spread in all circles. Balinese people used to be very friendly polite b. Differences in Mindset This difference in mindset produces a different point of view from each community. This can hamper the implementation of the coaching strategy carried out by the extension agent. Mindset is a way of assessing and making conclusions on something based on a certain point of view. Mindset differences are caused by a different number of points of view that are used as a basis or reason. The number of perspectives to think is influenced by emotion, knowledge, and experience. In overcoming some obstacles arising from differences in the mindset of Hindus, Pasraman, or other educational activities is carried out in implementing a coaching activity. It carried out so that society knows and understands the culture of each community. It hoped that the mindset of the culture and background of each community could be synchronized. individual, thus one person can define or interpret a word in different ways. A person’s perspective on what he heard, saw, and understood is shaped by his cultural background, family, education, and personal experiences. Therefore, it is very wise if someone can communicate their thoughts well with high flexibility, according to others and within oneself, thus communication barriers that occur can be minimized. The community’s mindset becomes an obstacle for the extension agents in carrying out the coaching strategy. Barriers to the frame of mind are obstacles to the formation that can occur due to differences in mindset. Different mindsets occur due to differences in backgrounds and cultures. from the community becomes an obstacle in carrying out the coach done by the extension agents. Environmental factors affect the running of the coaching process. This can be called a natural thing as well as an artificial thing, which means that is natural due to disturbances caused by natural disturbances. Artificial or deliberate factors, it is also one of the causes of the disturbance. Sociological factors, there are two types of association in community life. Types of association lead to differences in character. The difference in the types of association causes character difference, thus it can lead to different treatment in coaching. Anthropological factors, these obstacles occur because of differences in humans, such as in body posture, skin color, and culture. g This difference in mindset produces a different point of view from each community. This can hamper the implementation of the coaching strategy carried out by the extension agent. Vol. 6 No.1 May 2022 b. Differences in Mindset Hindu religious extension agents at the Ministry of Religious Affairs do not have time to focus on developing themselves as extension agents. This is caused by the concentration of the extension agents who are not only focused on their duties as an extension agent. The reality in the field shows that Hindu religious extension agents (especially non-Civil Servants extension agents) have a side job apart from being an extension agent. Other professions or jobs that are carried out by Hindu religious extension agents, such as staff in several village offices, teachers, entrepreneurs, and other jobs. This is due to the insufficient allowances of Hindu religious extension agents. This condition calls for non-civil servant Hindu extension agents to seek additional income through other professions. Sometimes the side profession outside of extension agents, demands a lot of time and good concentration. Thus, time and concentration on developing oneself as extension agents decrease hence perception is an interpretation of a certain thing. g p g Lack of ability to master the counseling audience. Extension agents who have dared to appear sometimes experience problems in the form of a lack of ability to master the audience. Nerves (communication anxiety), environmental or ecological are also experienced by religious extension workers. This is caused by the community’s mindset that is not fully understood and there are striking differences with what the religious extension agents understand in the community. Hindu religious counseling can be effective if there is a common meaning in the message received by the counseling participants. In the implementation of counseling activity, sometimes religious extension agents face obstacles. Obstacles in communication are Perception comes from the interpretation of each Vol. 6 No.1 May 2022 Vol. 6 No.1 May 2022 Vol. 6 No.1 May 2022 8 individual, thus one person can define or interpret a word in different ways. A person’s perspective on what he heard, saw, and understood is shaped by his cultural background, family, education, and personal experiences. Therefore, it is very wise if someone can communicate their thoughts well with high flexibility, according to others and within oneself, thus communication barriers that occur can be minimized. The community’s mindset becomes an obstacle for the extension agents in carrying out the coaching strategy. Barriers to the frame of mind are obstacles to the formation that can occur due to differences in mindset. Different mindsets occur due to differences in backgrounds and cultures. c. Social Environmental Factors The social environment is also called the social context. The social environment is defined as a physical atmosphere or a social atmosphere where humans live and interact hence it can develop. The social environment greatly influences various activities which are general activities carried out by extension agents in the Customary Villages in Tabanan, where these activities require support from the entire community. The social environment support from Customary Villages in Tabanan is greatly influential in all activities carried out by extension agents. Lack of interest and attention Balinese people used to be very friendly, polite, Vol. 6 No.1 May 2022 9 and very tight competition. It cannot be denied that conflicts always arise in human life, which is in the household, personal, group, social life, even in the life of the nation and state. A wider conflict between countries in the world as part of human civilization. Conflict arises as a consequence of social interaction. In fact that we have encountered various conflicts among customary villages that have been mentioned earlier. If we think about it conscientiously, the grave issue should not become a conflict. The village boundaries are made into conflict, where if examined more deeply, one of the elements that became the basis of the conflict is the problem of income or profit from the area. honest, hard-working, and resilient in facing challenges. In fact, at this time, many people have fallen into anarchic, sadistic, and impolite actions in facing problems. The most tragic thing is committing suicide. This humanitarian challenge should be balanced with spiritual enlightenment through various forms as outlined by Parisada Hindu Dharma Indonesia. The lack of extension agents caused the program implementation in Tabanan is less optimal. Especially in the current era of globalization which has a significant impact on the development of local indigenous cultures. Amid the intellectual progress, Hindus are accompanied by the development of modernization, where there are many cases of adat that emerged. The problems that arise show the feeling of Sagilik – saguluk paras paros sarpanaya, the coexistence is increasingly distant, more individualist, and materialistic. The resilience of traditional societies that strengthens culture is increasingly fragile. With the development of coaching roles, gradually, there will be joys and sorrows to be faced based on the concept of rwa bhineda, in which two elements are always sided by side. c. Social Environmental Factors The role of religious extension agents in fostering and providing services to the community often emerges the conflict in the community, both individual- individual and individual-community conflict. Conflict is defined as differences, contradictions, and disputes. honest, hard-working, and resilient in facing challenges. In fact, at this time, many people have fallen into anarchic, sadistic, and impolite actions in facing problems. The most tragic thing is committing suicide. This humanitarian challenge should be balanced with spiritual enlightenment through various forms as outlined by Parisada Hindu Dharma Indonesia. The lack of extension agents caused the program implementation in Tabanan is less optimal. Especially in the current era of globalization which has a significant impact on the development of local indigenous cultures. Amid the intellectual progress, Hindus are accompanied by the development of modernization, where there are many cases of adat that emerged. The problems that arise show the feeling of Sagilik – saguluk paras paros sarpanaya, the coexistence is increasingly distant, more individualist, and materialistic. The resilience of traditional societies that strengthens culture is increasingly fragile. With the development of coaching roles, gradually, there will be joys and sorrows to be faced based on the concept of rwa bhineda, in which two elements are always sided by side. The role of religious extension agents in fostering and providing services to the community often emerges the conflict in the community, both individual- individual and individual-community conflict. Conflict is defined as differences, contradictions, and disputes. Vol. 6 No.1 May 2022 d. Change in Work Culture Given that several extension agents need to present PowerPoint and others as a guide in speaking. Thus, LCD malfunction reduces the effectiveness and correctness of the material presented by extension agents. The transformation that occurs in modern society is a consequence of the fact that technology has acquired its autonomy. We do not face a human society, but a technological society. Technology consumption also means that someone consumes ideology, namely individualism. As a result, it is not surprising that someone who consumes technology can change become a selfish, egotistical, or asocial person. Thus it will lead to a bad relationship with other people. They may even not develop healthy social relationships, for example, exploiting other humans that means harmony as a reflection of a collectivist culture so that it causes alienation. understanding. One of the indicators used to determine a person’s understanding is to know the intent and purpose of the activities carried out. If there are obstacles within the person concerned during the understanding process, the next process will not be implemented, namely the obedience and compliance process. A person’s obedience and compliance process are greatly determined by several aspects, including external and internal elements. For example, external factors are a leading factor (whether government, customary, or family leaders) who are firm in implementing regulations, and many sanctions are given, thus the majority of members tend to implement them. Meanwhile, the internal factor is a factor of self-understanding of what will be implemented. If the obedience that appears comes from the external factors above, then the last stage of the institutionalization of socio- cultural values, namely the socio-cultural value appreciation stage that difficult to have. However, if the obedience that grows in the community is an understanding factor, then the communities’ respect for the socio-cultural values will be high. The appreciation process will look at how the community values an activity, which if the value given is positive (means beneficial) will tend to be repeated, then it will be preserved. On the other hand, if it is considered negative (means detrimental), it tends to be rejected. replacement of old technology with new technology. Changes in technology are often in the form of a steady change in institutionalized technology, both in society and in the market. Technology has many benefits, but technology can create problems for human life. d. Change in Work Culture LCD malfunction also made it difficult and at the same time reduced the confidence of extension agents to speaking. Given that several extension agents need to present PowerPoint and others as a guide in speaking. Thus, LCD malfunction reduces the effectiveness and correctness of the material presented by extension agents. The transformation that occurs in modern society is a consequence of the fact that technology has acquired its autonomy. We do not face a human society, but a technological society. Technology consumption also means that someone consumes ideology, namely individualism. As a result, it is not surprising that someone who consumes technology can change become a selfish, egotistical, or asocial person. Thus it will lead to a bad relationship with other people. They may even not develop healthy social relationships, for example, exploiting other humans that means harmony as a reflection of a collectivist culture so that it causes alienation. Vol. 6 No.1 May 2022 d. Change in Work Culture Culture is seen as the whole of human ideas and works that are accustomed to learning. Culture is also seen as human knowledge that is used to understand and interpret the environment and experiences as a basis for realizing behavior. Culture is a set of rules and norms that are shared by supporting community members, and if these norms are implemented, it can produce actions that can be accepted together (Haviland, 1988: 333). According to Koentjaraningrat (2005: 72- 73), culture is human social actions or patterns for human behavior. Thus, almost all the taken actions in social life that are not accustomed to learning are actions carried out due to a very limited physiological process. p y g p The process of institutionalizing the socio- cultural values of the organization at the Office of the Ministry of Religious Affairs in Tabanan Regency is inseparable from the role of the socialization of socio-cultural values itself, both government leaders, traditional leaders, or family leaders. Theoretically, the process of institutionalizing values towards society goes through several stages, including being known, understood, obeyed, and appreciated. The process of knowing the socio-cultural values of indigenous peoples through learning from parents from their childhood to adolescence. Indirectly, routine activities that are always carried out by parents will tend to be known, then perhaps learned by children. From the learning process, it will be improved into an Conflict management experts provide different definitions of conflict according to their respective expertise. Conflict is an inner atmosphere that contains anxiety because a conflict occurs between two or more motives. These contradictions and differences encourage people to engage in conflicting activities. Some types of conflicts, namely 1) Individual Conflict, 2) Group/organization and believer 3) conflict due to competition. In further structuring, customary villages experience conflicts in the community. Based on the results of monitoring, it shows that there are many sources of conflict in Tabanan in terms of ideology, politics, economy, socio- culture, ethnicity, religion, and race (Sara) due to differences in perspectives, understanding, Vol. 6 No.1 May 2022 10 replacement of old technology with new technology. Changes in technology are often in the form of a steady change in institutionalized technology, both in society and in the market. Technology has many benefits, but technology can create problems for human life. LCD malfunction also made it difficult and at the same time reduced the confidence of extension agents to speaking. e. Social Group Formation e. Social Group Formation Efforts to manage religious diversity in the context of realizing peace are not enough to rely solely on the notions of pluralism, multiculturalism, and religious pluralism, but can also be linked to local wisdom. Atmaja (2012) defines local wisdom as the result of human creativity in abstracting their social experiences in the form of ideas or concepts that are used as guidelines for action in realizing an idealized social order, namely a peaceful society. The social experiences they get are automatically related to diversity management, given that diversity is a necessity for humans. Balinese are familiar with various local wisdom (social creativities) which can guide on managing diversity in the context of strengthening pluralism, multiculturalism, and religious pluralism in realizing peace (Atmadja, 2018: 121). The public may focus on attending the counseling activity if the subject is seen as a senior, well-known, and educated figure. However, if the extension agent is seen as a junior, not very well known, then the community or the communicant does not have the focus and passion to attend the counseling. This condition becomes a challenge and an obstacle for non-Civil Servants Hindu religious extension agents. There are still many non- Civil Servants Hindu Religious Extension Agents who are young or junior. Thus, when non-Civil Servants Hindu extension agents want to provide counseling, they must be prepared with apathy from the community who is given counseling. Technological changes in the community can be seen in plain view from the presence of symptoms, namely the Value is an idea within humans hence it is abstract about good-bad, right-wrong, proper- Vol. 6 No.1 May 2022 Vol. 6 No.1 May 2022 11 inappropriate, and sublime-not-noble which is used as a recipe for action in the life of society or the state. The norms in society can be divided into several types, depending on the type of sanction and the strength or weakness of its binding power to the individual in the context of realizing the idealized ideal, namely peace. Peace can be achieved based on morality which has a much higher consideration of what is called truth and necessity. Morality can also be distinguished from the law that morality is not created or cannot be changed through legislative, executive, and judicial actions. REFERENCE Achmad Darodjat, 2015. Pentingnya Budaya kerja, Bandung: PT Refika Aditama. Arikunto, Suharsini. 2010. Prosedur Penelitian Kualitatif dan Satu Pendekatan Praktik. Yogyakarta: Rinaka Cipta. Bogdan dan Biklen (dalam Moleong, 1996) Metodologi Penelitian Kualitatif. Bandung: CV Remaja karya. Badudu-Zin. 2001. Pembinaan Karier Pegawai. Jakarta: Raja Gerafindo Persada e. Social Group Formation The sanctions imposed by morality are not like legal norms that involve physical coercion or threats, but are more internal, for example, guilt, shame, and so on. community development in Tabanan. The obstacles experienced were based on observations in the field. There were three obstacles in the implementation of the extension agent’s integration strategy, namely physical obstacles, differences in mindset, and social environmental factors. Vol. 6 No.1 May 2022 IV. CONCLUSION Chaplin, James P, 1989. Kamus Lengkap Psikologi, Terj. Kartini Kartono, Jakarta: Rajawali. The work culture integration of the Hindu religious extension agent with Parisada in community development in Tabanan. Work culture integration is a functional integration that can be formed from the rules of the work culture value system embedded in the Hindu religious extension agents with Parisada in Tabanan. This is inseparable from the role of socialization for the younger generation. The process of institutionalizing the cultural values of extension agents embedded in Hindu religious affairs at the Office of the Ministry of Religious Affairs in Tabanan Regency cannot be separated from the role of the cultural values socialization itself, both Hindu religious extension agents and Parisada, which are governed by government regulations and customary leaders. Gary Yulk, 2007. Kepemimpinan dalan Organisasi, Jakarta: Ineks. Hasibuan, SP, 2000. Manajemen Sumber Daya Manusia, Jakarta: PT. Bumi Aksara. Inspektorat Jenderal Departemen Agama, 2008. Modul Pengawasan dengan Pendekatan Agama, Jakarta: Inspektorat Jenderal Departemen Agama. Keputusan Menteri Pendayagunaan Aparatur Negara RI Nomor 25/KEP/ M.PAN/ 04/2002 Tentang Pedoman Pengembangan Budaya Kerja Aparatur Negar, Jakarta. Lincoln, Y.S. & Guba, E.G. 1985. Naturalistic Inquiry. Beverly Hills: SAGE Publikations, Inc There were several processes done in coaching carried out by the Hindu extension agents with Parisada in Tabanan, namely; program analysis, the coaching stage, and managing the coaching result stage. For the program analysis, first, the Hindu religious extension agents with Parisada made a work program; second, one provides coaching. Finally, the results of the coaching were evaluated on the condition of the people in the target area. Constraints on the cultural integrity of Hindu extension agents with Parisada in the Robins, SP, 1989. Prilaku Organisasi, Konsep Kontroversi, Aplikasi,Ed Indonesia, Jakarta: PT. Prenhallindo. Siagian, 2004. Teori Motivasi dan Aplikasi, Jakarta: Rineka Cipta. Sudiarta. 2009. Budaya Birokrasi dalam Kebijakan Pembangunan Pariwisata. Denpasar: UNHI Sugiono. 2006. Dasar-Dasar Metodologi Penelitian Kualitatif. Malang: PPS IKIP Malang. Sparingga, D. 2000. Kumpulan bahan mata Vol. 6 No.1 May 2022 Vol. 6 No.1 May 2022 12 Ajaran.- metode Penelitian Kualitatif, Surabaya: Fisip Unair. Ajaran.- metode Penelitian Kualitatif, Surabaya: Fisip Unair. Suhadi, 1.Suparno. 2003. Dasar-Dasar Metode Penelitian. Malang: Penerbit UM & LEMLIT UM. Suryadi, Sidharto,1995. Problem Pendidikan di Indonesia.Bandung: Remaja karya CV. Sugiyono. 2018. Metode Penelitian Kuantitatif, Kualitatif, dan R&D. Bandung: Alfabeta, CV. Sutrisno, Nanang. 2015. Vol. 6 No.1 May 2022 IV. CONCLUSION Transformasi Kultural Dalam Keberagamaan Umat Hindu di Kabupaten Banyuwang.Denpasar: UNHI Undang-Undang Nomor 20, Tahun 2003 tentang Sistem Pendidikan Nasional, Jakarta: Ekajaya. Wahyudi. 2009. Budaya Sekolah Unggul. Jurnal Cakrawala Pendidikan. 13
https://openalex.org/W2913382349	https://dugi-doc.udg.edu/bitstream/10256/18147/1/CapacityAdiposity.pdf	English	null	Capacity adiposity indices to identify metabolic syndrome in subjects with intermediate cardiovascular risk (MARK study)	PloS one	2,019	cc-by	8,933	☯These authors contributed equally to this work. * magomez@usal.es RESEARCH ARTICLE Background Obesity increases mortality, and is linked to cardiovascular diseases and metabolic syn- drome (MetS). Therefore, the purpose of this study was to analyze the ability of different adi- posity indices to identify subjects with MetS among people with intermediate cariovascular risk. Editor: Ying-Mei Feng, Beijing Key Laboratory of Diabetes Prevention and Research, CHINA Received: June 1, 2018 Accepted: December 16, 2018 Published: January 25, 2019 Copyright: © 2019 Gomez-Marcos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Ying-Mei Feng, Beijing Key Laboratory of Diabetes Prevention and Research, CHINA Editor: Ying-Mei Feng, Beijing Key Laboratory of Diabetes Prevention and Research, CHINA Received: June 1, 2018 Accepted: December 16, 2018 Published: January 25, 2019 Materials and methods Copyright: © 2019 Gomez-Marcos 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. The cross-sectional study involved 2478 subjects, recruited by the MARK study. Adiposity measures: general adiposity by body mass index (BMI), central adiposity by waist-to-height ratio (WHtR), fat mass percent by the Clı´nica Universidad de Navarra—body adiposity esti- mator (CUN-BAE), percentage of body fat and of visceral adipose tissue by body roundness index (BRI) and visceral obesity and general adiposity with body shape index (ABSI). The diagnosis of MetS was made in accordance with the criteria established in the international consensus of the Joint Scientific Statement National Cholesterol Education Program III. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN), the Carlos III Health Institute/European Regional Development Fund (ERDF) (MICINN, ISCIII/FEDER) (Red RedIAPP RD06/0018, Research Groups: RD16/0007/0003, RD16/0007/ 0004, RD16/0007/0008), the Health Research OPEN ACCESS Citation: Gomez-Marcos MA, Gomez-Sanchez L, Patino-Alonso MC, Recio-Rodriguez JI, Gomez- Sanchez M, Rigo F, et al. (2019) Capacity adiposity indices to identify metabolic syndrome in subjects with intermediate cardiovascular risk (MARK study). PLoS ONE 14(1): e0209992. https://doi. org/10.1371/journal.pone.0209992 Capacity adiposity indices to identify metabolic syndrome in subjects with intermediate cardiovascular risk (MARK study Manuel A. Gomez-MarcosID1,2,3, Leticia Gomez-Sanchez1,2, Maria C. Patino-Alonso1,2,4, Jose I. Recio-Rodriguez1,2,5, Marta Gomez-Sanchez1, Fernando Rigo6, Ruth Marti7, Cristina Agudo-Conde1,2, Jose A. Maderuelo-Fernandez1,2, Rafel Ramos7,8, Luis Garcia- Ortiz1,9☯, Emiliano Rodriguez-Sanchez1,2,3☯ 1 Institute of Biomedical Research of Salamanca (IBSAL), Primary Health Care Research Unit, the Alamedilla Health Center, Salamanca, Spain, 2 Health Service of Castilla y Leo´n (SACyL), Salamanca, Spain, 3 Department of Medicine, University of Salamanca, Salamanca, Spain, 4 Department of Statistics, University of Salamanca, Salamanca, Spain, 5 Department of Nursing and Physical Therapy, University of Salamanca, Salamanca, Spain, 6 San Agustı´n Health Care Center, Department of Health, Balearic Islands (IBSALUD), Palma de Majorca, Spain, 7 Girona Research Unit, Primary Care Research Institute Jordi Gol (IDIAP Jordi Gol), Biomedical Research Institute of Girona Dr. Josep Trueta (IDBGI), Girona, Spain, 8 Department of Medical Sciences, University of Girona, Girona, Spain, 9 Department of Biomedical Sciences and Diagnosis, University of Salamanca, Salamanca, Spain a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. magomez@usal.es ☯These authors contributed equally to this work. Introduction Obesity increases mortality [1], and is linked to cardiovascular diseases and metabolic syn- drome (MetS) [2, 3]. Similarly, MetS multiplies the risk of cardiovascular disease by 2 and the risk of mortality from all causes by 1.5 [2]. It is also known that the complications associated with obesity are due, above all, to excess adiposity rather than weight gain [4]. In this context, the most objective way to determine adiposity is by direct measurement of the body fat per- centage through computed tomography or magnetic resonance imaging [5]. However, the high cost and limited accessibility of these tests hinder their use in daily clinical practice. To alleviate these limitations, an indirect measurement of adiposity was designed in Spain, the CUN-BAE (Clı´nica Universidad de Navarra—Body Adiposity Estimator). This measure esti- mates the percentage of body fat, incorporating information on sex and age, and has shown a greater association with cardiovascular risk factors than the body mass index (BMI) [6]. Furthermore, classic adiposity indices such as the BMI or waist circumference (WC) [7] have important limitations. The BMI, for example, does not distinguish between lean mass and fat mass, nor does it discriminate the location of central/peripheral fat [8], while WC does not take into account the height and weight of the subject [9] and can over- or underestimate obesity in tall or short individuals [10]. An alternative which can mitigate this limitation is to use the waist-to-height ratio (WHtR) as an indicator of abdominal adiposity [11] since the association with cardiovascular risk factors is greater than with WC [12–14]. Due to the limitations of the classic measures used to assess obesity, new adiposity indices have been developed that take WC into account in addition to weight and/or height. Thus, in 2012 Krakauer et al [15] proposed the body shape index (ABSI), which allows visceral obesity and general adiposity to be estimated from easy-to-obtain clinical parameters (height, weight and WC), and which has been shown to predict mortality independently of BMI [15–17]. Like- wise, in 2013 Thomas et al [18] developed the body roundness index (BRI), which combines height and WC and was able to estimate the percentage of body fat and visceral adipose tissue. The BRI has shown greater predictive capacity for diabetes and hypertension than other mea- sures of adiposity [19, 20]. Conclusion All adiposity indices, except for ABSI, show an association with MetS and similar ability to detect subjects with MetS among people with intermediate cariovascular risk. Capacity adiposity indices to identify metabolic syndrome in males. In the logistic regression analysis, all adiposity factors, except ABSI, showed simi- lar OR values of MetS after adjusting for possible confounding factors. In the global analysis, the adiposity index that showed a highest OR of MetS was CUN-BAE (OR 5.50; 95% CI 4.27–7.09). In the analysis by gender, the highest ORs were BMI in males (OR 5.98; 95% CI 4.70–7.60) and both WHtR and BRI in females (OR 4.15; 95% CI 3.09–5.58). Fund (PI10/01088, PI10/02077, PI10/02043; PI13/ 01930), and the Regional Health Management of Castile and Leo´n (GRS 635/A/11; GRS 906/B/14). Competing interests: The authors have declared that no competing interests exist. Abbreviations: ABSI, adiposity with body shape index; ABSI, body shape index; AUROC, ROC curve; BMI, body mass index; BRI, body roundness index; CUN-BAE, Clı´nica Universidad de Navarra—body adiposity estimator; DQI, diet quality index; MARK, MediAte Risk management; METs, mean metabolic; ROC, Receiver-operating characteristic; SBP, systolic blood pressure equivalents; WC, Waist circumference; WHtR, waist-to-height ratio. Results The highest correlation coefficients were obtained by the glycemic components (HbA1c and FPG) of the MetS and ranged from 0.155 to 0.320. The exception was ABSI, which showed lower values in the global analysis and in the males. Values of the area under the ROC curve with the adiposity indices ranged from 0.773 with the BMI in males to 0.567 with ABSI 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Introduction The relationship between CUN-BAE and classic adiposity indices such as BMI and WHtR, and new indices such as ABSI and BRI has not been studied. Moreover, although there are publications on the association between indices of adiposity and MetS, there is no consensus on which is the best index to identify subjects with MetS [8, 12 – 15]. The objective of this study was to analyze the ability of different adiposity indices to identify subjects with MetS among people with intermediate cariovascular risk. 2 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome Ethics statement Before inclusion in the study, all participants were informed about its objectives, the tests that were to be performed and the need to sign consent in order to participate. The study was approved by the research ethics committees of the Jordi Gol Institute for Primary Care Research (IDIAP Jordi Gol) and the health areas of Salamanca and Palma de Mallorca. The study was carried out in accordance with the recommendations of the Declaration of Helsinki. Variables and measurement instruments A detailed description of the procedures for the collection of clinical data, anthropometric measurements and laboratory tests performed has been previously published [21]. Materials and methods Study design The results presented in this study correspond to a sub analysis of the data collected in the baseline visit of the improving interMediAte RisK management (MARK) study (NCT01428934) [21]. Trial Registration Clinical Trials.gov Identifier: https://clinicaltrials.gov/ct2/show/ NCT01428934. Registered 2 September 2011. Last updated September 8, 2017. The character- istics of the subjects and their selection process, as well as the methodology of the measure- ments made with the subjects included in the study have been described in detail in the study protocol [21] and in the manuscripts presented by the MARK study group [11, 22]. Characteristics and participants The selection of subjects in this multicentre study was carried out by random sampling of indi- viduals attending family medicine consultations in six primary care centers of three Spanish Autonomous Communities between July 2011 and June 2013 and meeting the inclusion crite- ria. The 2495 subjects recruited were aged 35 to 74 and had an intermediate cardiovascular risk as defined by one of the following criteria: 10-year coronary risk of 5–15% according to the REGICOR scale [23]; 10-year vascular mortality risk of 1–5% according to SCORE [24]; or moderate risk according to the guidelines of the European Society of Hypertension and Cardi- ology [25]. Exclusion criteria were: subjects with a history of atherosclerotic disease, terminal illness or being institutionalized at the time of the visit. This study examined 2478 of the 2495 subjects registered for the MARK study. We excluded 17 subjects who did not have any of the anthropometric measurements necessary to calculate the adiposity indices used in this study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Laboratory determinations Venous blood samples were taken between 08:00 and 09:00, after subjects had fasted for the previous 12 hours. Fasting plasma glucose, HbA1c, total serum cholesterol, HDL cholesterol and triglyceride levels were measured using automated standard enzymatic methods. Low- density lipoprotein (LDL) cholesterol was estimated using the Friedewald equation, LDL-Cho- lesterol = ((TC-(HDL-C+TG/5) for subjects with triglycerides levels < 400 mg/Dl. Diagnostic criteria of MetS Following the criteria established by the international consensus of the Joint Scientific State- ment National Cholesterol Education Program III [30], MetS was defined as the presence of three or more of the following five components: abdominal obesity, WC 88 cm in females and 102 cm in males; triglycerides 150 mg/dL or drug treatment for elevated triglycerides; high-density lipoprotein (HDL) cholesterol < 40 mg/dL in males or < 50 mg/dL in females; high blood pressure, systolic blood pressure (SBP) 130 mmHg or diastolic blood pressure (DBP) 85 mmHg or antihypertensive drug treatment and fasting plasma glucose (FPG) 100 mg/dL or drug treatment for elevated glucose. Office or clinical blood pressure Three SBP and DBP measurements were performed with a OMRON model M10-IT validated sphygmomanometer (Omron Health Care, Kyoto, Japan), with the mean of the last two mea- sures taken being registered. The measurement was made in accordance with the recommen- dations of the European Society of Hypertension. Adiposity indices Body weight and height. Two weight measurements were taken with an approved and calibrated electronic scale (accuracy ± 0.1 kg) (Seca 770, Medical scale and measurement sys- tems, Birmingham, United Kingdom). Height was measured twice with a stadiometer (Seca 222). As reference measures of weight and height the mean of the two respective measure- ments was used. The BMI was calculated by dividing the weight in kg by height in m2. A value of BMI 30 kg/m2 was taken to define obesity. Waist circumference and waist to height ratio. WC was measured following the 2007 recommendations of the Spanish Society for the Study of Obesity [26]. A value of WC 88 cm in females and 102 cm in males was taken to define abdominal obesity. The WHtR was calculated by dividing WC in cm by height in cm [7, 27]. A value of WHtR: 0.604 cm/cm in females and 0.617 cm/cm in males was taken to define obesity. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 3 / 16 Capacity adiposity indices to identify metabolic syndrome University of Navarra Clinic—Body Adiposity Estimator (CUN-BAE). The percentage of body fat was estimated following the recommendations of Gomez-Ambrosi et al [6] with the following formula, body fat percentage = -44.988 + (0.503 × age) + (10.689 × gender) + (3.172 × BMI)—(0.026 × BMI2) + (0.181 × BMI × gender)—(0.02 BMI × age)—(0.005 × BMI2 × gender) + (0.00021 × BMI2 × age) where age was in years, and gender was coded as 0 for males and 1 for females. A value of CUN-BAE 35 in females and 30 in males was taken to define increase in the percentage of body fat. Body roundness index (BRI). The BRI was calculated using the formula BRI ¼ 364:2 365:5X ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 ððWC=ð2pÞ 2=ð0:5heightÞ 2Þ q [28]. A body shape index (ABSI). A Body Shape Index (ABSI) was based on WC adjusted for height and weight: ABSI ¼ WC=BMI2=3XheightÞ 1=2 ABSI ¼ WC=BMI2=3XheightÞ 1=2 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Smoking status Smoking status (smoker/non-smoker) was recorded, with those who currently smoke or who quit smoking during the previous year considered smokers. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 4 / 16 Capacity adiposity indices to identify metabolic syndrome Alcohol consumption The amount of alcohol drunk in a week was recorded with a structured questionnaire and was measured in grams/week. Physical activity We use the Minnesota questionnaire to measure physical activity in free time[31]. The ques- tionnaire has been previously validated in both sexes in Spain. The questionnaire was collected by interviewers previously trained. They collected the physical activity carried out during the previous year, recording the type of activity and the duration of it. Each physical activity has a code of intensity, which is based on the quotient between the metabolic rate during the prac- tice of physical activity and the basal metabolic rate (MET). 1 MET is equivalent to approxi- mately 1 kcal / min of energy expenditure. Thus, we calculate the total energy expenditure in free time of total physical activity (EEPA_t) in kilocalories per week. Using the physical activity intensity code, we were also able to quantify the energy expenditure in physical activity (EEPA) according to the activity classification as intense, moderate, or light intensity as fol- lows: light intensity was below 4 METs, such as walking (EEPA light). Moderate PA intensity was 4–5.5 METs, such as brisk walking (EEPA moderate). Intense PA intensity was greater than or equal to 6 METs, such as jogging (EEPA intense). Thus, for each particular subject: EEPA total = EEPA light + EEPA moderate + EEPA intense [32]. Additionally, in accordance with the recommendations of the American Heart Association [33], we considered participants to be sedentary if they do not meet the recommendations of moderate-intensity aerobic PA practice for a minimum of 30 min on 5 days each week (EEPA moderate < 675 kcal/week) or high-intensity aerobic PA practice for a minimum of 20 min on 3 days each week (EEPA intense < 420 kcal/week) [32]. Recording diet The adherence to the Mediterranean diet questionnaire used in the PREDIMED study was administered [34]. This questionnaire with 14 items related to compliance with different aspects of the Mediterranean diet has been validated in Spain. A score 9 is required to be considered good compliance. All assessments were made within a period of 10 days. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Characteristics of the study subjects Table 1 shows the general characteristics of the population studied, globally and by gender. Males made up 61.3% of the sample, the median age was 62 (56–67) years. All the adiposity indexes showed higher values in the females except the ABSI that was higher in the males. The mean values of SBP, DBP and triglycerides were higher in males, and of total-cholesterol, LDL-cholesterol, HDL- and HA1c higher in females. The prevalence of MetS in the study par- ticipants was 45.2% (42.6% in men and 49.4% in women; p = 0.001). The percentage of subjects with hypertension was higher in men, and the percentages of subjects with diabetes mellitus, obesity and sedentary behavior were higher in females. Statistical analysis Continuous variables were presented as means ± standard deviation for normally distributed variables or medians (interquartile ranges) for the skewed variables. Statistical normality was tested using the Kolmogorov-Smirnov test. The means of two groups were compared using Student’s t-test or Mann-Wittney U test. All categorical variables were presented as numbers and proportions. Chi-square and Fisher’s exact tests were used for analysis of proportion. The Spearman rho correlation coefficient was calculated to measure the CUN-BAE rela- tionships with other measures of adiposity, as well as the correlation between measures of adi- posity with the components that make up the MetS. Receiver-operating characteristic (ROC) analyses were performed to examine the ability of the different measures of adiposity to diagnose MetS. The area under the ROC curve (AUROC) and the 95% confidence intervals (CIs) were computed to compare the discrimina- tive power of each adiposity index. 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome The Kappa coefficient was used to analyze the agreement between the different adiposity indices, taking the optimal cut points calculated in the ROC curves to identify the presence or absence of MetS. To estimate the association of the presence of adiposity, defined by the different adiposity indices, with the presence de MetS, three logistic regression models were applied: model 1 adjusted for age (years) and gender (0 = male and 1 = female); model 2 further adjusted for smoking status (0 = non-smoker and 1 = smoker), physical activity status (0 = sedentary and 1 = active), adherence to the Mediterranean diet (0 = non-adherence and 1 = adherence) and alcohol consumption en gr/week; and model 3 further adjusted for antihypertensive drugs (0 = no and 1 = yes), lipid lowering drugs (0 = no and 1 = yes) and antidiabetic drugs (0 = no and 1 = yes). Analyses were performed with the subjects overall and by gender. Data were analyzed using SPSS Statistics for Windows version 23.0 (IBM Corp, Armonk, NY). Values of p < 0.05 were considered statistically significant. Correlations between adiposity indices and MetS components Table 2 shows the Spearman correlation coefficients between adiposity indices and MetS com- ponents, global and stratified by gender. The highest correlation coefficients were obtained by the glycemic components (Hb A1c and FPG) of the MetS and ranged from 0.155 to 0.320. The exception was ABSI, which showed lower values in the global analysis and in the males. Correlations of CUN-BAE with other indices of global adiposity and by gender The following Spearman correlation coefficients between the CUN-BAE and the different adi- posity measurements were recorded: ρ = 0.621 with BMI; ρ = 0.616 with WHtR and BRI and ρ = -0.149 with ABSI. All coefficients increased when calculated by gender, except in the case of CUN-BAE with ABSI. Fig 1 also shows the dispersion diagrams by gender between CUN- BAE and BMI (Fig 1A), WHtR (Fig 1B), BRI (Fig 1C) and ABSI (Fig 1D). Capacity of the adiposity indices to predict MetS using AUROC analysis Table 3 and Fig 2, Fig 2A (global), Fig 2B (males) and Fig 2C (females) show the AUROC val- ues and the optimal cut-off points between MetS and the anthropometric measures, both glob- ally and by gender. AUROC values were similar in all adiposity indices studied except in the case of ABSI, where they were lower. The highest values of the Youden index were obtained with the BMI (0.416) among males; and with the WHtR and the BRI in both the global analysis (0.380 in both cases), and among females (0.368 in both cases). 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome Table 1. Baseline characteristics and of study subjects. Variables Global n = 2478 Males n = 1520 (61.3%) Females n = 958(38.7%) p Value Lifestyle factors (median IR o number and %) Age (years) 62 (56–67) 62 (55–68) 62 (57–67) 0.252 Smoking 248 (37.7) 195 (29.8) 214 (32.5) <0.001 Total physical activity (MET) 1800 (750–3293) 2064 (917–3836) 600 (750–2514) 0.011 Anthropometric measures (median IR) Height (m) 1.65 (1.57–1.71) 1.70 (1.65–1.74) 1.56 (1.52–1.60) <0.001 Weight (kg) 78.2 (69.9–88.0) 82.0 (74.8–91.0) 70.8 (62.2–81.0) <0.001 WC (cm) 100 (97–107) 102 (96–108) 100 (97–107) <0.001 Adiposity index (median IR) CUN-BAE (%) 35.8 (34.4–41.4) 30.8(28.3–33.8) 42.8(39.4–46.9) <0.001 BMI (kg/m2) 28.7 (26.3–31.7) 28.3 (26.3–33.8) 28.7 (26.3–31.7) <0.001 WHtR (cm/cm) 0.61 (0.57–0.66) 0.60 (0.57–0.64) 0.62 (0.57–0.67) <0.001 BRI 5.6 (4.7–6.7) 5.5 (4.7–6.4) 5.9 (4.7–7.2) <0.001 ABSI 0.083 (0.080–0.086) 0.084 (0.082–0.086) 0.082 (0.079–0.086) <0.001 Cardiovascular risk factors (median IR) SBP (mmHg) 136 (125–147) 137 (127–146) 133 (122–145) <0.001 DBP (mmHg) 84 (78–91) 85 (79–92) 82 (76–89) <0.001 Total-Cholesterol (mg/dl) 224 (198–250) 220 (193–245) 232 (205–260) <0.001 LDL- Cholesterol (mg/dl) 140 (116–163) 138 (115–161) 142 (117–167) 0.010 HDL-Cholesterol (mg/dl) 48 (41–56) 46 (40–54) 50 (44–58) <0.001 Triglycerides (mg/dl) 124 (124–174) 126 (191–179) 121 (92–166) 0.026 FPG (mg/dl) 98 (88–115) 98 (88–114) 97 (87–117) 0.451 HA1c (%) 5.8 (5.4–6.3) 5.7 (5.4–6.3) 5.8 (5.5–6.5) <0.001 Prevalence of cardiovascular risk factors, number and %) Hypertension 1798 (72.6) 1169 (76.9) 629 (65.7) <0.001 Diabetes Mellitus 843 (34.0) 491 (32.3) 352 (37.7) 0.013 MetS 1120 (45.2) 647 (42.6) 473 (49.4) 0.001 High BMI 907 (36.6) 512 (33.7) 395 (41.2) <0.001 High WC 1559 (62.9) 800 (52.6) 759 (79.2) <0.001 High WHtR 2396 (96.7) 1478 (97.2) 918 (95.8) 0.037 High CUN-BAE 2312 (93.3) 1404 (92.4) 908 (94.8) 0.011 Sedentary 1097 (44.3) 576 (37.9) 521 (54.4) <0.001 Drug treatment, n (%) Antihypertensive drugs 1275 (51.5) 765 (50.3) 510 (53.2) 0.086 Antidiabetic drugs 514 (20.7) 288 (18.9) 226 (23.6) 0.003 Lipid-lowering drugs 672 (27.1) 385 (25.3) 287 (30.0) 0.007 Table 1. Baseline characteristics and of study subjects. Continuous variables were presented as medians (interquartile ranges) for the skewed variables. Categorical variables were presented as numbers and proportions. Continuous variables were presented as medians (interquartile ranges) for the skewed variables. Categorical variables were presented as numbers and proportions. We consider High BMI 30 kg/m2; High WC: WC 88 cm in females and 102 cm in males; High WHtR: 0.604 cm/cm in females and 0.617 cm/cm in males; High CUN-BAE 35 in females and 30 in males. Abbreviations: IR, interquartile range. MET, basal metabolic rate. BMI, body mass index. WC, Waist circumference. CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. WHtR, waist-to-height ratio. BRI, body roundness index. ABSI, a body shape index. SBP, systolic blood pressure. DBP, diastolic blood pressure. LDL, low density lipoprotein. HDL, high density lipoprotein. FPG, fasting plasma glucose. HbA1C, glycosylated hemoglobin. MetS, Metabolic syndrome. CVD RF, cardiovascular risk factors https://doi.org/10.1371/journal.pone.0209992.t001 Association analysis In the logistic regression analysis, all the adiposity indices, except for ABSI, show a similar probability of presenting MetS associated with the presence of obesity. The OR in the global analysis ranges from 5.50 (4.27–7.09) with CUN-BAE to 4.57 (3.81–5.49) with BMI. With males it is between 5.98 (4.70–7.60) with BMI and 5.51 (4.32–7.02) with CUN-BAE and with females between 4.15 (3.06–5.63) with CUN-BAE, 4.15 (3.09–5.58) WHtR and BRI, and 4.14 (3.07–5.58) with BMI (Table 4). Capacity adiposity indices to identify metabolic syndrome Table 2. Spearman correlation coefficient between adiposity index and components of metabolic syndrome. CUN-BAE BMI WHtR BRI ABSI Global n = 2478 SBP -0.028 0.101 0.101 0.082 0.059 DBP -0.025 0.160 0.070 0.062 0.049 FPG 0.155 0.260 0.279 0.214 0.062 HbA1c 0.212 0.241 0.287 0.243 0.064 HDL-Cholesterol 0.030 -0.207 -0.135 -0.110 -0.005 Triglycerides 0.078 0.215 0.164 0.107 -0.026 Males n = 1520 SBP 0.128 0.115 0.115 0.100 0.027 DBP 0.124 0.178 0.088 0.093 -0.062 FPG 0.197 0.196 0.264 0.204 0.047 HbA1c 0.256 0.242 0.255 0.221 0.061 HDL-Cholesterol -0.196 -0.222 -0.139 -0.141 -0.105 Triglycerides 0.139 0.191 0.115 0.086 -0.097 Females n = 958 SBP 0.118 0.099 0.121 0.117 0.111 DBP 0.133 0.151 0.079 0.120 0.074 FPG 0.273 0.253 0.302 0.218 0.231 HbA1c 0.277 0.265 0.320 0.236 0.254 HDL-Cholesterol -0.189 -0.211 -0.191 -0.162 -0.143 Triglycerides 0.246 0.255 0.249 0.180 0.180 Table 2. Spearman correlation coefficient between adiposity index and components of metabolic syndrome. Adiposity index with highest correlation coefficient for each variable in bold. Abbreviations: CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. BMI, body mass index. WHtR, waist-to-height ratio. BRI, body roundness index. ABSI, a body shape index. SBP, systolic blood pressure. DBP, diastolic blood pressure. FPG, fasting plasma glucose. HbA1C, glycosylated hemoglobin. HDL, high density lipoprotein. https://doi.org/10.1371/journal.pone.0209992.t002 The degree of agreement of the five adiposity indices analyzed for the purpose of identifying subjects with MetS are shown in S1 Table (additional file 1). In the stratified analysis, all values of the kappa index, except those corresponding to ABSI, are higher than 0.65, with the perfect correlation between BRI and WHtR standing out. In the global analysis, the degree of agree- ment between BMI, BRI and WHtR is also higher than 0.65; with CUN-BAE it is 0.40 and the maximum value reached with ABSI is 0.27. Abbreviations: CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. BMI, body mass index. WHtR, waist-to-height ratio. BRI, body roundness index. ABSI, a body shape index. SBP, systolic blood pressure. DBP, diastolic blood pressure. FPG, fasting plasma glucose. HbA1C, glycosylated hemoglobin. HDL, high density lipoprotein. We consider High BMI 30 kg/m2; High WC: WC 88 cm in females and 102 cm in males; High WHtR: 0.604 cm/cm in females and 0.617 cm/cm in males; High CUN-BAE 35 in females and 30 in males. Abbreviations: IR, interquartile range. MET, basal metabolic rate. BMI, body mass index. WC, Waist circumference. CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. WHtR, waist-to-height ratio. BRI, body roundness index. ABSI, a body shape index. SBP, systolic blood pressure. DBP, diastolic blood pressure. LDL, low density lipoprotein. HDL, high density lipoprotein. FPG, fasting plasma glucose. HbA1C, glycosylated hemoglobin. MetS, Metabolic syndrome. CVD RF, cardiovascular risk factors 7 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Adiposity index with highest correlation coefficient for each variable in bold. rsidad de Navarra—Body Adiposity Estimator. BMI, body mass index. WHtR, waist-to-height ratio. BRI, body roundness or each variable in bold. Discussion The present study, conducted with Spanish subjects at intermediate cardiovascular risk, sug- gests that both the association of the adiposity indices with MetS and their ability to identify PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 8 / 16 Capacity adiposity indices to identify metabolic syndrome Fig 1. Correlation between CUN-BAE and BMI (Fig 1A), WHtR (Fig 1B), BRI (Fig 1C) and ABSI (Fig 1D) stratified by gender. Vertical lines indicate cut-offs for defining MetS according to CUN-BAE (31.22 males and 41.95 females respectively) and horizontal lines indicate cut-offs for defining MetS according to BMI (28.96 males and 28.02 females respectively), WHtR (0.611 males and 0.615 females respectively), BRI (5.69 males and 5.77 females respectively) and ABSI (0.0835 males and 0.0816 females respectively). In the lower part of the figure coefficient global Spearman correlation and by gender. p <0.001 Abbreviations: CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. WHtR, waist-to-height ratio. BRI, body roundness index. ABSI, a body shape index. https://doi.org/10.1371/journal.pone.0209992.g001 Fig 1. Correlation between CUN-BAE and BMI (Fig 1A), WHtR (Fig 1B), BRI (Fig 1C) and ABSI (Fig 1D) Fig 1. Correlation between CUN-BAE and BMI (Fig 1A), WHtR (Fig 1B), BRI (Fig 1C) and ABSI (Fig 1D) stratified by gender. Vertical lines indicate cut-offs for defining MetS according to CUN-BAE (31.22 males and 41.95 females respectively) and horizontal lines indicate cut-offs for defining MetS according to BMI (28.96 males and 28.02 females respectively), WHtR (0.611 males and 0.615 females respectively), BRI (5.69 males and 5.77 females respectively) and ABSI (0.0835 males and 0.0816 females respectively). In the lower part of the figure coefficient global Spearman correlation and by gender. p <0.001 Abbreviations: CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. WHtR, waist-to-height ratio. BRI, body roundness index. ABSI, a body shape index. https://doi.org/10.1371/journal.pone.0209992.g001 MetS subjects is similar with all indices analyzed, both in the global analysis and by gender, apart from ABSI. The probability of developing MetS associated with the presence of obesity ranged in the global analysis with the best-fit model from 5.50 with CUN-BAE to 4.57 with BMI, being lower in females than in males. These results are confirmed in the ROC curve analysis. The results of this study are new and may be of important clinical relevance since it is the first study that compares the capacity of five adiposity indices to identify subjects with MetS in a population with intermediate cardiovascular risk. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome Table 3. Adjusted area under receiver operating characteristic curve and cut-off points, sensitivity and specificity for the various adiposity index and metabolic syndrome. ROC (95% CI) Cut-off Sensitivity (%) Specificity (%) Youden index Global n = 2478 CUN-BAE 0.684 (0.663–0.704) 33.15 0.700 0.592 0.292 BMI 0.748 (0.729–0.767) 28.73 0.692 0.680 0.372 WHtR 0.752 (0.733–0.771) 0.61 0.695 0.685 0.380 BRI 0.752 (0.733–0.771) 5.67 0.695 0.685 0.380 ABSI 0.569 (0.546–0.591) 0.08 0.550 0.552 0.102 Males n = 1520 CUN-BAE 0.761 (0.737–0.785) 31.22 0.674 0.701 0.375 BMI 0.773 (0.749–0.796) 28.96 0.702 0.714 0.416 WHtR 0.761 (0.738–0.785) 0.61 0.675 0.7175 0.392 BRI 0.761 (0.738–0.785) 5.69 0.675 0.7175 0.392 ABSI 0.567 (0.538–0.596) 0.08 0.592 0.501 0.093 Females n = 958 CUN-BAE 0.714 (0.682–0.747) 41.95 0.748 0.588 0.336 BMI 0.718 (0.686–0.750) 28.02 0.740 0.606 0.346 WHtR 0.735 (0.704–0.767) 0.61 0.702 0.666 0.368 BRI 0.735 (0.704–0.767) 5.77 0.702 0.666 0.368 ABSI 0.569 (0.554–0.626) 0.08 0.622 0.538 0.160 Adiposity index with the highest and area down the curve value in bold. postmenopausal females showed that BMI had greater predictive power than WHtR when identifying MetS. postmenopausal females showed that BMI had greater predictive power than WHtR when identifying MetS. The AUROC results obtained by the adiposity indices for identifying subjects with MetS coincide with those published in other studies [4, 5, 7]. Similarly, several studies [5, 38, 39], although not all [39], have reported that ABSI was a weak indicator of subjects with MetS. Furthermore, previous studies conducted in Asian and American populations [38,39] showed that the relationship of the different components of MetS with the adiposity indices analyzed was stronger in females than in males [19, 40, 41], data that coincide in part with the Fig 2. Receiver operating characteristic curve of CUN-BAE, BMI, WC, BRI and ABSI to identify subjects with metabolic syndrome, (A) global, (B) males and (C) females. Areas under the ROC curves are summarized in Table 3. Abbreviations: CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. BMI, body mass index. WC, Waist circumference. BRI, body roundness index. ABSI, a body shape index. https://doi.org/10.1371/journal.pone.0209992.g002 Fig 2. Receiver operating characteristic curve of CUN-BAE, BMI, WC, BRI and ABSI to identify subjects with metabolic syndrome, (A) global, (B) males and (C) females. Areas under the ROC curves are summarized in Table 3. Abbreviations: CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. BMI, body mass index. WC, Waist circumference. Discussion The results suggest that, except for ABSI, they may all be useful in clinical practice for identifying subjects at higher risk of developing MetS. Over recent years, several studies have assessed the capacity of different adiposity indices to identify subjects with MetS, without obtaining conclusive results. The results coincide with those published in other studies conducted with American adolescents or in the general oriental population in which BMI, WC and WHtR behaved similarly in identifying subjects with MetS [4, 5, 35]. However, other studies conducted with oriental adults [30, 36], or over- weight/obese children and adolescents [30, 36] have shown that WHtR had greater power to identify subjects with MetS, but these did not include CUN-BAE nor ABSI among the indices analyzed. On the other hand, the Women’s Health Initiative Study [37] involving 2672 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 9 / 16 Adiposity index with the highest and area down the curve value in bold. Abbreviations: ROC, receiver operating characteristic. CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. BMI, body mass index. WHtR, waist-to- height ratio. BRI, body roundness index. ABSI, a body shape index. https://doi.org/10.1371/journal.pone.0209992.t003 BRI, body roundness index. ABSI, a body shape index. https://doi.org/10.1371/journal.pone.0209992.g002 10 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome Table 4. Adjusted odds ratios of metabolyc syndrome by adiposity index. a. Model 1. Odds Ratio Adjusted for Age and Gender Global n = 2478 Males n = 1520 Females n = 958 OR (95% CI) p value OR (95% CI) p value OR (95% CI) p value CUN-BAE 5.90 (4.63–7.51) <0.001 5.29 (4.22–6.63) <0.001 4.42 (3.34–5.85) <0.001 BMI 4.70 (3.96–5.58) <0.001 5.83 (4.66–7.29) <0.001 4.38 (3.33–5.77) <0.001 WHtR 5.30 (4.44–6.32) <0.001 5.93 (4.71–7.47) <0.001 4.78 (3.64–6.30) <0.001 BRI 5.32 (4.46–6.34) <0.001 5.99 (4.75–7.54) <0.001 4.78 (3.64–6.30) <0.001 ABSI 1.78 (1.50–2.10) <0.001 1.66 (1.34–2. 05) <0.001 1.96 (1.51–2.54) <0.001 b. Model 2. Odds Ratio Adjusted for Age, Gender, Smoking Status, Alcohol consumption, Adherence to the Mediterranean diet and Physical Activity Status CUN-BAE 5.79 (4.54–7.39) <0.001 5.28 (4.21–6.24) <0.001 4.18 (3.14–5.56) <0.001 BMI 4.60 (3.87–5.46) <0.001 5.81 (4.64–7.28) <0.001 4.13 (3.12–5.46) <0.001 WHtR 5.21 (4.36–6.23) <0.001 5.96 (4.72–7.53) <0.001 4.54 (3.43–6.01) <0.001 BRI 5.23 (4.37–6.25) <0.001 6.02 (4.76–7.60) <0.001 4.54 (3.43–6.01) <0.001 ABSI 1.78 (1.50–2.10) <0.001 1.66 (1.33–2. 05) <0.001 1.96 (1.51–2.55) <0.001 c. Model 3. Odds Ratio Adjusted for Age, Gender, Smoking Status, Alcohol consumption, Adherence to the Mediterranean diet, Physical Activity Status, Antihypertensive drugs, Antidiabetic drugs and Lipid-lowering drugs CUN-BAE 5.50 (4.27–7.09) <0.001 5.51 (4.32–7.02) <0.001 4.15 (3.06–5.63) <0.001 BMI 4.57 (3.81–5.49) <0.001 5.98 (4.70–7.60) <0.001 4.14 (3.07–5.58) <0.001 WHtR 5.02 (4.16–6.06) <0.001 5.93 (4.63–7.58) <0.001 4.15 (3.09–5.58) <0.001 BRI 5 04 (4 18 6 09) <0 001 5 96 (4 66 7 63) <0 001 4 15 (3 09 5 58) <0 001 Table 4. Adjusted odds ratios of metabolyc syndrome by adiposity index. j g , , g , p , , y y , Antihypertensive drugs, Antidiabetic drugs and Lipid-lowering drugs CUN-BAE 5.50 (4.27–7.09) <0.001 5.51 (4.32–7.02) <0.001 4.15 (3.06–5.63) <0.001 BMI 4.57 (3.81–5.49) <0.001 5.98 (4.70–7.60) <0.001 4.14 (3.07–5.58) <0.001 WHtR 5.02 (4.16–6.06) <0.001 5.93 (4.63–7.58) <0.001 4.15 (3.09–5.58) <0.001 BRI 5.04 (4.18–6.09) <0.001 5.96 (4.66–7.63) <0.001 4.15 (3.09–5.58) <0.001 ABSI 1.69 (1.41–2.02) <0.001 1.65 (1.31–2. 07) <0.001 1.81 (1.37–2.39) <0.001 Adiposity index with the highest significant OR value in bold. Adiposity index with the highest significant OR value in bold. In the analysis in males and in females the gender was not used as adjustment variable Abbreviations: OR, odds ratio. CI, confidence interval. CUN-BAE, Clı´nica Universidad de Navarra—Body Adiposity Estimator. BMI, body mass index. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 WHtR, waist-to- height ratio. BRI, body roundness index. ABSI, a body shape index. Cut points used with the different adiposity index: Global: CUN-BAE: 33.15, BMI: 28.73, WHtR: 0.613, BRI: 5.67 and ABSI: 0.0834; Males: 31.22, BMI: 28.96, WHtR: 0.611, BRI: 5.69 and ABSI: 0.0835 and females: 41.95, BMI: 28.02, WHtR: 0.615, BRI: 5.77 and ABSI: 0.0816. https://doi.org/10.1371/journal.pone.0209992.t004 results of the present study. The reason for this sex difference is unclear, although differences in anatomy, physiology, metabolism, and sex hormones may offer a partial explanation [36]. There are similar gender differences with the optimal cut-off points for the five adiposity indi- ces analyzed, which suggests that gender-specific reference values should be used to identify subjects with a higher risk of MetS. Regarding the adiposity indices less frequently studied in identifying subjects with MetS, we must note that the CUN-BAE estimates the percentage of body fat and it is the excess body fat which is chiefly responsible for the complications associated with obesity [42, 43]. Although in our study CUN-BAE is not superior to other measures in predicting MetS, there are previ- ous studies that suggest that the determination of body fat percentage can better predict MetS [12, 44]. The BRI was developed to measure body fat and the percentage of visceral adipose tis- sue by using WC in relation to height, which allows the estimation of the shape of the human body as an ellipse or oval [18]. The BRI correlates with MetS components and identifies MetS as other adiposity indices do. The equal predictive power (identical areas under the ROC curve) of BRI and WHtR which this study found among females has been described in previ- ous studies [19, 20, 45]. The Spearman coefficient revealed an extremely close relationship between the two indices (r = 1). Finally, ABSI was created as a quantitative measure to estimate the health of body shape regardless of body size, so ABSI predicts premature mortality better than BMI or WC [15]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 11 / 16 Capacity adiposity indices to identify metabolic syndrome Our results suggest that, compared to the other four indices used in this study, ABSI should not be used to predict MetS. A possible explanation for the non-concordant findings between our data and those of Krakauer et al. Conclusions All adiposity indices, except for ABSI, show an association with MetS and similar ability to detect subjects with MetS among people with intermediate cardiovascular risk. Supporting information S1 Table. Degree of agreement between the adiposity indices to identify subjects with MetS. Kappa index. (DOC) S1 Table. Degree of agreement between the adiposity indices to identify subjects with MetS. Kappa index. S1 File. Data file. (SAV) S1 File. Data file. (SAV) [15], are the different end point variables (mortality and MetS), the characteristics of the subjects analyzed (general population and subjects of interme- diate cardiovascular risk) and height, which was 1.70 meters in the study by Krakauer et al. [15], while in ours it was 1.65 meters, indicating that height could influence ABSI’s ability to identify MetS. Future studies should therefore further investigate the limits of ABSI and the impact of body height on the calculation of ABSI. The main limitation of our study is its transversal design, which does not allow us to estab- lish causal relationships or their direction. Secondly, the population in this study was ethnically homogeneous, since all the patients in this study were Caucasians at intermediate cardiovascu- lar risk, thus potentially limiting the generalizability of our findings. However, its strong points include the large sample analyzed and the fact that as adjustment variables we have used those which according to other authors [19] can influence the different adiposity indices, such as age, gender, sociodemographic variables, dietary intake, physical activity and consumption of alcohol and tobacco, which could affect the strength of the associations. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Acknowledgments We are grateful to all professionals participating in the MARK study. Lead author for this group: Rafel Ramos, Research Unit, Primary Health Care, Girona, Jordi Gol Institute for Primary Care Research (IDIAP Jordi Gol), Catalonia, Spain, E-mail: rramos.girona.ics@gencat.net. Coordinating Center: Rafel Ramos, Ruth Martı´, Dı´dac Parra- mon, Anna Ponjoan, Miquel Quesada, Maria Garcia-Gil, Martina Sidera and Lourdes Camo´s, Research Unit, Primary Health Care, Jordi Gol Institute for Primary Care Research (IDIAP Jordi Gol), C/Maluquer Salvador, 11, 17002-Girona, Catalonia, Spain. Fernando Montesinos, Ignacio Montoya, Carlos Lo´pez, Anna Agell, Nu´ria Pagès of the Primary Care Services, Girona, Catalan Institute of Health (ICS), Catalonia, Spain. Irina Gil, Anna Maria-Castro of the Pri- mary Care Services, Girona, Institut d’Assistència Sanitaria (IAS), Catalonia, Spain. Fernando Rigo, Guillermo Frontera, Antònia Rotger, Natalia Feuerbach, Susana Pons, Natividad Garcia, John Guillaumet, Micaela Llull and Mercedes Gutierrez of the Health Center Primary Care San Augustı´n, Ibsalut Balears, Spain. Cristina Agudo-Conde, Leticia Go´mez-Sanchez, Carmen Castaño-Sanchez, Carmela Rodriguez-Martı´n, Benigna Sanchez-Salgado, Angela de Cabo- Laso, Marta Go´mez-Sa´nchez, Emiliano Rodriguez-Sanchez, Jose Angel Maderuelo-Fernandez, Emilio Ramos-Delgado, Carmen Patino-Alonso, Jose I Recio-Rodriguez, Manuel A Gomez- Marcos and Luis Garcia-Ortiz, Primary Care Research Unit of The Alamedilla, Salamanca, Spain, Castilla and Leo´n Health Service–SACYL. 12 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome Author Contributions Conceptualization: Manuel A. Gomez-Marcos, Jose I. Recio-Rodriguez, Marta Gomez-San- chez, Fernando Rigo, Rafel Ramos, Emiliano Rodriguez-Sanchez. Data curation: Leticia Gomez-Sanchez, Maria C. Patino-Alonso, Jose I. Recio-Rodriguez, Fer- nando Rigo, Rafel Ramos. Formal analysis: Manuel A. Gomez-Marcos, Maria C. Patino-Alonso, Luis Garcia-Ortiz. Funding acquisition: Manuel A. Gomez-Marcos, Fernando Rigo, Rafel Ramos. Investigation: Manuel A. Gomez-Marcos, Maria C. Patino-Alonso, Marta Gomez-Sanchez, Cristina Agudo-Conde, Jose A. Maderuelo-Fernandez, Emiliano Rodriguez-Sanchez. Methodology: Manuel A. Gomez-Marcos, Maria C. Patino-Alonso, Jose A. Maderuelo-Fer- nandez, Luis Garcia-Ortiz, Emiliano Rodriguez-Sanchez. Project administration: Manuel A. Gomez-Marcos, Marta Gomez-Sanchez, Fernando Rigo. Resources: Jose I. Recio-Rodriguez, Marta Gomez-Sanchez, Ruth Marti, Cristina Agudo- Conde. Software: Jose I. Recio-Rodriguez, Ruth Marti, Jose A. Maderuelo-Fernandez, Luis Garcia- Ortiz, Emiliano Rodriguez-Sanchez. Supervision: Manuel A. Gomez-Marcos, Leticia Gomez-Sanchez, Cristina Agudo-Conde, Luis Garcia-Ortiz. Validation: Ruth Marti, Rafel Ramos, Emiliano Rodriguez-Sanchez. Validation: Ruth Marti, Rafel Ramos, Emiliano Rodriguez-Sanchez. Visualization: Leticia Gomez-Sanchez, Ruth Marti, Rafel Ramos. Writing – original draft: Manuel A. Gomez-Marcos, Emiliano Rodriguez-Sanchez. Writing – review & editing: Manuel A. Gomez-Marcos, Leticia Gomez-Sanchez, Jose I. Recio-Rodriguez, Marta Gomez-Sanchez, Ruth Marti, Cristina Agudo-Conde, Jose A. Maderuelo-Fernandez, Rafel Ramos, Luis Garcia-Ortiz, Emiliano Rodriguez-Sanchez. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 References 1. Cameron AJ, Magliano DJ, Shaw JE, Zimmet PZ, Carstensen B, Alberti KG, et al. The influence of hip circumference on the relationship between abdominal obesity and mortality. Int J Epidemiol. 2012; 41(2):484–94. Epub 2012/01/24. https://doi.org/10.1093/ije/dyr198 PMID: 22266094. 2. Mottillo S, Filion KB, Genest J, Joseph L, Pilote L, Poirier P, et al. The metabolic syndrome and cardio- vascular risk a systematic review and meta-analysis. J Am Coll Cardiol. 2010; 56(14):1113–32. Epub 2010/09/25. https://doi.org/10.1016/j.jacc.2010.05.034 PMID: 20863953. 3. Slagter SN, van Vliet-Ostaptchouk JV, van Beek AP, Keers JC, Lutgers HL, van der Klauw MM, et al. Health-Related Quality of Life in Relation to Obesity Grade, Type 2 Diabetes, Metabolic Syndrome and Inflammation. PLoS One. 2015; 10(10):e0140599. Epub 2015/10/17. https://doi.org/10.1371/journal. pone.0140599 PMID: 26474291. 4. Romero-Corral A, Lopez-Jimenez F, Sierra-Johnson J, Somers VK. Differentiating between body fat and lean mass-how should we measure obesity? Nat Clin Pract Endocrinol Metab. 2008; 4(6):322–3. Epub 2008/04/03. https://doi.org/10.1038/ncpendmet0809 PMID: 18382423. 5. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cho- lesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972; 18(6):499–502. Epub 1972/06/01. PMID: 4337382. 6. Gomez-Ambrosi J, Silva C, Catalan V, Rodriguez A, Galofre JC, Escalada J, et al. Clinical usefulness of a new equation for estimating body fat. Diabetes Care. 2012; 35(2):383–8. Epub 2011/12/20. https:// doi.org/10.2337/dc11-1334 PMID: 22179957. 13 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome 7. Oliveros E, Somers VK, Sochor O, Goel K, Lopez-Jimenez F. The concept of normal weight obesity. Prog Cardiovasc Dis. 2014; 56(4):426–33. Epub 2014/01/21. https://doi.org/10.1016/j.pcad.2013.10. 003 PMID: 24438734. 7. Oliveros E, Somers VK, Sochor O, Goel K, Lopez-Jimenez F. The concept of normal weight obesity. Prog Cardiovasc Dis. 2014; 56(4):426–33. Epub 2014/01/21. https://doi.org/10.1016/j.pcad.2013.10. 003 PMID: 24438734. 8. Nevill AM, Stewart AD, Olds T, Holder R. Relationship between adiposity and body size reveals limita- tions of BMI. Am J Phys Anthropol. 2006; 129(1):151–6. Epub 2005/11/05. https://doi.org/10.1002/ajpa 20262 PMID: 16270304. 9. Nishida C, Ko GT, Kumanyika S. Body fat distribution and noncommunicable diseases in populations: overview of the 2008 WHO Expert Consultation on Waist Circumference and Waist-Hip Ratio. Eur J Clin Nutr. 2010; 64(1):2–5. Epub 2009/11/26. https://doi.org/10.1038/ejcn.2009.139 PMID: 19935820. 10. Ashwell M, Gunn P, Gibson S. Waist-to-height ratio is a better screening tool than waist circumference and BMI for adult cardiometabolic risk factors: systematic review and meta-analysis. Obes Rev. 2012; 13(3):275–86. Epub 2011/11/24. References https://doi.org/10.1111/j.1467-789X.2011.00952.x PMID: 22106927. 11. Gomez-Sanchez L, Garcia-Ortiz L, Patino-Alonso MC, Recio-Rodriguez JI, Fernando R, Marti R, et al. Association of metabolic syndrome and its components with arterial stiffness in Caucasian subjects of the MARK study: a cross-sectional trial. Cardiovasc Diabetol. 2016; 15(1):148. Epub 2016/10/26. https://doi.org/10.1186/s12933-016-0465-7 PMID: 27776526. 12. Lam BC, Koh GC, Chen C, Wong MT, Fallows SJ. Comparison of Body Mass Index (BMI), Body Adi- posity Index (BAI), Waist Circumference (WC), Waist-To-Hip Ratio (WHR) and Waist-To-Height Ratio (WHtR) as predictors of cardiovascular disease risk factors in an adult population in Singapore. PLoS One. 2015; 10(4):e0122985. Epub 2015/04/17. https://doi.org/10.1371/journal.pone.0122985 PMID: 25880905. 13. Langenberg C, Sharp SJ, Schulze MB, Rolandsson O, Overvad K, Forouhi NG, et al. Long-term risk of incident type 2 diabetes and measures of overall and regional obesity: the EPIC-InterAct case-cohort study. PLoS Med. 2012; 9(6):e1001230. Epub 2012/06/09. https://doi.org/10.1371/journal.pmed. 1001230 PMID: 22679397. 14. Lee CM, Huxley RR, Wildman RP, Woodward M. Indices of abdominal obesity are better discriminators of cardiovascular risk factors than BMI: a meta-analysis. J Clin Epidemiol. 2008; 61(7):646–53. Epub 2008/03/25. https://doi.org/10.1016/j.jclinepi.2007.08.012 PMID: 18359190. 15. Krakauer NY, Krakauer JC. A new body shape index predicts mortality hazard independently of body mass index. PLoS One. 2012; 7(7):e39504. Epub 2012/07/21. https://doi.org/10.1371/journal.pone. 0039504 PMID: 22815707. 16. Thomson CA, Garcia DO, Wertheim BC, Hingle MD, Bea JW, Zaslavsky O, et al. Body shape, adiposity index, and mortality in postmenopausal women: Findings from the Women’s Health Initiative. Obesity (Silver Spring). 2016; 24(5):1061–9. Epub 2016/03/19. https://doi.org/10.1002/oby.21461 PMID: 26991923. 17. Dhana K, Kavousi M, Ikram MA, Tiemeier HW, Hofman A, Franco OH. Body shape index in comparison with other anthropometric measures in prediction of total and cause-specific mortality. J Epidemiol Community Health. 2016; 70(1):90–6. Epub 2015/07/15. https://doi.org/10.1136/jech-2014-205257 PMID: 26160362. 18. Thomas DM, Bredlau C, Bosy-Westphal A, Mueller M, Shen W, Gallagher D, et al. Relationships between body roundness with body fat and visceral adipose tissue emerging from a new geometrical model. Obesity (Silver Spring). 2013; 21(11):2264–71. Epub 2013/03/23. https://doi.org/10.1002/oby. 20408 PMID: 23519954. 19. Tian S, Zhang X, Xu Y, Dong H. Feasibility of body roundness index for identifying a clustering of cardio- metabolic abnormalities compared to BMI, waist circumference and other anthropometric indices: the China Health and Nutrition Survey, 2008 to 2009. Medicine. 2016; 95(34):e4642. Epub 2016/08/26. https://doi.org/10.1097/MD.0000000000004642 PMID: 27559964. 20. Chang Y, Guo X, Chen Y, Guo L, Li Z, Yu S, et al. References PLoS One. 2012; 7(7):18. 30. Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, et al. Harmonizing the meta- bolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epide- miology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation. 2009; 120(16):1640–5. Epub 2009/10/07. https://doi.org/10.1161/ CIRCULATIONAHA.109.192644 PMID: 19805654. 31. Taylor HL, Jacobs DR Jr., Schucker B, Knudsen J, Leon AS, Debacker G. A questionnaire for the assessment of leisure time physical activities. J Chronic Dis. 1978; 31(12):741–55. Epub 1978/01/01. PMID: 748370. 32. Redondo A, Subirana I, Ramos R, Solanas P, Sala J, Masia R, et al. [Trends in leisure time physical activity practice in the 1995–2005 period in Girona]. Revista espanola de cardiologia. 2011; 64 (11):997–1004. Epub 2011/09/29. https://doi.org/10.1016/j.recesp.2011.06.020 PMID: 21945092. 33. Haskell WL, Lee IM, Pate RR, Powell KE, Blair SN, Franklin BA, et al. Physical activity and public health: updated recommendation for adults from the American College of Sports Medicine and the American Heart Association. Circulation. 2007; 116(9):1081–93. Epub 2007/08/03. https://doi.org/10. 1161/CIRCULATIONAHA.107.185649 PMID: 17671237. 34. Schroder H, Fito M, Estruch R, Martinez-Gonzalez MA, Corella D, Salas-Salvado J, et al. A short screener is valid for assessing Mediterranean diet adherence among older Spanish men and women. J Nutr. 2011; 141(6):1140–5. Epub 2011/04/22. https://doi.org/10.3945/jn.110.135566 PMID: 21508208. 35. Zhao M, Bovet P, Ma C, Xi B. Performance of different adiposity measures for predicting cardiovascular risk in adolescents. Sci Rep. 2017; 7:43686. Epub 2017/03/07. https://doi.org/10.1038/srep43686 PMID: 28262726. 36. Zhang ZQ, Deng J, He LP, Ling WH, Su YX, Chen YM. Comparison of various anthropometric and body fat indices in identifying cardiometabolic disturbances in Chinese men and women. PLoS One. 2013; 8(8):e70893. Epub 2013/08/21. https://doi.org/10.1371/journal.pone.0070893 PMID: 23951031. 37. Kabat GC, Heo M, Van Horn LV, Kazlauskaite R, Getaneh A, Ard J, et al. Longitudinal association of anthropometric measures of adiposity with cardiometabolic risk factors in postmenopausal women. Ann Epidemiol. 2014; 24(12):896–902. Epub 2014/12/03. https://doi.org/10.1016/j.annepidem.2014.10.007 PMID: 25453348. 37. Kabat GC, Heo M, Van Horn LV, Kazlauskaite R, Getaneh A, Ard J, et al. Longitudinal association of anthropometric measures of adiposity with cardiometabolic risk factors in postmenopausal women. Ann Epidemiol. 2014; 24(12):896–902. Epub 2014/12/03. https://doi.org/10.1016/j.annepidem.2014.10.007 PMID: 25453348. 38. Behboudi-Gandevani S, Ramezani Tehrani F, Cheraghi L, Azizi F. References A body shape index and body roundness index: two new body indices to identify diabetes mellitus among rural populations in northeast China. BMC Public Health. 2015; 15:794. Epub 2015/08/20. https://doi.org/10.1186/s12889-015-2150-2 PMID: 26286520. 21. Marti R, Parramon D, Garcia-Ortiz L, Rigo F, Gomez-Marcos MA, Sempere I, et al. Improving interMedi- Ate risk management. MARK study. BMC Cardiovasc Disord. 2011; 11:61. Epub 2011/10/14. https:// doi.org/10.1186/1471-2261-11-61 PMID: 21992621. 22. Gomez-Sanchez L, Garcia-Ortiz L, Patino-Alonso MC, Recio-Rodriguez JI, Feuerbach N, Marti R, et al. Glycemic markers and relation with arterial stiffness in Caucasian subjects of the MARK study. PLoS One. 2017; 12(4):e0175982. Epub 2017/04/18. https://doi.org/10.1371/journal.pone.0175982 PMID: 28414819. 23. Marrugat J, D’Agostino R, Sullivan L, Elosua R, Wilson P, Ordovas J, et al. An adaptation of the Fra- mingham coronary heart disease risk function to European Mediterranean areas. J Epidemiol 14 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome Community Health. 2003; 57(8):634–8. Epub 2003/07/29. https://doi.org/10.1136/jech.57.8.634 PMID: 12883073. 24. Conroy RM, Pyorala K, Fitzgerald AP, Sans S, Menotti A, De Backer G, et al. Estimation of ten-year risk of fatal cardiovascular disease in Europe: the SCORE project. Eur Heart J. 2003; 24(11):987–1003. Epub 2003/06/06. PMID: 12788299. 25. Mancia G, Fagard R, Narkiewicz K, Redon J, Zanchetti A, Bohm M, et al. 2013 ESH/ESC Guidelines for the management of arterial hypertension: the Task Force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). J Hypertens. 2013; 31(7):1281–357. Epub 2013/07/03. PMID: 23817082. 26. Salas-Salvado J, Rubio MA, Barbany M, Moreno B. [SEEDO 2007 Consensus for the evaluation of overweight and obesity and the establishment of therapeutic intervention criteria]. Med Clin (Barc). 2007; 128(5):184–96. Epub 2007/02/15. PMID: 17298782. 27. Browning LM, Hsieh SD, Ashwell M. A systematic review of waist-to-height ratio as a screening tool for the prediction of cardiovascular disease and diabetes: 0.5 could be a suitable global boundary value. Nutr Res Rev. 2010; 23(2):247–69. Epub 2010/09/08. https://doi.org/10.1017/S0954422410000144 PMID: 20819243. 28. DM T, Bredlau C Fau—Bosy-Westphal A, Bosy-Westphal A Fau—Mueller M, Mueller M Fau—Shen W, Shen W Fau—Gallagher D, Gallagher D Fau—Maeda Y, et al.—Relationships between body round- ness with body fat and visceral adipose tissue emerging from a new geometrical model. Obesity. 2013; 21(11):2264–71. https://doi.org/10.1002/oby.20408 PMID: 23519954 29. NY K, JC K. A new body shape index predicts mortality hazard independently of body mass index. References Could "a body shape index" and "waist to height ratio" predict insulin resistance and metabolic syndrome in polycystic ovary syndrome? Eur J Obstet Gynecol Reprod Biol. 2016; 205:110–4. Epub 2016/09/01. https://doi.org/10.1016/j.ejogrb. 2016.08.011 PMID: 27579518. 38. Behboudi-Gandevani S, Ramezani Tehrani F, Cheraghi L, Azizi F. Could "a body shape index" and "waist to height ratio" predict insulin resistance and metabolic syndrome in polycystic ovary syndrome? Eur J Obstet Gynecol Reprod Biol. 2016; 205:110–4. Epub 2016/09/01. https://doi.org/10.1016/j.ejogrb. 2016.08.011 PMID: 27579518. 39. Haghighatdoost F, Sarrafzadegan N, Mohammadifard N, Asgary S, Boshtam M, Azadbakht L. Assess- ing body shape index as a risk predictor for cardiovascular diseases and metabolic syndrome among Iranian adults. Nutrition. 2014; 30(6):636–44. Epub 2014/05/08. https://doi.org/10.1016/j.nut.2013.10. 021 PMID: 24800666. 39. Haghighatdoost F, Sarrafzadegan N, Mohammadifard N, Asgary S, Boshtam M, Azadbakht L. Assess- ing body shape index as a risk predictor for cardiovascular diseases and metabolic syndrome among Iranian adults. Nutrition. 2014; 30(6):636–44. Epub 2014/05/08. https://doi.org/10.1016/j.nut.2013.10. 021 PMID: 24800666. 15 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 Capacity adiposity indices to identify metabolic syndrome 40. Oka R, Miura K, Sakurai M, Nakamura K, Yagi K, Miyamoto S, et al. Impacts of visceral adipose tissue and subcutaneous adipose tissue on metabolic risk factors in middle-aged Japanese. Obesity (Silver Spring). 2010; 18(1):153–60. Epub 2009/06/06. https://doi.org/10.1038/oby.2009.180 PMID: 19498348. 41. Fox CS, Massaro JM, Hoffmann U, Pou KM, Maurovich-Horvat P, Liu CY, et al. Abdominal visceral and subcutaneous adipose tissue compartments: association with metabolic risk factors in the Framing- ham Heart Study. Circulation. 2007; 116(1):39–48. Epub 2007/06/20. https://doi.org/10.1161/ CIRCULATIONAHA.106.675355 PMID: 17576866. 42. Waters DL, Ward AL, Villareal DT. Weight loss in obese adults 65years and older: a review of the con- troversy. Exp Gerontol. 2013; 48(10):1054–61. Epub 2013/02/14. https://doi.org/10.1016/j.exger.2013. 02.005 PMID: 23403042. 43. Porter Starr KN, Bales CW. Excessive Body Weight in Older Adults. Clin Geriatr Med. 2015; 31(3):311– 26. Epub 2015/07/22. https://doi.org/10.1016/j.cger.2015.04.001 PMID: 26195092. 44. Yamashita K, Kondo T, Osugi S, Shimokata K, Maeda K, Okumura N, et al. The significance of measur- ing body fat percentage determined by bioelectrical impedance analysis for detecting subjects with car- diovascular disease risk factors. Circ J. 2012; 76(10):2435–42. Epub 2012/07/13. PMID: 22784998. 45. Maessen MF, Eijsvogels TM, Verheggen RJ, Hopman MT, Verbeek AL, de Vegt F. Entering a new era of body indices: the feasibility of a body shape index and body roundness index to identify cardiovascu- lar health status. PLoS One. 2014; 9(9):e107212. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209992 January 25, 2019 References Epub 2014/09/18. https://doi.org/10.1371/journal. pone.0107212 PMID: 25229394. 16 / 16
https://openalex.org/W2214059621	https://jsi.cs.ui.ac.id/index.php/jsi/article/download/377/258	Indonesian	null	STUDI FAKTOR PENENTU PENERIMAAN DAN PENGGUNAAN SISTEM INFORMASI AKUNTANSI PADA LEMBAGA PERKREDITAN DESA	Jurnal Sistem Informasi/Jurnal sistem informasi (Journal of information system)	2,014	cc-by-sa	4,320	Abstrak Penelitian ini mengkaji penerimaan sistem informasi akuntansi berbasis komputer pada LPD di Bali, mengadopsi Technology Acceptance Model (TAM). Modifikasi dilakukan pada faktor eksternal, karena faktor ini sangat ditentukan oleh latar belakang LPD sebagai lembaga keuangan milik desa adat yang bersifat tradisional. Penelitian menggunakan pendekatan kuantitatif non-eksperimen dan bersifat ex- post facto. Pengumpulan data dilakukan dengan metode survei, menggunakan kuesioner sebagai alat pengumpul data. Sampel penelitian diambil secara acak proporsional (proportional random sampling) dari karyawan LPD yang ada di Kabupaten Badung dan Kota Denpasar. Operasional LPD dalam pemrosesan transaksi, pelaporan keuangan dan pelaporan manajemen belum sepenuhnya memanfaatkan sistem informasi akuntansi berbasis komputer. Sementara itu, model hubungan struktural yang dikembangkan berdasarkan kajian teoritis belum sepenuhnya didukung oleh data empiris. Pengeruh faktor eksternal terhadap penggunaan sesungguhnya sistem informasi akuntansi berbasis komputer di lingkungan LPD bersifat tidak nyata. Perlu dilakukan kajian lanjutan dengan merevisi model serta mempertimbangkan indikator-indikator tambahan sebagai bagian dari variabel eksternal agar dapat memberikan gambaran yang lebih komprehensif. Kata Kunci: sistem informasi akuntansi, teknologi informasi, technology acceptance model, lembaga perkreditan desa, model persamaan struktural. kan informasi akuntansi dari sudut pandang strate- gis [1]. Abstract This study examines the acceptance of computer-based accounting information systems at the Village Micro Financial Institutions (Lembaga Perkreditan Desa or LPD), by adopting the Technology Acceptance Model (TAM). Modification of the model performed on external factors, because these factors are determined by the LPD’s background as a traditional financial institution owned by indigenous villages. The study uses a non-experimental quantitative approach and ex-post facto research. Data collected by survey method, using a questionnaire. The respondents were selected randomly proportional from employees LPD in Badung regency and Denpasar city. The operational transaction processing, financial and management reporting at LPD is not fully utilizing computer- based accounting information system. Meanwhile, the structural model developed based on the theoretical study is not well supported by empirical data. The total effect of external factors on the actual use of computer-based accounting information systems in the LPD is not significant. Further research is needed, with revise the model and consider additional indicators as part of the external variables in order to provide a comprehensive model. Keywords: accounting information systems, information technology, technology acceptance model, village micro financial institutions (LPD), structural equation model. 46 Journal of Information Systems, Volume 10, Issue 1, April 2014 kan oleh Davis mengadopsi Theory of Reasoned Action (TRA). Persepsi kemudahan menggunakan (perceived ease of use) dan persepsi keberman- faatan (perceived usefulness) merupakan dua indi- kator kunci yang menentukan penerimaan indivi- dual terhadap teknologi informasi. Pembentukan persepsi kebermanfaatan dan kemudahan menggu- nakan, menentukan sikap terhadap penggunaan (attitude towards using technology), pada giliran- nya membentuk minat perilaku menggunakan tek- nologi informasi (behavioral intention to use) dan akhirnya mempengaruhi penggunaan teknologi in- formasi (actual technology use) [5]. al. menyebutkan manfaat dari sistem informasi akuntansi yang terkomputerisasi di antaranya: me- ningkatkan ketepatan waktu dalam penyajian infor- masi akuntansi, meningkatkan akurasi pencatatan transaksi, pengolahan data dan penyajian informasi menjadi lebih cepat, serta pelaporan bagi pihak eksternal menjadi lebih baik dan kredibel [2]. Keberhasilan penerapan teknologi informasi di samping ditentukan aspek perangkat keras dan perangkat lunak, juga ditentukan oleh pengguna teknologi tersebut. Hasil-hasil penelitian menun- jukkan kegagalan penerapan teknologi informasi lebih disebabkan oleh faktor perilaku penggunanya [3], [4]. Terlebih, pada lembaga usaha di mana sumber daya manusianya tidak secara khusus di- persiapkan untuk menggunakan teknologi infor- masi. Aspek keprilakuan dalam konteks manusia sebagai pengguna teknologi informasi menjadi penting diperhatikan dalam penerapan teknologi informasi [5]. Model TAM yang dikembangkan oleh Davis telah banyak dikaji ulang, direplikasi dan dikem- bangkan oleh peneliti-peneliti lainnya [6], [7], [8], [9], [10]. Penelitian aspek keperilakuan dalam pe- manfaatan teknologi informasi terus berkembang seperti yang dilakukan oleh Adamson & Shine [11], Barnett et al. [12], Fagan et al. [13], Jones et al. [14], dan Ilias & Nazirah [4]. Hasil-hasil pene- litian tersebut menunjukkan model TAM secara konsisten dan valid dapat menjelaskan tentang perilaku penerimaan teknologi informasi. Sejak tahun 1984, di Bali mulai dikembang- kan lembaga keuangan mikro yang dinamakan Lembaga Perkreditan Desa (LPD). Data per Agus- tus 2013 menunjukan jumlah LPD di Bali menca- pai 1.421 dari jumlah desa yang ada yakni 1.480. Total aset yang dikelola mencapai Rp 9,8 triliun lebih (postbali.com, 25 November 2013). Mening- katnya jumlah aset yang dikelola oleh setiap LPD, diikuti dengan meningkatnya volume transaksi keuangan. Dukungan teknologi informasi dalam operasional usaha LPD menjadi hal yang sangat penting. Pengamatan empirik di lapangan menun- jukkan, penggunaan teknologi informasi pada LPD masih sangat rendah. Kondisi ini tidak terlepas dari LPD sebagai lembaga keuangan yang berangkat dari organisasi tradisional, dikelola oleh sumber daya manusia dengan tingkat pengetahuan dalam bidang teknologi informasi yang terbatas. 46 Journal of Information Systems, Volume 10, Issue 1, April 2014 Beberapa peneliti juga mengembangkan mo- del TAM dengan menambahkan beberapa variabel eksternal yang menjadi penentu dari persepsi ke- bermanfaatan dan kemudahan menggunakan. Vari- abel-variabel eksternal yang digunakan seperti va- riabel-variabel individual, organisasi, kultur, dan karakteristik tugas [15], [16], [17], [18], [19], [20]. g Penelitian ini mengkaji faktor penentu pene- rimaan dan penggunan sistem informasi akuntansi berbasis komputer pada LPD di Bali, mengadopsi Technology Acceptance Model yang dikembang- kan oleh Davis [5]. Konstruk utama dalam peneli- tian sebelumnya yang meliputi persepsi keberman- faatan, persepsi kemudahan menggunakan, dan si- kap terhadap penggunaan teknologi informasi tetap dipertahankan. Modifikasi dilakukan pada faktor eksternal, mengingat faktor ini dipengaruhi oleh la- tar belakang LPD sebagai lembaga keuangan yang berangkat dari lembaga adat dan bersifat tradisio- nal. Berkenaan dengan hal tersebut permasalahan utama yang ingin diungkap adalah apakah faktor- faktor eksternal berpengaruh terhadap persepsi ke- bermanfaatan dan kemudahan menggunakan sis- tem informasi akuntansi, serta bagaimana implika- sinya terhadap penggunaan sesungguhnya sistem informasi akuntansi. Supaya teknologi informasi dapat diterima dengan baik di lingkungan LPD, maka perilaku pengguna perlu diubah. Merubah perilaku peng- guna tidak dapat dilakukan secara langsung, tetapi dilakukan lewat penentu-penentu perilaku tersebut. Kajian aspek keperilakuan dalam penerimaan dan penggunaan teknologi informasi pada dunia usaha telah berkembang seiring dengan meningkatnya kecenderungan dunia usaha memanfaatkan tekno- logi informasi dalam mendukung aktifitas bisnis- nya. Berbagai model telah dikembangkan untuk mengkaji penentu-penentu perilaku yang mempe- ngaruhi penerimaan atau penggunaan teknologi in- formasi, seperti Theory of Reasoned Action (TRA), Theory of Planned Behavior (TPB), serta Techno- logy Acceptance Model (TAM). 1. Pendahuluan Saat ini, teknologi infomasi sudah menjadi kebutu- han dasar bagi setiap perusahaan dalam menjalan- kan aktifitas bisnisnya. Kombinasi sistem informa- si akuntansi (SIA) dengan teknologi informasi te- lah menjelma menjadi alat bantu utama dalam pe- ngelolaan dan pengendalian terkait keuangan peru- sahaan. Kemajuan teknologi informasi telah mem- buka peluang untuk menghasilkan dan memanfaat- Penggunaan sistem informasi akuntansi ber- basis komputer telah membawa peluang bagi peru- sahaan untuk melakukan fungsi akuntansi secara lebih efektif dan efisien. Dampak terbesar dari pe- nerapan teknologi informasi pada SIA adalah ke- mampuan perusahaan untuk mengembangkan dan menggunakan sistem komputerisasi dalam mela- cak dan merekam transaksi keuangan. Ghasemi et 45 46 Journal of Information Systems, Volume 10, Issue 1, April 2014 2. Metodologi Penelitian ini menggunakan pendekatan kuantitatif non-eksperimen dan bersifat ex-post facto. Pe- ngumpulan data dilakukan dengan metode survei, menggunakan kuesioner sebagai alat pengumpul data. Sampel penelitian diambil secara acak pro- Technology Acceptance Model (TAM) meru- pakan salah satu model yang banyak digunakan dalam penelitian untuk mengkaji perilaku peneri- maan teknologi informasi. TAM yang dikembang- I Made Suarta, et al., Faktor Penentu Penerimaan 47 Gambar 1. Kerangka konseptual penelitian I Made Suarta, et al., Faktor Penentu Penerimaan 47 Gambar 1. Kerangka konseptual penelitian porsional dari karyawan LPD di Kabupaten Ba- dung dan Kota Denpasar. tertutup menggunakan skala Likert lima tingkat. Keseluruhan instrumen penelitian terdiri atas 25 butir pertanyaan. Butir-butir pertanyaan variabel persepsi kebermanfaatan (perceived usefulness), persepsi kemudahan menggunakan (perceived ease of use), sikap menggunakan sistem informasi akun- tansi (attitude toward using), minat menggunakan sistem informasi akuntansi (intention to use), di- kembangkan dan diadaptasi dari Davis [5] (Tabel 1). Model penelitian dikembangkan mengguna- kan persamaan struktural (structural equation mo- del) yang menggambarkan hubungan-hubungan li- near di antara variabel-variabel laten. Variabel la- ten merupakan konsep abstrak, hanya dapat dia- mati secara tidak langsung melalui efeknya pada variabel teramati. Model hubungan di antara vari- abel-variabel laten seperti pada Gambar 1. Kuesi- oner penelitian disusun dalam bentuk pernyataan TABEL 1 BUTIR-BUTIR PERTANAAN YANG DIKEMBANGKAN Persepsi kebermanfaatan sistem informasi akuntansi (perceived usefulness) 1. Sistem informasi akuntansi menjadikan pekerjaan terasa lebih mudah 2. Sistem informasi akuntansi meningkatkan kualitas pekerjaan 3. Sistem informasi akuntansi meningkatkan produktivitas pekerjaan 4. Dengan sistem informasi akuntansi, penyelesaian pekerjaan menjadi lebih cepat 5. Secara kesluruhan, sistem informasi akuntansi mampu meningkatkan kinerja Persepsi kemudahan menggunakan sistem informasi akuntansi (perceived ease of use) 1. Mempelajari penggunaan sistem informasi akuntansi sangat mudah 2. Interaksi pemakai dengan sistem informasi akuntansi sangat jelas 3. Instruksi-instruksi dalam sistem informasi akuntansi mudah dipahami 4. Secara keseluruhan, sistem informasi akuntansi sangat mudah dioperasikan Sikap menggunakan sistem informasi akuntansi (attitude toward using) 1. Menggunakan sistem informasi akuntansi merupakan gagasan yang sangat baik 2. Menggunakan sistem informasi akuntansi membuat pekerjaan lebih menarik 3. Bekerja dengan sistem informasi akuntansi menyenangkan 4. Sistem informasi akuntansi memotivasi saya untuk bekerja lebih baik 5. Saya suka bekerja dengan sistem informasi akuntansi Minat menggunakan sistem informasi akuntansi (Intention to Use) 1. Saya tertarik untuk mempelajari penggunaan sistem informasi akuntansi 2. Saya berniat menggunakan sistem informasi akuntansi dalam bidang pekerjaan saya 3. Saya berniat menggunakan sistem informasi akuntansi dalam pembuatan laporan keuangan 4. 3. Hasil dan Pembahasan Penelitian ini mengindikasikan penggunaan sesungguhnya sistem informasi akuntansi di LPD masih rendah. Hal ini ditandai oleh masih ba- nyaknya responden (45.8%) menggunakan kurang dari setengah waktu kerjanya dalam sehari berin- teraksi dengan sistem informasi akuntansi. Karya- wan yang waktu kerjanya banyak digunakan untuk berinteraksi dengan sistem informasi akuntansi a- dalah karyawan bagian front-line seperti teller, dan karyawan bagian back-office seperti bagian keua- ngan dan akuntansi. Secara kuantitatif, jumlah kar- yawan LPD pada posisi ini pada umumnya tidak banyak. 48 Journal of Information Systems, Volume 10, Issue 1, April 2014 48 Journal of Information Systems, Volume 10, Issue 1, April 2014 48 Journal of Information Systems, Volume 10, Issue 1, April 2014 Butir-butir pertanyaan berkaitan dengan pe- nggunaan sesungguhnya sistem informasi akun- tansi dikembangkan dari indikator-indikator peng- gunaan SIA (Tabel 2). Sementara butir-butir per- tanyaan variabel eksternal dikembangkan dengan memperhatikan karakteristik LPD yang berbasis pada masyarakat adat dan bersifat tradisional. Fak- tor eksternal yang diidentifikasi meliputi: (1) latar belakang LPD sebagai lembaga adat dan bersifat tradisional, serta (2) kultur sumber daya manusia pengelola LPD. kuensi penggunaan sistem informasi akuntansi da- lam kategori sering dan sangat sering. Sebaliknya, terdapat 166 responden (73.8%) responden yang frekuensi penggunaannya dalam kategori tidak per- nah hingga jarang. Sementara itu, keterampilan karyawan LPD dalam menggunakan sistem infor- masi akuntansi sudah cukup baik. Sebanyak 140 responden (62.3%) mempunyai keterampilan da- lam kategori terampil dan sangat terampil. Hanya 18.3% yang menyatakan dirinya tidak atau kurang terampil. TABEL 2 p TABEL 2 PENGGUNAAN SESUNGGUHNYA SISTEM INFORMASI AKUNTANSI (N=225) Indikator Penggunaan SIA Frekuensi % Waktu yang digunakan untuk berinteraksi dengan sistem informasi akuntansi (jam/hari kerja) <1 jam 6 2.7 1 – 2 jam 30 13.3 3 – 4 jam 67 29.8 4 – 5 jam 103 45.8 >5 jam 19 8.4 Frekuensi penggunaan sistem informasi akuntansi Tidak pernah 15 6.7 Sangat jarang 29 12.9 Jarang 122 54.2 Sering 50 22.2 Sangat sering 9 4.0 Keterampilan dalam menggunakan sistem informasi akuntansi Tidak terampil 6 2.7 Kurang terampil 35 15.6 Cukup terampil 44 19.6 Terampil 116 51.6 Sangat terampil 24 10.7 Uji validitas instrumen penelitian dilakukan menggunakan korelasi Pearson Product Moment dengan α=5%. Hasil uji menunjukkan validitas yang signifikan dengan koefisien product moment bervariasi antara 0.516 – 0.845. Sedangkan uji re- liabilitas dilakukan dengan menggunakan Cron- bach’s Alpha. Hasil uji raliabilitas menunjukkan nilai Cronbach’s Alpha bervariasi antara 0.509 – 0.816. TABEL 2 PENGGUNAAN SESUNGGUHNYA SISTEM INFORMASI AKUNTANSI (N=225) Hasil penelitian dianalisis menggunakan mo- del persamaan struktural dengan bantuan program LISREL 8.51 for Windows. Analisis model persa- maan struktural dimaksudkan untuk menganalisis serangkaian hubungan di antara variabel-variabel penelitian secara simultan. Kesesuaian model struktural dievaluasi menggunakan beberapa fit in- dex (indeks kesesuaian), seperti: Chi-Square Sta- tistic (Π2), Goodness of Fit Index (GFI), Adjusted Goodness of Fit Index (AGFI), Non-normed Fit Index (NNFI), Comparative Fit Index (CFI), Root Mean Square Residual (RMSR), dan the Root Mean Square Error of Approximation (RMSEA). 2. Metodologi Saya berniat mengusulkan penggunaan sistem informasi akuntansi dalam operasional LPD TABEL 1 BUTIR-BUTIR PERTANAAN YANG DIKEMBANGKAN Persepsi kebermanfaatan sistem informasi akuntansi (perceived usefulness) 1. Sistem informasi akuntansi menjadikan pekerjaan terasa lebih mudah 2. Sistem informasi akuntansi meningkatkan kualitas pekerjaan 3. Sistem informasi akuntansi meningkatkan produktivitas pekerjaan 4. Dengan sistem informasi akuntansi, penyelesaian pekerjaan menjadi lebih cepat 5. Secara kesluruhan, sistem informasi akuntansi mampu meningkatkan kinerja Persepsi kemudahan menggunakan sistem informasi akuntansi (perceived ease of use) 1. Mempelajari penggunaan sistem informasi akuntansi sangat mudah 2. Interaksi pemakai dengan sistem informasi akuntansi sangat jelas 3. Instruksi-instruksi dalam sistem informasi akuntansi mudah dipahami 4. Secara keseluruhan, sistem informasi akuntansi sangat mudah dioperasikan Sikap menggunakan sistem informasi akuntansi (attitude toward using) 1. Menggunakan sistem informasi akuntansi merupakan gagasan yang sangat baik 2. Menggunakan sistem informasi akuntansi membuat pekerjaan lebih menarik 3. Bekerja dengan sistem informasi akuntansi menyenangkan 4. Sistem informasi akuntansi memotivasi saya untuk bekerja lebih baik 5. Saya suka bekerja dengan sistem informasi akuntansi Minat menggunakan sistem informasi akuntansi (Intention to Use) 1. Saya tertarik untuk mempelajari penggunaan sistem informasi akuntansi 2. Saya berniat menggunakan sistem informasi akuntansi dalam bidang pekerjaan saya 3. Saya berniat menggunakan sistem informasi akuntansi dalam pembuatan laporan keuangan 4. Saya berniat mengusulkan penggunaan sistem informasi akuntansi dalam operasional LPD Penggunaan Sistem Informasi Akuntansi di Lingkungan LPD Sistem informasi akuntansi yang dimaksudkan da- lam penelitian ini adalah sistem yang berbasis pada teknologi informasi. Konstruk perilaku (behavior) dalam TAM merupakan tindakan yang dilakukan oleh seseorang. Dengan kata lain, perilaku (beha- vior) merupakan penggunaan sesungguhnya (actu- al usage) dari sistem informasi tersebut. Penggu- naan sesungguhnya dari sistem informasi akuntansi di lingkungan LPD diukur menggunakan indikator- indikator: (1) waktu yang digunakan untuk berinte- raksi dengan sistem informasi akuntansi, (2) freku- ensi penggunaannya, dan (3) keterampilan dalam menggunakan sistem informasi. Deskripsi penggu- naan sesungguhnya sistem informasi akuntansi di lingkungan LPD disajikan pada Tabel 2. y Frekuensi penggunaan sistem informasi akun- tansi termasuk dalam kategori sangat rendah. Hasil ini sejalan dengan waktu yang digunakan berinte- raksi dengan sistem informasi akuntansi, di mana cukup banyak karyawan yang berinteraksi dengan sistem informasi akuntansi kurang dari 4 jam seha- ri. Temuan ini juga didukung oleh tingkat keteram- pilan karyawan LPD, di mana hampir 40% karya- wan LPD dalam kategori tidak terampil hingga cu- kup terampil. Upaya penggunaan sistem informasi Terdapat 122 responden (54.2%) yang meng- gunakan waktu kerjanya dalam sehari untuk berin- teraksi dengan sistem informasi akuntansi lebih da- ri 4 jam. Sebanyak 59 responden (26.2%) yang fre- I Made Suarta, et al., Faktor Penentu Penerimaan 49 Gambar 2. Model lintasan dengan estimasi standardized solution Gambar 2. Model lintasan dengan estimasi standardized solution akuntansi di lingkungan LPD secara maksimal, perlu didukung oleh berubahnya perilaku karya- wan dalam memanfaatkan sistem informasi baru. Perubahan perilaku karyawan ditandai oleh kemau- an untuk selalu belajar dan meningkatkan keteram- pilan dalam menggunakan sistem informasi akun- tansi berbasis komputer. tem informasi akuntansi berbasis komputer di ling- kungan LPD tidak ditentukan oleh variabel ekster- nal yang diidentifikasi dari: (1) latar belakang LPD sebagai lembaga adat dan bersifat tradisional, serta (2) kultur sumber daya manusia pengelola LPD. Penelitian sebelumnya menunjukkan bahwa varia- bel-variabel eksternal secara konsisten signifikan, namun ada juga beberapa di antaranya yang tidak signifikan [21]. Analisis Model Persamaan Struktural TABEL 3 RINGKASAN STATISTIK ANALISIS JALUR Jalur Koefisien Jalur Nilai-t Signifikansi (t≥1,96) EXT PEU 0.14 2.17 Signifikan EXT PU -0.11 -1.58 Non signifikan PEU PU 0.16 2.42 Signifikan PEU ATT 0.09 1.77 Non signifikan PU ATT 0.55 13.45 Signifikan PU INT 0.06 0.57 Non signifikan PU ACT 0.30 4.53 Signifikan ATT INT 0.12 -1.34 Non signifikan INT ACT 0.01 -0.22 Non signifikan Keterangan: EXT = Variabel Eksternal; PEU = Persepsi kemudahan menggunakan; PU = Persepsi kebermanfaatan; AT = Sikap menggunakan; INT = Minat menggunakan; ACT = Penggunaan sesungguhnya TABEL 3 Model persamaan struktural menggambarkan hu- bungan-hubungan linear di antara variabel-variabel laten. Sebuah hubungan di antara variabel-variabel laten serupa dengan sebuah persamaan regresi line- ar di antara variabel-variabel laten tersebut. Bebe- rapa persamaan regresi linear membentuk sebuah persamaan simultan varabel-variabel laten. Model persamaan struktural dianalisis dengan bantuan program LISREL 8.51 for Windows. Hasil analisis model persamaan struktural yang menunjukkan hubungan kausal di antara va- riabel-variabel penelitian menghasilkan diagram lintasan basic model dengan estimasi standardized solution berikut nilai koefisien pada setiap jalur (Gambar 2). Angka-angka yang ada pada setiap ja- lur disebut sebagai koefisien struktural, menunjuk- kan pengaruh langsung (direct effect) antara varia- bel laten dengan variabel laten lainnya. Jalur lain yang bersifat signifikan adalah di an- tara persepsi kemudahan menggunakan (PEU) de- ngan persepsi kebermanfaatan (PU), persepsi ke- bermanfaatan (PU) dengan sikap terhadap meng- gunakan sistem informasi akuntansi (ATT), serta jalur di antara persepsi kebermanfaatan (PU) de- ngan penggunaan sistem informasi sesungguhnya (ACT). Sebagian dari temuan penelitian ini sejalan dengan hasil-hasil penelitian sebelumnya, namun sebagian lagi ada yang tidak selaras [22], [23]. Se- cara keseluruhan faktor eksternal berpengaruh ti- Signifikansi hubungan di antara variabel die- valuasi menggunakan nilai koefisien jalur (γ atau β) dan nilai-t (T-Value). Nilai koefisien jalur dan nilai-t dari hubungan-hubungan yang ada sebagai- mana dihipotesiskan sebelumnya dirangkum dalam Tabel 3. Hubungan antara variabel eksternal (EXT) dengan persepsi kemudahan menggunakan (PEU) bersifat signifikan. Sementara hubungan antara va- riabel eksternal dengan persepsi kebermanfaatan (PU) bersifat tidak signifikan. Kebermanfaatan sis- 50 Journal of Information Systems, Volume 10, Issue 1, April 2014 dak nyata terhadap penggunaan sesungguhnya sis- tem informasi akuntansi. Evaluasi terhadap berbagai fit index menun- jukkan nilai-nilai RMSR, GFI dan AGFI berada di atas ambang batas yang ditentukan. Berdasarkan ketiga fit index tersebut, dapat dinyatakan terdapat kecocokan antara data empiris dengan model yang diestimasi. Analisis Model Persamaan Struktural Beberapa fit index lainnya mempunyai nilai di bawah ambang batas yang ditentukan. Da- lam analisis model struktural memang tidak ada alat uji statistik tunggal untuk mengukur atau me- nguji hipotesis mengenai model struktural. Umum- nya digunakan berbagai jenis fit index (indeks ke- sesuaian) untuk mengukur derajat kesesuaian anta- ra model yang dihipotesiskan dengan data empirik [24]. Hasil ini memberikan makna bahwa model yang dibangun berdasarkan kajian teoritis belum sepenuhnya didukung oleh data empiris, karena be- berapa fit index menghasilkan nilai yang termasuk kategori tingkat kecocokan kurang baik. Kecocokan model dievaluasi menggunakan sejumlah kriteria goodness of fit [24]. Tabel 4 me- nyajikan ringkasan statistik tujuh fit index yang digunakan untuk mengevaluasi kecocokan model. Chi-square () digunakan untuk menguji sebera- pa dekat kecocokan antara matrik kovarian sampel dengan matrik kovarian model. Nilai Χ2 yang ren- dah sehingga menghasilkan significance level lebih besar atau sama dengan 0.05 (p≥0.05) menunjuk- kan hipotesis nol diterima dan matrik input yang diprediksi dengan yang sebenarnya tidak berbeda secara statistik. Root Mean Square Error of Appro- ximation (RMSEA) mewakili nilai rerata error dari model. Model dengan nilai RMSEA ≤ 0.05 menun- jukkan kecocokan model yang sangat baik. TABEL 4 RINGKASAN STATISTIK FIT MODEL Fit Index Nilai Rekomendasi Hasil Analisis Chi-square / degrees of freedom ≤3.00 30.44/6 = 6.74 RMSEA ≤0.06 or ≤0.08 0.135 NNFI ≥0.90 0.63 CFI ≥0.90 0.85 RMSR ≤0.10 0.027 GFI ≥0.90 0.96 AGFI ≥0.80 0.85 GFI = goodness-of-fit index; AGFI = adjusted goodness-of- fit index; NNFI = non-normed fit index; CFI = comparative fit index; RMSR = root mean square residual; RMSEA = root mean square error of approximation. TABEL 4 RINGKASAN STATISTIK FIT MODEL TABEL 4 TABEL 4 Referensi [1] Grande, E. U., Estébanez, R. P., & Colomina, C. M. (2011). The Impact of Accounting Information Systems (AIS) on Performance Measures: Empirical Evidence in Spanish SMEs. The International Journal of Digital Accounting Research, 11: 25 – 43. [13] Fagan, M., Neill, S., & Woolridge, B. (2008). Exploring the intention to use computers: An empirical investigation of the role of intrinsic motivation, extrinsic motivation and perceived ease of use. Journal of Computer Information Systems, 48(3): 31-37. [2] Ghasemi, M., Shafeiepour, V., Aslani, M., & Barvayeh, E. (2011). The Impact of Information Technology (IT) on Modern Accounting Systems. Procedia - Social and Behavioral Sciences, 28: 112 – 116. [14] Jones, C. M., McCarthy, R. V., Halawi, L., & Mujtaba, B. (2010). Utilizing the Technology Acceptance Model to Assess the Employee Adoption of Information Systems Security Measures. Issues in Information Systems, XI(1): 9 – 16. [3] Petter, S., DeLone, W., & McLean, E. (2008). Measuring information systems success: models, dimensions, measures, and interrelationships. European Journal of Information Systems, 17: 236–263. [15] Agarwal, R., & Prasad, J. (1999). Are Individual Differences Germane to the Acceptance of New Information technologies?. Decision Sciences, 30(2): 361 – 391. [4] Ilias, A., & Nazirah, N. (2013). Factor Affecting the Computerised Accounting System (CAS) Usage in Public Sector. Journal of Internet Banking and Commerce, 18(1): 1 – 19. [16] Gefen, D., & Straub, D.W. (1997). Gender Difference in the Perception and Use of Email: An Extention to the Technology Acceptance Model. MIS Quarterly, 12(4): 389 – 400. [5] Davis, F. D. (1989). Perceived usefulness, perceived ease of use, and user acceptance of information technology. MIS Quarterly, 13(3): 319-340. [17] Igbaria, M., Zinatelli, N., Cragg, P., & Cavaye, A.L.M. 1997. Personal Computing Acceptance Factors in Small Firms: A Structural Equation Modelling. MIS Quarterly, 21(3): 279-305. [6] Davis, F.D., Bagozzi, R.P., & Warshaw, P.R. (1989). User acceptance of computer technology: A comparison of two theoretical models. Management Science, 35(8): 982- 1003. [18] Karahanna, E., & Limayem, M. (2000). E-mail and V-mail usage: Generalizing Across Technologies. Journal of Organizational Computing and Electronic Commerce, 10(1): 49 – 66. [7] Adams, D.A., Nelson, R.R., & Todd, P.A. (1992). Perceived Usefulness, Ease of Use, and Usage of Information Technology: A Replication. MIS Quarterly, 16(2): 227-247. [19] Moon, J., & Kim, Y. (2001). Extending the TAM for a World Wide Web Context. Information and Management, 38(4): 217 – 230. [8] Davis, F.D. & Venkatesh, V. 4. Kesimpulan Berdasarkan indikator-indikator penggunaan se- sungguhnya dari sistem informasi akuntansi di lingkungan LPD ditemukan tingkat penggunaan sistem informasi akuntansi berbasis komputer ma- sih rendah. Hasil penelitian mengindikasikan ope- rasional LPD dalam pemrosesan transaksi, pelapo- ran keuangan dan pelaporan manajemen belum se- penuhnya memanfaatkan sistem informasi akun- tansi berbasis komputer. Hubungan antara variabel eksternal dengan persepsi kemudahan menggunakan, persepsi ke- mudahan menggunakan dengan persepsi keber- manfaatan, persepsi kebermanfaatan dengan sikap terhadap menggunakan sistem informasi akuntan- si, serta persepsi kebermanfaatan dengan penggu- naan sistem informasi sesungguhnya menunjukkan hasil yang signifikan. Sementara jalur lainnya me- nunjukkan hasil yang tidak signifikan. Pengaruh total faktor eksternal terhadap penggunaan sistem informasi akuntansi menunjukkan hasil yang tidak signifikan. Evaluasi kecocokan model mengguna- kan beberapa kriteria goodness of fit ditemukan fit index RMSR, GFI dan AGFI berada di atas ambang batas yang ditentukan. Hasil ini memberikan mak- na bahwa secara keseluruhan model yang dibangun berdasarkan kajian teoritis belum sepenuhnya di- dukung oleh data empiris. Non-Normed Fit Index (NNFI) merupakan ukuran kecocokan inkremental, yaitu memban- dingkan model yang diusulkan dengan model dasar (null model). Kisaran nilainya antara 0 sampai 1, dimana nilai NNFI ≥ 0.9 menunjukkan kecocokan model yang sangat baik. Comparative Fit Index (CFI) juga merupakan ukuran kecocokan inkre- mental. Nilai CFI ≥ 0.9 menunjukkan kecocokan model yang sangat baik. Root Mean Square Resi- dual (RMSR) mewakili nilai rerata residual yang diperoleh dari mencocokkan matrik varian-kova- rian dari model yang dihipotesiskan dengan matrik varian-kovarian dari data sampel. Goodness-of-Fit Index (GFI) merupakan ukuran kecocokan absolut, membandingkan model yang dihipotesiskan de- ngan tidak ada model sama sekali. Adjusted Good- ness-of-Fit Index (AGFI) merupakan perluasan da- ri GFI yang disesuaikan dengan rasio antara degree of freedom dari null model dengan degree of free- dom dari model yang dihipotesiskan. Nilai GFI dan AGFI berkisar antara 0 (poor fit) sampai 1 (perfect fit). Direkomendasikan untuk melakukan kajian lanjutan dengan mempertimbangkan indikator-in- dikator tambahan sebagai bagian dari variabel eks- ternal agar dapat memberikan gambaran yang lebih komprehensif. Model yang dibangun juga perlu di- kaji ulang dengan mempertimbangkan fokus pe- manfaatan sistem informasi akuntansi berbasis komputer. I Made Suarta, et al., Faktor Penentu Penerimaan 51 [12] Barnett, T., Kellermanns, F., Pearson, A., & Pearson, R. (2006/2007). Measuring information system usage: Replication and extensions. The Journal of Computer Information Systems, 47(2): 76-85. 52 Journal of Information Systems, Volume 10, Issue 1, April 2014 Multivariate data analysis. 6th edtion. New Jersey: Pearson Education, Inc. Referensi (1996). A critical assessment of potential measurement biases in the technology acceptance model: three experiments. International Journal Human- Computer Studies, 45: 19-45. [20] Lee, Y., Kozar, K.A., & Larsen, K.R.T. (2003). The Technology Acceptance Model: Past, Future, Present. Communications of the Association for Information Systems, 12: 752- 780. [9] Venkatesh, V., & Davis, F.D. (2000). A Theoretical Extension of the Technology Acceptance Model: Four Longitudinal Field Studies. Management Science, 46(2): 186– 204. [21] Legris, P., Ingham, J., & Collerette, P. (2002). ”Why do People Use Information Technology? A Critical Review of the Technology Acceptance Model”. Information & Management, 40: 191 – 204. [10] Venkatesh, V., & Morris, M. G. (2000). “Why Don’t Men Ever Stop to Ask For Directions? Gender, Social Influence, and Their Role in Technology Acceptance and Usage Behavior,” MIS Quarterly, 24(1): 115-139. [22] Venkatesh, V., Morris, M.G., Davis, G.B., & Davis, F.D. (2003). User Acceptance of Information Technology: Toward a Unified View. MIS Quarterly, 27(3): 425-478. [11] Adamson, I., & Shine, J. (2003). Extending the new technology acceptance model to measure the end user information systems satisfaction in a mandatory environment: a bank's treasury. Technology Analysis and Strategic Management, 15(4): 441-455. [23] Sriwidharmanely & Syafrudin, V. (2012). An Empirical Study of Accounting Software Acceptance among Bengkulu City Students. 52 Journal of Information Systems, Volume 10, Issue 1 Asian Journal of Accounting and Governance, 3: 99–112 [24] Hair, Jr., J.F., Black, W.C., Babin, B.J., Anderson, R.E., & Tatham, R.L. (2006). 52 Journal of Information Systems, Volume 10, Issue 1, April 2014 Asian Journal of Accounting and Governance, 3: 99–112 [24] Hair, Jr., J.F., Black, W.C., Babin, B.J., Anderson, R.E., & Tatham, R.L. (2006). Multivariate data analysis. 6th edtion. New Jersey: Pearson Education, Inc. Multivariate data analysis. 6th edtion. New Jersey: Pearson Education, Inc.
https://openalex.org/W2744763593	https://bmccomplementalternmed.biomedcentral.com/track/pdf/10.1186/s12906-017-1894-x	English	null	Polarity directed optimization of phytochemical and in vitro biological potential of an indigenous folklore: Quercus dilatata Lindl. ex Royle	BMC complementary and alternative medicine	2,017	cc-by	12,912	Abstract Background: Plants have served either as a natural templates for the development of new chemicals or a phytomedicine since antiquity. Therefore, the present study was aimed to appraise the polarity directed antioxidant, cytotoxic, protein kinase inhibitory, antileishmanial and glucose modulatory attributes of a Himalayan medicinal plant- Quercus dilatata. Methods: Total phenolic and flavonoid contents were determined colorimetrically and various polyphenols were identified by RP-HPLC analysis. Brine shrimp lethality, SRB and MTT assays were employed to test cytotoxicity against Artemia salina and human cancer cell lines respectively. Antileishmanial activity was determined using standard MTT protocol. Glucose modulation was assessed by α-amylase inhibition assay while disc diffusion assay was used to establish protein kinase inhibitory and antifungal spectrum. Background: Plants have served either as a natural templates for the development of new chemicals or a phytomedicine since antiquity. Therefore, the present study was aimed to appraise the polarity directed antioxidant, cytotoxic, protein kinase inhibitory, antileishmanial and glucose modulatory attributes of a Himalayan medicinal plant- Quercus dilatata. Methods: Total phenolic and flavonoid contents were determined colorimetrically and various polyphenols were identified by RP-HPLC analysis. Brine shrimp lethality, SRB and MTT assays were employed to test cytotoxicity against Artemia salina and human cancer cell lines respectively. Antileishmanial activity was determined using standard MTT protocol. Glucose modulation was assessed by α-amylase inhibition assay while disc diffusion assay was used to establish protein kinase inhibitory and antifungal spectrum. Results: Among 14 extracts of aerial parts, distilled water-acetone extract demonstrated maximum extract recovery (10.52% w/w), phenolic content (21.37 ± 0.21 μg GAE/mg dry weight (DW)), total antioxidant capacity (4.81 ± 0.98 μg AAE/mg DW) and reducing power potential (20.03 ± 2.4 μg/mg DW). On the other hand, Distilled water extract proficiently extracted flavonoid content (4.78 ± 0.51 μg QE/mg DW). RP-HPLC analysis revealed the presence of significant amounts of phenolic metabolites (0.049 to 15.336 μg/mg extract) including, pyrocatechol, gallic acid, catechin, chlorogenic acid, p-coumaric acid, ferulic acid and quercetin. Highest free radical scavenging capacity was found in Methanol-Ethyl acetate extract (IC50 8.1 ± 0.5 μg/ml). In the brine shrimp toxicity assay, most of the tested extracts (57%) showed high cytotoxicity. Among these, Chloroform-Methanol extract had highest cytotoxicity against THP-1 cell line (IC50 3.88 ± 0.53 μg/ml). About 50% of the extracts were found to be moderately antiproliferative against Hep G2 cell line. Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 DOI 10.1186/s12906-017-1894-x Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 DOI 10.1186/s12906-017-1894-x Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 DOI 10.1186/s12906-017-1894-x © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Polarity directed optimization of phytochemical and in vitro biological potential of an indigenous folklore: Quercus dilatata Lindl. ex Royle Madiha Ahmed1, Humaira Fatima1, Muhammad Qasim2, Bilquees Gul2 and Ihsan-ul-Haq1* * Correspondence: ihsn99@yahoo.com; ihaq@qau.edu.pk 1Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan Full list of author information is available at the end of the article Collection and extraction Collection and extraction Aerial parts of Q. dilatata were collected during September 2013 from Murree hills, Pakistan and identified by Prof. Dr. Rizwana Aleem Qureshi, Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, Pakistan. Voucher specimen (PHM-490) was ar- chived in the herbarium of medicinal plants, Department of Pharmacy, Quaid-i-Azam University, Islamabad. The plant was thoroughly washed under running water and shade dried with active ventilation at ambient temperature for three weeks. The dried plant material was pulverised to fine powder and stored in air-tight containers till further use. The powdered plant (40 g) was subjected to sonication aided maceration for 24 h at room temperature using ana- lytical grade solvents (400 ml) in 1000 ml Erlenmeyer flask. The extracts were concentrated by vacuum evaporation in rotary evaporator (Buchi, Switzerland) and dried in vacuum oven (Yamato, Japan) at 45 °C to obtain final crude extract. The experiment was run in triplicate. The solvents included n-Hexane (NH), Chloroform (C), Acetone (A), Ethyl acetate + Acetone (EtA), Ethyl acetate (Et), Chloroform + Ethanol (CE), Chloroform + Methanol (CM), Ethanol + Ethyl acetate (EEt), Methanol + Ethyl acetate (MEt), Ethanol (E), Distilled water + Acetone (DA), Methanol (M), Distilled water + Methanol (DM) and Distilled water (D). A ratio of 1:1 was used for the preparation of binary solvent systems. The extracts were stored at −30 °C for further testing. Quercus dilatata Lindl. ex Royle (Synonym; Quercus floribunda Lindl. Camus), known commonly as Holly Oak and locally as Bunj or Barungi, belongs to family Fagaceae. The genus Quercus encompassing around 400 species is distributed in America, temperate Europe, Asia and sub-tropical Africa. In Pakistan this evergreen tree is abundant in the Himaliyan mountains specially in Dir, Chitral, Swat, Hazara, Tirah, Kurram Agency, Murree hills and Azad Kashmir [3]. Powdered form of its fruits are utilized for the eradication of gonorrhoea and urinary tract infections in district Swat [4]. Leaves and seeds are utilized against sore mouth and throat in Lawat district [5]. Seeds are also believed to be astrin- gent, diuretic and are employed in diarrhoea, indigestion and asthma in Poonch Valley [6]. Extraction efficiency and bioactivity of herbal extracts can be optimized by varying extraction solvent polarity [7]. Solvents and reagents Solvents (n-Hexane, chloroform, acetone, ethyl acetate, methanol, ethanol and DMSO), gallic acid, quercetin, aluminium chloride (AlCl3), potassium acetate, 2,2-diphe- nyl-1-picrylhydrazyl (DPPH), ascorbic acid, sulfuric acid (H2SO4), ammonium molybdate, monosodium dihydro- gen phosphate (NaH2PO4), trichloroacetic acid (TCA), potassium ferricyanide, ferric chloride (FeCl3), standard antibiotics (cefixime, ciprofloxacin), standard antifungal (clotrimazole), trypton soy broth (TSB), α-amylase enzyme (from Bacillus subtilis), acarbose, phosphate buffer (PB), Phytochemical analysis y y Stock solution of each crude extract in DMSO (4 mg/ml) was prepared for phytochemical analysis. Collection and extraction In this study, multiple mono and binary solvent systems of escalating polarities were applied to find out most proficient system for the extraction of antioxidant, cytotoxic, protein kinase inhibitory, antidiabetic and/or antimicrobial potential of Q. dilatata. To the best of our knowledge, protein kinase inhibitory, antileishmanial, antidiabetic and cytotoxic (against THP-1 and Hep G2 human cancer cell lines) activities of Q. dilatata extracts are being reported for the first time here. Extract recovery The dried extracts were weighed to calculate % recovery of crude extracts by the following formula. Extract recovery %w=w ð Þ ¼ A=40 ð Þ 100 Extract recovery %w=w ð Þ ¼ A=40 ð Þ 100 Where; A = weight of crude extract obtained after drying. Abstract Methanol extract exhibited considerable protein kinase inhibitory activity against Streptomyces 85E strain (28 ± 0.35 mm bald phenotype at 100 μg/disc; MIC = 12.5 μg/ disc) while, Chloroform extract displayed maximum antidiabetic activity (α-amylase inhibition of 21.61 ± 1.53% at 200 μg/ml concentration). The highest antileishmanial potential was found in Ethyl acetate-Acetone extract (12.91 ± 0.02% at 100 μg/ml concentration), while, Q. dilatata extracts also showed a moderate antifungal activity. Conclusion: This study proposes that multiple-solvent system is a crucial variable to elucidate pharmacological potential of Q. dilatata and the results of the present findings prospects its potential as a resource for the discovery of novel anticancer, antidiabetic, antileishmanial and antioxidant agents. Keywords: Antileishmanial, Antioxidant, Cytotoxicity, Hep G2, Protein kinase inhibition, THP-1 leukemia cell line * Correspondence: ihsn99@yahoo.com; ihaq@qau.edu.pk 1Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan Full list of author information is available at the end of the article Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 2 of 16 Background Folin–Ciocalteu reagent, RPMI-1640 medium, Medium 119, DMEM and sea salt were purchased from Sigma (Sigma-Aldrich Germany). Sabouraud dextrose agar (SDA) was purchased from Oxoid England; Tween-20 from Merck-Schuchardt, USA. Medium ISP4 was prepared in lab while doxorubicin was purchased from Merck (Darmstadt, Germany). Bioactive phytoconstituents include an array of compounds (e.g., tannins, lignans, coumarins, quinones, stilbenes, xanthones, phenolic acids, flavones, flavonols, catechins, anthocyanins, and proanthocyanins) that could delay or deter the inception of diseases such as cancer and diabetes [1]. They have also contributed to the pharmacopeia of the world for the provision of new and effective agents against infectious diseases such as leishmaniasis [2]. Mounting the credibility of traditional medicinal plants requires consoli- dation of scientific data to fill the research based evidence gaps. Therefore, the present study is designed to appraise the antioxidant, cytotoxic, kinases inhibitory, antileishma- nial and antidiabetic properties of a folklore medicinal plant; Quercus dilatata. Determination of total flavonoid content (TFC) Total flavonoid content was estimated by aluminium chloride colorimetric method with minor modifications [10, 11]. Briefly 20 μl from each test sample stock solu- tion, 10 μl of aluminium chloride (10% w/v in H2O), 10 μl of 1.0 M potassium acetate and 160 μl of distilled water were added in 96 well plate which was incubated at room temperature for 30 min. The absorbance of the plate was measured at 415 nm using microplate reader. A calibration curve (y = 0.0268× + 0.00764, R2 = 0.9851) of quercetin was drawn at final concentrations of 2.5, 5, 10, 20, 40 μg/ml and the resultant flavonoid content is expressed in microgram equivalents of quercetin per milligram dry weight (μg QE/mg DW). Total antioxidant capacity (TAC) Phosphomolybde- num based colorimetric assay was employed to determine total antioxidant capacity and is expressed as the number of microgram equivalents of ascorbic acid per milligram of dry plant weight (μg AAE/mg DW) [10, 16]. An aliquot of 0.1 ml of each test extract (4 mg/ml DMSO) and positive control (ascorbic acid, 4 mg/ml DMSO) was mixed with 0.9 ml of reagent (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate solution in H2O). Blank contained 0.9 ml of reagent solution and 0.1 ml of DMSO without extract. All tubes were kept in water bath for 90 min at 95 °C and then cooled to room temperature from which 200 μl of each sample was transferred to 96 well plate and the absorbance of each sample was measured at 630 nm using micro- plate reader (Biotech USA, microplate reader Elx 800). A calibration curve (y = 0.0212× + 0.0926, R2 = 0.9913) of ascorbic acid was drawn at final con- centrations of 100, 50, 25, 12.5, 6.25, 3.12 μg/ml and the experiment was performed in triplicate. Determination of total phenolic content (TPC) Folin-Ciocalteu (FC) reagent method was used to estimate total phenolic content [8, 9]. An aliquot of 20 μl from stock solution and 90 μl of FC reagent in 96 well plate was incubated for 5 min at room temperature followed by the Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 3 of 16 addition of 90 μl of sodium carbonate solution. Absorbance of the assay plate was recorded at 630 nm using microplate reader (Biotech USA, microplate reader Elx 800). A calibration curve (y = 0.0136× + 0.0845, R2 = 0.9861) was obtained under the same operating con- ditions using gallic acid (6.25–50 μg/ml) as a positive standard. The assay was performed in triplicate and the results are expressed as microgram gallic acid equivalent per milligram dry weight (μg GAE/mg DW). concentrations of 200, 66.66, 22.22 and 7.41 μg/ml, were mixed with 180 μl of DPPH solution (9.2 mg/100 ml methanol) in 96 well plate. After incubating the plate for 30 min at 37 °C, absorbance was recorded at 517 nm. Percent free radical scavenging activity (%FRSA) was calculated by using the formula: %FRSA ¼ 1–Abs=Abc ð Þ 100 Where Abs is the absorbance of test sample, whereas Abc is the absorbance of negative control containing the DMSO instead of sample. Ascorbic acid was used as positive control and the assay was performed in tripli- cate. Afterwards IC50 of samples with significant radical scavenging efficiency (>50%) was also calculated. RP-HPLC quantitative analysis High performance liquid chromatography was per- formed according to previously described procedure [12, 13]. Dried extracts (0.5 g) were dissolved in methanol (62.5%) and 6 M HCl solution. After purging ni- trogen for few sec, samples were refluxed for 2 h. Filtered extracts were adjusted to 100 ml (with methanol) and re- filtered through 0.45 μm membrane filter (Millex-HV) be- fore injecting into HPLC. The HPLC system (Shimadzu LC-20AT) was equipped with auto-sampler (SIL-20A), column oven (CTO-20A), and diode array detector (SPD- M20A). Analytical column- Nucleosil C18, 5 μm 100 A° (250 × 4.60 mm, Phenomenex) coupled with a guard col- umn (KJO-4282, Phenomenex) was used. Mobile phase was composed of 1% acetic acid solution and 70% metha- nol. Gradient program by Araruna et al. [14] was used with a flow rate of 0.8 ml/min. Phenolic compounds were identified by comparing retention time and UV–vis spec- tra of chromatographic peaks with that of authentic refer- ence standards at 280 nm. Total reducing power (TRP) Standard potassium ferri- cyanide colorimetric assay was performed to estimate the reducing power of different solvent extracts [10, 17]. An aliquot of 200 μl of test extracts (4 mg/ml DMSO) was mixed with 400 μl of each phosphate buffer (0.2 mol/l, pH 6.6) and potassium ferricyanide (1% w/v in H2O). The mixture was incubated for 20 min at 50 °C followed by addition of 400 μl of trichloroacetic acid (10% w/v in H2O) and centrifuged at 3000 rpm at room temperature for 10 min. The upper layer of solution (500 μl) was mixed with distilled water (500 μl) and 100 μl of FeCl3 (0.1% w/v in H2O). From this mixture, 200 μl was transferred to 96 well plate and the absorbance of the reaction mixture was measured at 630 nm. Blank was prepared by adding 200 μl of DMSO to the aforesaid reaction mixture instead of extract. A Cytotoxicity against Hep G2 cell line y y g p The cytotoxic potential of extracts towards Hep G2 cancer cell line (RBRC-RCB1648) was determined by using SRB colorimetric assay as described previously [19]. Briefly, Hep G2 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% v/v FBS, 100 IU/ml penicillin G sodium, 100 μg/ml streptomycin sulphate and 0.25 μg/ml amphotericin B. The plate was then incubated in humidified atmosphere at 37 °C and 5% CO2 for 72 h to obtain a confluence of approximately 60– 70%. Old medium was replaced with fresh medium and the cells were incubated for another 24 h after which they were trypsonised and diluted to get an assay density of 1 × 105 cells/ml. An aliquot of 180 μl from aforemen- tioned culture was then transferred to each well of 96 well plate having 20 μl of test samples (containing 1% DMSO in PBS) to have final concentration of 20 μg/ml. Doxo- rubicin (20–0.08 μg/ml) and 1% v/v DMSO in PBS instead of test sample were employed as positive and negative controls respectively. The culture plate was then incu- bated at 37 °C for 72 h in CO2 incubator. The incubation was stopped with the addition of 50 μl of cold 20% w/v TCA for 1 h at 4 °C for cell fixation. The fixed cells were washed 4 times with tap water, air dried and stained with 50 μl of 0.057% w/v SRB in 1% w/v acetic acid for 30 min at room temperature. Wells were then washed 4 times with 1% v/v acetic acid and the plates were dried over- night. Bound dye was solubilized in 200 μl 10 mM Tris base, pH 10, for 1 h. Optical density was measured on a micro-plate reader (Biotech USA, microplate reader Elx 800) at 515 nm and percent survival was determined. In each case, a zero-day control was performed by adding an equivalent number of cells to sixteen wells, incubating at 37 °C for 1 h and processing as described above. Percent of cell growth inhibition was calculated using the formula: Cytotoxicity against THP-1 human leukemia cell line The in vitro cytotoxicity evaluation of extracts against human leukemia (THP-1) cell line (ATCC # TIB-202) was performed using standard protocol [18]. Cytotoxicity against Hep G2 cell line Concisely, leukemia cells were grown in complete growth medium [RPMI-1640 buffered with 2.2 g/l NaHCO3 and supple- mented with 10% v/v heat inactivated foetal bovine serum (HIFBS); pH 7.4] in a humidified carbon dioxide incubator (37 °C, 5% CO2). 10 μl of test sample contain- ing 1% DMSO in PBS was added in wells of microtitre plate to have a final concentration of 20 μg/ml. Subsequently, an aliquot of 190 μl of THP-1 cells (seed- ing density of 1 × 104 cells per ml) was transferred to each well. The culture was incubated at 37 °C for 72 h in humidified CO2 (5%) incubator (Panasonic, Japan MCO-18 AC-PE). Serial concentrations of fluorouracil and vincristine were employed as positive controls whereas 1% DMSO in PBS served as negative control. Afterwards, 20 μl of pre-filter sterilized MTT solution (4 mg/ml in distilled H2O) was added and plates were again incubated at 37 °C for 4 h in humidified CO2 (5%) incubator. After incubation supernatant was removed carefully by multi-channel pipette without disturbing coloured formazan sediments, which were equivalent to amount of live cells. To dissolve the formazan sediments %Inhibition ¼ 100–½ ODcellsþsamples–ODday 0 = ODcellsþ1%DMSO–ODday0 100 Antioxidant assays Free radical scavenging activity (FRSA) The FRSA of the crude extracts was evaluated by monitoring their capability to quench the stable 2, 2-diphenyl-1-picrylhy- drazyl (DPPH) free radical [8, 15]. Briefly, 20 μl of four different dilutions of each test sample to have final Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 4 of 16 Page 4 of 16 calibration curve (y = 0.038× + 0.7484, R2 = 0.9967) of ascorbic acid was obtained at final concentrations of 100, 50, 25, 12.5, 6.25, 3.12 μg/ml and the resultant re- ducing power of each sample is expressed as μg AAE/mg DW. The assay was performed in triplicate. 100 μl of DMSO was added in each well, the plate was kept aside for 1 h to ensure full dissolution and the ab- sorbance was measured at 540 nm using microplate reader. Samples showing more than 50% cell mortality at 20 μg/ml were further analysed at lower concentrations i.e. 10, 5, 2.5 and 1.25 μg/ml and the assay was per- formed in triplicate. IC50 was calculated by using table curve 2D v5.01 software. Brine shrimp lethality assay A 24 h lethality test was performed in a 96 well plate against brine shrimp (Artemia salina) larvae as de- scribed previously [8]. Eggs of A. salina (Ocean star, USA) were incubated for 24–48 h hatching period under light and warmth (30–32 °C) in simulated sea water (38 g/l supplemented with 6 mg/l dried yeast) in a spe- cially designed two-compartment plastic tray. The ma- ture phototropic nauplii (10) were then harvested with the help of Pasteur pipette and transferred to each well of plate. Corresponding volume of each extract contain- ing ≤1% DMSO in sea water at final concentrations of 200, 100, 50 and 25 μg/ml was transferred to the wells containing sea water and shrimp larvae. The final vol- ume in each well was kept 300 μl. Positive and negative control wells included serial concentrations of doxorubi- cin and 1% DMSO in sea water respectively. After 24 h incubation, live shrimps were counted and the percent- age of deaths was determined. Median lethal concentra- tion (LC50) was calculated using table curve 2D v5.01 software. The experiment was run in triplicate. %α–amylase inhibition ¼ Os–On ð Þ= Ob–On ð Þ 100 where On = Absorbance of negative control, Os = Absorbance of sample and Ob = Absorbance of blank well. α-Amylase inhibition assay d b l f Antidiabetic potential of test extracts was determined by α-amylase inhibition assay following the standard proto- col with minor modification [23]. The reaction mixture containing 15 μl phosphate buffer (pH 6.8), 25 μl α- amylase enzyme (0.14 U/ml), 10 μl sample (4 mg/ml DMSO) and 40 μl starch solution (2 mg/ml in potassium phosphate buffer) was incubated at 50 °C for 30 min in 96 well plate followed by addition of 20 μl of 1 M HCl to stop the reaction. Afterwards 90 μl of iodine reagent (5 mM iodine, 5 mM potassium iodide) was added to each well. Negative control was prepared without plant extracts whereas blank was prepared without extracts and amylase enzyme; each being replaced by equal quan- tities of buffer. Acarbose (250 μM) was used as positive control. Absorbance of reaction plate after incubation was measured at 540 nm. Activity was expressed as per- cent α-amylase inhibition/mg dry extract and calculated by the following equation: Cytotoxicity against isolated lymphocytes Lymphocytes were isolated using formerly described pro- cedure with slight modifications [20]. Informed consent was taken from volunteers and protocol was followed Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 5 of 16 according to international ethical guidelines after approval from the ethical committee of the Quaid-i-Azam Univer- sity (IRB-QAU-116). A volume of 3 mL of blood was col- lected from a healthy donor by venipuncture and diluted (1:1) with PBS. It was layered over 2 mL Histopaque-1077 and centrifuged at 800×g for 20 min. The buffy coat was aspirated into 5 mL of PBS and centrifuged at 350 rpm for 4 min to pellet the lymphocytes. The pellet was suspended in 1 mL of RPMI-1640 and cell density was adjusted to get 1 × 105 cells/ml. For cytotoxicity determination, 20 μl of samples (20 μg/ml) or vincristine or 1% DMSO in PBS and 180 μl of lymphocyte suspension were incubated in 96-well plate at 37 °C for 24 h in humidified 5% carbondi- oxide incubator (Panasonic, Japan MCO-18 AC-PE). Phytohaemagglutinin (PHA) was added in medium to stimulate lymphocyte growth. Afterwards, MTT assay was performed as described above. H2O) was added and plates were again incubated at 24 °C for 4 h. After incubation supernatant was removed carefully without disturbing coloured formazan sediments. To dis- solve the formazan sediments, 100 μl of DMSO was added in each well, the plate was kept aside for 1 h to ensure full dissolution and the absorbance was measured at 540 nm using microplate reader. Samples showing more than 50% cell mortality at 100 μg/ml were further analysed at lower concentrations i.e. 33.3, 11.1, 3.7 and 1.23 μg/ml. IC50 was calculated by using table curve 2D v5.01 software. Protein kinase inhibition assay The protein kinase inhibition assay was performed by observing hyphae formation in purified isolates of Strep- tomyces 85E strain [21]. Microbial lawn was formed by spreading spores and mycelia fragments (100 μl) of refreshed culture of Streptomyces on plates containing minimal ISP4 medium under sterile conditions. An ali- quot of 5 μl of each extract (20 mg/ml of DMSO) was loaded onto sterile 6 mm filter paper discs. The impreg- nated sterile discs each having 100 μg/disc of extract were placed on the surface of the plates seeded with Streptomyces 85E. Surfactin (5 μl of 4 mg/ml of DMSO) and DMSO impregnated discs served as positive and negative controls respectively. The plates were incubated for 72–96 h at 30 °C (time required for hyphae forma- tion in Streptomyces 85E). Results were noted for the presence of bald or clear zone of inhibition around sam- ples and control discs. %α–amylase inhibition ¼ Os–On ð Þ= Ob–On ð Þ 100 %α–amylase inhibition ¼ Os–On ð Þ= Ob–On ð Þ 100 Antifungal assay The antifungal activity of test extracts was evaluated by agar disc diffusion method performed in triplicate [24]. The spores of test fungal strains [Aspergillus fumigatus (FCBP- 66), Mucor species (FCBP-0300), Aspergillus niger (FCBP-0198) and Aspergillus flavus (FCBP-0064)] were harvested in 0.02% v/v Tween 20 solution in H2O and their turbidity was adjusted according to McFarland 0.5 turbidity standard. Then 100 μl of each harvested fungal strain was swabbed on plates containing Sabouraud Dextrose agar. Sterile filter paper discs impregnated with 5 μl of test extracts (20 mg/ml of DMSO), positive control (Clotrimazole, 4 mg/ml of DMSO) and negative control (DMSO) were placed on seeded plates and in- cubated for 24–48 h at 28 °C. Afterwards, the average diameter (mm) of growth inhibition zone around the The in vitro antileishmanial evaluation of test extracts was carried out by employing MTT colorimetric assay as de- scribed previously [22]. A 6–7 days incubated culture of Leishmania tropica kwh 23 promastigotes was used. Concisely, parasites were grown in Medium 199 supple- mented with 10% foetal bovine serum (FBS), 100 μg/ml streptomycin sulphate and 100 IU/ml penicillin G at 24 °C. An aliquot of 180 μl of promastigote culture at a pre- adjusted seeding density of 1 × 106 promastigotes/ml was transferred to each well of 96 well plate having 20 μl of test samples (containing ≤1% DMSO in PBS) at concentration of 100 μg/ml. Amphotericin B (0.33–0.004 μg/ml) and 1% DMSO in PBS instead of test sample were employed as positive and negative controls respectively. The culture plate was then incubated at 24 °C for 72 h after which 20 μl of pre-filter sterilized MTT solution (4 mg/ml in distilled Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 6 of 16 Page 6 of 16 samples as well as control treated discs was measured and recorded. phytoconstituents [25]. This in turn also affects the biological evaluation of extracts. We observed a decreasing trend in extract yield along with decreasing polarity of extraction solvent. Thus, the use of sonication aided macer- ation with wide ranging solvent polarities in our study is a critical parameter in extraction yield optimization and its correlation with biological activities. This observation is in agreement with the previous report where effects of differ- ent extraction solvents, used in two extraction methods, on the total polyphenol contents of Q. Antifungal assay coccifera fruits were studied and it was found that solvents with different polarities had significant effects on antioxidant activity [26]. The differences in the extract yields can be attributed to variable solubility of phytoconstituents based on their varied chemical composition in the plant [22]. Phytochemical analysis In vitro phytochemical and antioxidant assays were per- formed by drawing a dose–response (calibration) curve of the reference compounds at multiple doses. The cal- culation of the sample was made in triplicate by using the equation drawn from the above curve. Fig. 1 Percent extract recovery of Q. dilatata using mono and binary (1:1) solvents for extraction. Values are presented as mean ± standard error from triplicate investigation. NH: n-hexane, C: Chloroform, A: Acetone, EtA: Ethyl acetate + Acetone, Et: Ethyl acetate, CE: Ethanol + Chloroform, CM: Chloroform + Methanol, EEt: Ethanol + Ethyl acetate, MEt: Methanol + Ethyl acetate, E: Ethanol, DA: Distilled water + Acetone, M: Methanol, DM: Distilled water + Methanol, D: Distilled water Results and discussion Effect of extraction solvent on the extract yields y The percentage recovery of extracts prepared by employing a total of 14 mono and binary solvent systems of escalating polarity has been summarized in Fig. 1. Maximum amount of extract was recovered when DA was used as the extraction solvent with an extract yield of 10.52% w/w followed by D (6.97% w/w) and A (6.75% w/w) extracts respectively. On the other hand, it was least (0.9% w/w) in case of NH extract. An extraction procedure with superlative efficiency with respect to time/yield ratio is fundamental to accurately quantify the Statistical analysis Data were expressed as mean ± SD. The results obtained for phytochemical and cytotoxic assays were analysed statistically by one-way analysis of variance (ANOVA using the statistical package PASW Statistics 18). TPC 2 Correlation of total phenolic and flavonoid content with antioxidant potential determining assays scavenging activity in the plants and act through scavenging or chelating process [39]. Thus, the current flavonoid deter- mination in polar extracts of Q. dilatata suggests it to be a valuable natural antioxidant. strengthening the antioxidant defences [35]. There- fore, the presence of significant amounts of phenolic content in Q. dilatata proposes it a natural hub of the aforementioned pharmacological attributes. scavenging activity in the plants and act through scavenging or chelating process [39]. Thus, the current flavonoid deter- mination in polar extracts of Q. dilatata suggests it to be a valuable natural antioxidant. TPC The total gallic acid equivalent phenols in Q. dilatata ex- tracts ranged from 21.37 ± 0.21 to 0.12 ± 0.01 μg GAE/mg DW with the highest content quantified in the DA extract. The phenolic content decreased in accordance with the following order; DA > D > DM > MEt > A > EtA > M > Et = E > CM > EEt > C > CE > NH (Fig. 3). Since, the po- larities of polyphenols range from polar to non-polar, thus a wide range of solvents is required for their extraction. It has been observed that polar binary solvent systems are more efficient than mono solvent systems in the extrac- tion of polyphenolic compounds which is in agreement with the results of previous study [27]. Thus polarity plays a key role in increasing solubility of phenols. A significant relationship between antioxidant capacity, reducing power and total phenolic content was found in this exploration, indicating that phenolic compounds are the major contributors to the antioxidant properties of this plant (Fig. 2). High levels of polyphenols are also reported in other species of Quercus such as Q. robur, Q. cocci- fera [26, 28]. The HPLC-MS data of phenolic fraction of cork from Q. suber identified 15 phenolic compo- nents, with ellagic acid, followed by gallic and proto- catechuic acids as the most abundant compounds [29]. The plant phenolics possess diverse biological activities such as anthelmintic [30], antifungal [31], antibacterial [32], antitumor [33], antiviral [34], anti- oxidant [35] and hepatoprotective [36]. The antioxi- dant properties have an important role in combating oxidative stress, cytotoxicity and cell death by scaven- ging free radicals or chelating trace elements thereby Fig. 1 Percent extract recovery of Q. dilatata using mono and binary (1:1) solvents for extraction. Values are presented as mean ± standard error from triplicate investigation. NH: n-hexane, C: Chloroform, A: Acetone, EtA: Ethyl acetate + Acetone, Et: Ethyl acetate, CE: Ethanol + Chloroform, CM: Chloroform + Methanol, EEt: Ethanol + Ethyl acetate, MEt: Methanol + Ethyl acetate, E: Ethanol, DA: Distilled water + Acetone, M: Methanol, DM: Distilled water + Methanol, D: Distilled water Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 7 of 16 Fig. 2 Correlation of total phenolic and flavonoid content with antioxidant potential determining assays Fig. TFC The total flavonoid content of Q. dilatata aerial parts in terms of μg QE/mg DW are presented in Fig. 3. Among all the extracts, highest flavonoid content of 4.78 ± 0.51 μg QE/mg DW was recorded in aqueous extract followed by MEt extract (3.76 ± 0.16 μg QE/mg DW). Lowest content was observed when NH was used alone with the value of 0.09 ± 0.01 μg QE/mg DW. The flavonoid content de- creased with polarity in the following order; D > MEt > Et > EtA > E > M > A > DA > EEt > CM > DM > C > CE > NH. The genus Quercus is reported to be rich in flavonoid polyphenols as Brossa et al. [37] established that major con- stituents in Q. ilex leaves are flavanols and flavonols while Q. petraea and Q. robur are also found to be rich in flavo- noids [38]. The compounds such as flavonoids, which hold hydroxyls groups, are responsible for the free radical Quantitative analysis of polyphenols as well as chro- matographic fingerprinting of bioactive samples was done by RP-HPLC profiling (using 18 standards), after comparing their chromatographs with those of standards (Fig. 4). Quantitative determination of 11 detected polyphenols has been presented in Table 1. Maximum contents of gallic acid, chlorogenic acid and p-coumaric acid were expressed in DA extract (5.282, 15.336 and 1.026 μg/mg extract respectively), while highest contents of pyrocatechol, catechin, coumarin and quercetin were observed in MEt extract (0.361, 2.291, 0.368 and 0.969 μg/mg extract respect- ively). In addition, maximum contents of vanillic acid, ferulic acid, rutin and kaempferol were observed in Et extract (1.271, 0.701, 1.181 and 0.291 μg/mg extract, Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 8 of 16 Fig. 3 TPC (Total phenolic content μg GAE/mg DW), TFC (Total flavonoid content μg QE/mg DW), TAC (Total antioxidant capacity μg AAE/mg DW), TRP (Total reducing power μg AAE/mg DW) and %FRSA (radical scavenging activity) of Q. dilatata in different solvents. Values are presented as mean ± Standard deviation from triplicate investigation Fig. 3 TPC (Total phenolic content μg GAE/mg DW), TFC (Total flavonoid content μg QE/mg DW), TAC (Total antioxidant capacity μg AAE/mg DW), TRP (Total reducing power μg AAE/mg DW) and %FRSA (radical scavenging activity) of Q. dilatata in different solvents. Values are presented as mean ± Standard deviation from triplicate investigation respectively). TFC Various solvents systems have been employed for the isolation of polyphenols. Their extraction efficiency is mainly effected by the choice of solvents and method of extraction [40]. Our results clearly indicate an increase in levels of detected polyphenols with ascending polarity of extraction solvents. Highest amounts of phenolic com- pounds were detected in DA mixture, which is supported by previous report in which aqueous acetone mixture (70%) was found to be most effective for isolation of maximum amounts of condensed tannins from peas [41]. In terms of bioactivities, it is evident from the results that highest anti- oxidant potential shown by DA and MEt extracts might be attributed to the presence of hydroxycinnamic acids (chlorogenic acid, coumarin, p-coumarin), hydroxybenzoic acids (gallic acid) as well as flavonols (quercetin) and flavan-3- ols (catechin). Previous reports also support this hypothesis in which phenolics have been considered as strong candidates with potential antioxidant activity [42, 43]. In addition to their antioxidant properties, these phenolic compounds have also shown chemo- preventive and cytotoxic effects both in in vitro and in vivo models. Anthocyanins, kaempferol, quercetin, esters of cou- maric acid and ellagic acid were found to be inhibitors of human oral (KB, CAL-27), breast (MCF-7), colon (HT-29, HCT-116), and prostate (LNCaP, DU-145) tumor cell lines in a dose-dependent manner [44]. Similarly, polyphenol constituents such as epigallocatechin-gallate (EGCG) and theaflavin have been demonstrated as potent anticancer agents in various animal models [45]. These studies suggest the possible role of polyphenols for the induction of cyto- toxic potential of Q. dilatata extracts in the present study. Chromatograms of standards as well as phenols detected in the samples have been presented in Fig. 4. Biological evaluation Antioxidant assays FRSA The percent free radical scavenging activity (% FRSA) of test samples, evaluated by measuring the discoloration of DPPH solution is shown in Fig. 3. The assay protocol relies on the conversion of stable purple coloured DPPH radical to its yellow-coloured diphenyl Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 9 of 16 Fig. 4 RP-HPLC chromatograms of standard compounds (a) and DA (b), D (c), MEt (d), CM (e), M (f) and Et (g) extracts of Quercus dilatata showing phenolic compounds Hydroquinone (1), Pyrocatechol (2), Gallic acid (3), Resorcinol (4), Catechin (5), Chlorogenic acid (6), Caffeic acid (7), Vanillic acid (8), p-Coumaric acid (9), Ferulic acid (10), Sinapic acid (11), Coumarin (12), Salicylic acid (13), Trans cinnamic acid (14), Rutin (15), Quercetin (16), Ellagic acid (17) and Kaempferol (18). Values represented by bold letters are maximum phenolic contents detected in test samples Fig. 4 RP-HPLC chromatograms of standard compounds (a) and DA (b), D (c), MEt (d), CM (e), M (f) and Et (g) extracts of Quercus dilatata showing phenolic compounds Hydroquinone (1), Pyrocatechol (2), Gallic acid (3), Resorcinol (4), Catechin (5), Chlorogenic acid (6), Caffeic acid (7), Vanillic acid (8), p-Coumaric acid (9), Ferulic acid (10), Sinapic acid (11), Coumarin (12), Salicylic acid (13), Trans cinnamic acid (14), Rutin (15), Quercetin (16), Ellagic acid (17) and Kaempferol (18). Values represented by bold letters are maximum phenolic contents detected in test samples [3]. Previously, the FRSA of Q. subar and Q. coccifera extracts, evaluated using this assay was found to be considerably higher than BHT and comparable to that of BHA (3-t-butyl-4-hydroxyanisole), quercetin and ascor- bic acid [29, 47]. picryl hydrazine molecule by accepting electron or hydrogen radical from the donor antioxidant. The DPPH molecule is regarded as a stable free radical owing to the presence of a delocalized spare electron over the entire molecule that gives a characteristic absorption band at 517 nm [46]. Highest free radical scavenging efficiency was exhibited by MEt extract (IC50 = 8.1 ± 0.5 μg/ml) followed by A extract (IC50 = 9.1 ± 0.89 μg/ml). The scavenging effect of plant extracts on the DPPH radical decreased in the following order: MEt > A > EtA > CM > DM > DA > M > D > E > EEt > Et > C > CE > NH. Biological evaluation Antioxidant assays The radical scavenging efficiency of MEt, A, EtA, CM, DM and DA extracts is comparable, but the high extrac- tion yields obtained by DA extract opens good perspec- tives for the exploitation of this extract in nutraceutical applications. The current scavenging results also support the previous findings where the polar ethyl acetate fraction of Q. dilatata exhibited the highest antiradical potential (IC50 = 38.02 μg/ml) while the least quenching effect was manifested by the non-polar n-hexane fraction TAC The total antioxidant capacity (TAC) of various solv- ent soluble extracts of Q. dilatata is summarized in Fig. 3. The assay is based on antioxidant mediated reduction of Mo (VI) to Mo (V) resulting in the formation of green coloured phosphate/Mo (V) complex [22]. The total anti- oxidant capacity of the extracts was found to decrease in order of DA extract (4.81 ± 0.98 μg AAE/mg DW) > D > EtA > A > DM > MEt > E > CM > Et > M > EEt > C > CE > NH extract with the value of 0.34 ± 0.10 μg AAE/mg DW. The presence of a significant correlation between TAC and TPC (R2 = 0.7004) and a non-significant correlation between TAC and TFC (R2 = 0.3746) suggests that phenols other than flavonoids are the major contributors towards the antioxidant activity of Q. dilatata crude extracts (Fig. 2). Ahmed et al. Biological evaluation Antioxidant assays BMC Complementary and Alternative Medicine (2017) 17:386 Page 10 of 16 Table 1 Phenolic composition (μg/mg extract) of six different Quercus dilatata extracts Standards Retention time (min) Samples Et CM MEt DA M D Hydroquinone 10.07 – – – – – – Pyrocatechol 12.06 0.049 0.013 0.361 0.232 0.111 0.017 Gallic acid 15.24 0.534 0.499 2.822 5.282 2.112 1.382 Resorcinol 17.48 – – – – – – Catechin 23.61 – 0.150 2.291 1.380 0.808 0.095 Chlorogenic acid 25.80 1.009 2.699 9.478 15.336 7.122 5.162 Caffeic acid 27.51 – – – – – – Vanillic acid 28.21 1.271 – – – – – p-Coumaric acid 30.16 0.534 0.146 0.232 1.026 0.339 0.036 Ferulic acid 34.87 0.701 0.046 0.461 0.173 0.192 – Sinapinic acid 38.48 – – – – – – Coumarin 44.85 0.105 0.105 0.368 – 0.149 – Salicylic acid 50.05 – – – – – – Trans cinnamic acid 54.44 – – – – – – Rutin 58.79 1.181 0.094 – 0.245 0.407 – Quercetin 63.13 0.164 0.060 0.969 0.616 0.085 0.059 Ellagic acid 67.35 – – – – – – Kaempferol 73.06 0.291 0.069 0.284 – 0.129 – - Not detected Table 1 Phenolic composition (μg/mg extract) of six different Quercus dilatata extracts Previously the total antioxidant activity of methanol extract was reported as 64 mg equivalent of BHT/g of Q. infectoria nutgalls which is quite high in comparison to the antioxi- dant capacity of the methanolic extract of Q. dilatata aerial parts (2.20 ± 0.25 μg AAE/mg DW) as determined in the current analysis [39]. However, overall yield of aerial parts per plant is also much higher than the nutgalls. This still buttress the potential of this plant as a source of natural antioxidants. inhibition and 1115 mM Fe+2/g respectively [48]. The re- ducing properties are generally associated with the pres- ence of reductones which have been allied to the antioxidant action through breakage of the free radical chain by donating a hydrogen atom. Consequently, a dir- ect correlation have been observed between the antioxi- dant capacity and reducing power of certain plant extracts which is in agreement with the results of our study [49]. y y Brine shrimp lethality assay TRP Figure 3 shows the reductive power of various solv- ent extracts of Q. dilatata. It was observed that the max- imum extraction efficiency in terms of highest reducing power was achieved in the DA extract (20.03 ± 2.4 μg AAE/mg DW). The total reducing capacity of test extracts was found to decrease in order of DA > MEt > A > DM > D > EtA > E > CM > M > Et > EEt > C > CE > NH. A significant relationship between TRP and TPC (R2 = 0.9338) while a non-significant correlation between TRP and TFC (R2 = 0.2331) is observed in this study (Fig. 2). Therefore, it is suggested that phenolic compounds might be the major contributors to the reducing proper- ties of this plant. The various antioxidant potential assays of medicinal plants used in diet therapy during postpar- tum healthcare in Rajasthan, India revealed that among all the plants tested, the Q. infectoria extract possessed high- est FRSA and reducing potential of 90.20% of DPPH Cytotoxicity prospective of the plant was tested against brine shrimp larvae to establish its bioactivity profile. The degree of lethality was found to be directly propor- tional to the concentration of test extracts when ana- lyzed by using serial dilution method. Out of 14 organic extracts screened for cytotoxic activity, 21.42% of the ex- tracts demonstrated LC50 value below 50 μg/ml and were categorized as highly cytotoxic while 57.14% of them were considered as moderately cytotoxic (LC50 value ≥50 but ≤200 μg/ml). The remaining 21.42% of the extracts had LC50 values >200 μg/ml and were consid- ered to have a weak cytotoxic activity under the experi- mental conditions. The results from screening of Q. dilatata extracts against A. salina larvae are shown in Table 2. The positive control, doxorubicin demonstrated an LC50 5.93 μg/ml. Among all the individual extracts Page 11 of 16 Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Table 2 Cytotoxicity and protein kinase inhibition of different solvent extracts of Q. y y Brine shrimp lethality assay dilatata Samples Brine shrimp cytotoxicity (μg/ml) THP-1 cytotoxicity (μg/ml) Hep G2 cytotoxicity (μg/ml) Isolated lymphocytes Protein kinase inhibition (μg/disc) % mortality LC50 % inhibition IC50 % inhibition IC50 % inhibition IC50 Diameter (mm) at 100 μg/disc MIC 200 20 20 20 Clear zone Bald zone NH 33.6 ± 2.5a >200 22.32 ± 2.41c > 20 46.73 ± 0.85 a >20 8.76 ± 1.12 >20 – 7 ± 0.15c – C 80.00 ± 0.49b 96.45 ± 0.98 14.24 ± 1.77a > 20 43.20 ± 1.56 a >20 10.43 ± 2.56 >20 – 25 ± 0.2a 12.5 A 42.5 ± 0.94b >200 82.43 ± 1.68 3.88 ± 0.53 41.20 ± 1.56 a >20 14.43 ± 1.21 >20 – 8 ± 0.47 – EtA 80.0 ± 0.47a 77.7 ± 1.14 38.42 ± 2.13b > 20 38.03 ± 2.28 b >20 12.43 ± 1.43 >20 – 7 ± 0.39c – Et 50.0 ± 1.25c >200 47.25 ± 2.99 > 20 41.32 a ± 2.56 b >20 15.21 ± 1.22 >20 – 27 ± 0.47a 12.5 CE 91.5 ± 0.94 c 49.62 ± 0.22 19.56 ± 3.22 > 20 9.72 ± 0.87 >20 13.87 ± 2.12 >20 – 9 ± 0.52b – CM 100.0 ± 1.70a 34.54 ± 0.16 21.56 ± 2.87 > 20 28.27 ± 0.85 >20 6.65 ± 1.54 >20 – 7 ± 0.21 – EEt 100.0 ± 0.94 81.4 ± 0.17 43.12 ± 3.18 > 20 42.79 ± 0.76 a >20 5.86 ± 1.01 >20 – 9 ± 0.15 – MEt 80.30 ± 2.36 69.9 ± 0.18 71.5 ± 4.11 5.59 ± 0.25 23.51 ± 1.54 >20 14.76 ± 2.22 >20 – 10 ± 0.41b – E 100.0 ± 0.94a 86.62 ± 1.35 82.52 ± 1.45b 4.95 ± 0.53 42.30 ± 1.12 a >20 12.87 ± 1.12 >20 – 26 ± 0.42a 12.5 DA 82.40 ± 1.25 49.1 ± 1.18 62.85 ± 2.45 9.24 ± 0.53 26.21 ± 1.21 >20 16.34 ± 4.45 >20 – 8 ± 0.35 – M 91.50 ± 1.70 71.0 ± 0.74 22.53 ± 0.83a > 20 45.50 ± 1.43 a >20 18.45 ± 3.98 >20 – 28 ± 0.35a 12.5 DM 80.50 ± 1.70 92.3 ± 1.70 42.54 ± 2.45 > 20 9.64 ± 0.76 c >20 22.54 ± 5.16 >20 – 8 ± 0.35 – D 90.0 ± 1.70 78.4 ± 1.70 48.54 ± 0.83a > 20 – >20 12.11 ± 1.15 >20 – 9 ± 0.55 – Doxorubicin 100 5.93 98 ± 0.18 5.1 5-Florouracil 100 5 Vincristine 100 8.1 73.45 ± 1.56 6.66 ± 0.22 Surfactin 30 ± 1.02 DMSO – – – 1% DMSO in PBS/sea water – – – – – Values are presented as mean ± standard deviation of triplicate analysis. y y Brine shrimp lethality assay –: no activity. a-c means difference is highly significant, slightly significant, significant at p < 0.05 Page 12 of 16 Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 1 cell line cytotoxicity (R2 = 0.0542) or TFC and THP-1 cell cytotoxicity (R2 = 0.3288). 1 cell line cytotoxicity (R2 = 0.0542) or TFC and THP-1 cell cytotoxicity (R2 = 0.3288). 1 cell line cytotoxicity (R2 = 0.0542) or TFC and THP-1 cell cytotoxicity (R2 = 0.3288). 1 cell line cytotoxicity (R2 = 0.0542) or TFC and THP-1 cell cytotoxicity (R2 = 0.3288). 1 cell line cytotoxicity (R2 = 0.0542) or TFC and THP-1 cell cytotoxicity (R2 = 0.3288). CM extract was found to be the most cytotoxic exhibit- ing the LC50 34.54 ± 0.16 μg/ml signifying the effective- ness of a moderately polar binary solvent system against a highly polar or non-polar solvent system. A working concentration of <1% in DMSO was employed to pre- pare test mixtures as it is a safer solvent in brine shrimp lethality test compared to others such as Tween 20 [50]. Brine shrimp lethality assay is a simple and high throughput cytotoxicity test of bioactive chemicals. It is based on the killing ability of test compounds of a simple zoological organism, the brine shrimps. Artemia nauplii are used extensively in research and toxicology due to the commercial availability of dried cysts from which live test material can be hatched at will. This assay draws extrapolations on the safety of the plant extracts and further illustrates trends of their biological activities. In bioactivity evaluation of the plant extracts by brine shrimp bioassay, an LC50 value of less than 1000 μg/ml is considered to be cytotoxic [51]. In our study, 100% of the screened extracts revealed LC50 values <1000 μg/ml suggesting the presence of cytotoxic bioactive compounds responsible for the observed deaths. Cytotoxicity against THP-1 cell line y y g Advances in prevention and treatment of cancer requires continuous development of novel and improved chemo- therapeutic and chemopreventive agents. The plant based anticancer drug discovery such as vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinote- can accentuate the need for their further exploration as fundamental [52]. Keeping in view the prodigious cytotoxic potential illustrated by brine shrimp lethality assay; the plant extracts were further screened for an in vitro cytotoxic activity using human leukemia (THP-1 ATCC# TIB-202) cell line (Table 2). Samples were found to be more cytotoxic at higher concentrations as compared to lower concentrations. Among all the test extracts, most promin- ent inhibition was shown by A extract exhibiting 82.43 ± 1.68% inhibition at 20 μg/ml concentration and an IC50 3.88 ± 0.53 μg/ml which is comparable to the standard drugs 5-florouracil and vincristine with IC50 5 μg/ml and 8.1 μg/ml respectively. Previously methanol extract of Q. dilatata aerial parts demonstrated a noteworthy antitumor activity exhibiting 84.78% tumour inhibition at a concentra- tion of 1000 μg/ml in potato disc antitumor assay [53]. Previously water infusions of mature and fresh Q. resinosa leaves were evaluated for antioxidant activity and genotoxic effects on HeLa cells. Results showed that fresh leaves infu- sions increase the oxidative process and other damage to DNA and may serve as a potential source of phenolics with anticancer activity [54]. In the present analysis, there was no significant correlation observed between TPC and THP- Cytotoxicity against Hep G2 cell line y y The cytotoxicity of Q. dilatata crude extracts against Hep G2 human hepatoma cell line was determined as percent inhibition at 20 μg/ml concentration in compari- son to the anticancer drug doxorubicin (Table 2). A moderate antiproliferative activity was exhibited by 50% of the test extracts with IC50 46.73 ± 0.85– 38.03 ± 2.28%. The highest inhibition was exhibited by the non-polar NH extract (46.73 ± 0.85%) followed by M > C > EEt > E > Et > EtA > CM > DA > MEt > DM > A = D. Hepatoma is amongst the two major forms of primary liver cancers and is the most common wide- spread cancer in the world which is preceded by the oc- currence of hepatocellular damage via ROS and the generation of chronic inflammation [55]. Therefore, the antioxidant stature and cytotoxicity of Q. dilatata ex- tracts as revealed in the present exploration suggests it to be a worthy hit for the identification of lead com- pounds against hepatoma. In the present analysis, there was no significant correlation observed between TPC and Hep G2 cell cytotoxicity (R2 = 0.128) or TFC and Hep G2 cell cytotoxicity (R2 = 0.0542). Cytotoxicity against isolated lymphocytes y y g y p y Cytotoxicity of sample extracts was also tested against iso- lated lymphocytes at the same concentration employed for cytotoxicity assessment against THP-1 and Hep G2 cell lines i.e. 20 μg/ml to compare effect at same concentration (Table 2). The results showed that none of the extracts was cytotoxic against the isolated lymphocytes indicating se- lective response against cancer cell lines which is benefi- cial in targeting cancer cells while limiting damage to normal cells. Vincristine, the positive control employed in the assay exhibited significant cytotoxicity against normal lymphocytes with IC50 6.66 ± 0.22 μg/ml. The selective in- hibition of THP-1 and Hep G2 cells indicate prospective of Q. dilatata as candidate for anticancer drug development. Protein kinase inhibition assay Protein kinase inhibition assay The results of zones of protein kinase inhibition re- corded for the test samples are presented in Table 2. A direct relationship was observed between concentrations and activity. Among all the extracts, a noteworthy inhib- ition zone of 28 ± 0.35 mm bald phenotype (MIC = 12.5 μg/ml) was formed around M extract loaded disc followed by Et (27 ± 0.47 mm bald) and E extracts (26 ± 0.42 mm bald). These results are comparable with 30 mm bald zone of surfactin. The non-toxic effect of DMSO (negative control) was confirmed by the absence of growth inhibition zone. In recent years, there has been a tremendous surge for the discovery of protein kinase Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 13 of 16 Page 13 of 16 enzyme inhibitors especially from plants. Protein phosphor- ylation at serine/threonine and tyrosine residues by protein kinases is one of the major mechanisms regulating bio- logical processes including apoptosis, cell proliferation, cell differentiation, and metabolism. Deregulated phosphoryl- ation at serine/threonine and tyrosine residues by protein kinases resulting from genetic alterations acquired early in tumorigenesis are often the cause of cancer. In this respect, inhibition of protein kinases has emerged as a promising target for cancer treatment [21]. Protein kinase activity is critical for the aerial hyphae formation of Streptomyces and this prerequisite was exploited in the present study as kinase inhibitory effect of extracts represents their possible anticancer potential. this was confined to northern sphere of Pakistan but now it is widely spreading throughout the country with cutaneous and visceral leishmaniasis being the major threats [57]. The World Health Organization advocates the use of traditional medicine for the treatment of tropical diseases such as leishmaniasis due to their established safe use in humans [58]. Previously, Q. insignis ethanol bark extract showed promising activity against L. amazonensis promastigotes with low cytotoxic activity and it was suggested as a worthy candidate for further phytochemical exploration [2]. y In the present study the C extract showed profound kin- ase inhibitory activity, cytotoxicity against shrimps and antileishmanial activity but did not exhibit any significant anti-proliferative potential against THP-1 or Hep G2 cell lines. The observed lethality against shrimps and promas- tigotes might be due to the inhibition of various kinases mandatory for cell survival. Protein kinase inhibition assay On the other hand among all the analyzed samples, E extract displayed significant cyto- toxicity against shrimps and THP-1 leukemia cell line as well as protein kinase inhibition; therefore, mechanistic studies are required to extrapolate these activities for its possible anticancer role. In the present analysis, there was no significant correlation observed between TPC and anti- promastigote activity (R2 = 0.0077) or TFC and antileish- manial potential (R2 = 0.0165). Antileishmanial potential Antileishmanial capability of different extracts of Q. dilatata has been evaluated for the first time in the current study. From the screening performed, EtA and A extracts exhibited remarkable and comparable leish- manicidic potential with IC50 12.91 ± 0.02 μg/ml and 14.40 ± 0.01 respectively and it was found to be absent in the polar extracts (Fig. 5). Amphotericin B, the posi- tive control exhibited IC50 0.01 μg/ml. Although the antipromastigote activity of both the extracts is compar- able but the percentage yield of A extract is more than EtA extract suggesting it as the most efficient solvent system for the reclamation of leishmanicidic potential of Q. dilatata in terms of yield and bioactivity. Leishmania- sis that represents a significant disease burden especially in the developing countries requires exploration of new less toxic chemotherapeutic agents due to variable ef- fectiveness of the current treatments, their associated side effects and absence of any vaccine [56]. Previously α-Amylase inhibition assay flavus NH 7 ± 0.24 8 ± 1.85 7 ± 1.21 7.5 ± 0.45 C 7.5 ± 1.0 7 ± 0.25 7.5 ± 1.2 7.5 ± 0.25 A 7.5 ± 0.56 7.5 ± 1.21 7.5 ± 1.5 7.5 ± 0.32 EtA 7 ± 0.45 8 ± 0.56 7.5 ± 0.78 6.5 ± 0.41 Et 8 ± 0.52 8 ± 1.40 7 ± 0.25 8 ± 0.35 CE 7.5 ± 0.45 7 ± 0.35 8 ± 0.50 7.5 ± 0.30 CM 7 ± 0.35 8 ± 0.40 7 ± 0.57 7 ± 0.25 EEt 7.5 ± 1.2 7 ± 0.35 7.5 ± 0.20 7 ± 0.65 MEt 8 ± 0.40 7 ± 0.65 8 ± 1.10 7.5 ± 0.67 E 8 ± 0.45 7.5 ± 1.43 8 ± 1.0 9 ± 0.98 DA 7 ± 0.40 7 ± 1.50 7 ± 1.67 8 ± 0.55 M 7 ± 0.50 9 ± 1.54 8 ± 0.76 7.5 ± 0.69 DM 7.5 ± 0.5 8 ± 0.78 7.5 ± 0.80 6.5 ± 0.55 D 8 ± 0.55 7.5 ± 0.45 9 ± 0.75 7 ± 0.40 Clotrimazole 23 ± 0.12 24 ± 0.98 22 ± 1.03 24 ± 1.01 DMSO – – – – aZone of inhibition including the diameter of disc (6 mm). In each disc, the sample size was 100 μg per disc (5 μl) in disc diffusion assay. –: no activity. Values are presented as mean ± standard error from triplicate investigation i.e. 21.61 ± 1.53, 16.43 ± 2.23, 5.78 ± 1.23 and 3.69 ± 1.21% respectively. IC50 values were not calculated due to comparatively moderate inhibition (<50%) of sub- ject enzyme. Acarbose, the positive control inhibited 80.34 ± 1.12% of α-amylase enzyme’s activity and demon- strated an IC50 33.73 ± 0.12 μg/ml. The hallmark in dia- betes control is the management of blood glucose level which may be achieved through the use of oral hypoglycaemic agents, insulin secretagogues, and carbohydrate hydrolysing enzyme inhibitors. Plants continue to play an important role in the treatment of diabetes, particularly in developing countries where most of the people have limited resources and do not have access to modern treatment. Even in the developed countries there is a shift to the use of alternative ap- proaches to treat diabetes, such as plant-based medicines owing to the side effects associated with the use of insulin and oral hypoglycaemic agents [59]. α-Amylase inhibition assay All the fourteen extracts of varying polarities prepared from Q. dilatata were subjected to standard chromogenic α-amylase inhibition assay and the results are presented in Fig. 6. In our present study, C, EEt, CM and Et extracts were found to have some acarbose like antidiabetic activity Fig. 6 α-amylase inhibition by Q. dilatata extracts. The IC50 of acarbose (positive control) = 33.73 ± 0.12 μg/ml. Experiment was performed in triplicate and values are presented as mean ± standard deviation Fig. 6 α-amylase inhibition by Q. dilatata extracts. The IC50 of acarbose (positive control) = 33.73 ± 0.12 μg/ml. Experiment was performed in triplicate and values are presented as mean ± standard deviation Fig. 5 Antileishmanial potential of Q. dilatata extracts. Values are presented as mean ± Standard deviation from triplicate investigation. IC50 > 100 μg/ml Fig. 6 α-amylase inhibition by Q. dilatata extracts. The IC50 of acarbose (positive control) = 33.73 ± 0.12 μg/ml. Experiment was performed in triplicate and values are presented as mean ± standard deviation Fig. 5 Antileishmanial potential of Q. dilatata extracts. Values are presented as mean ± Standard deviation from triplicate investigation. IC50 > 100 μg/ml Fig. 6 α-amylase inhibition by Q. dilatata extracts. The IC50 of acarbose (positive control) = 33.73 ± 0.12 μg/ml. Experiment was performed in triplicate and values are presented as mean ± standard deviation Fig. 6 α-amylase inhibition by Q. dilatata extracts. The IC50 of acarbose (positive control) = 33.73 ± 0.12 μg/ml. Experiment was performed in triplicate and values are presented as mean ± standard deviation Fig. 5 Antileishmanial potential of Q. dilatata extracts. Values are presented as mean ± Standard deviation from triplicate investigation. IC50 > 100 μg/ml Fig. 5 Antileishmanial potential of Q. dilatata extracts. Values are presented as mean ± Standard deviation from triplicate investigation. IC50 > 100 μg/ml Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Page 14 of 16 Table 3 Antifungal activity of Q. dilatata extracts tested against filamentous fungi Samples aDiameter of growth inhibition zone at 100 μg/disc A. fumigatus F. Solani A. niger A. Abbreviations DMSO: Dimethyl sulfoxide; DPPH: 2, 2-diphenyl-1-picrylhydrazyl; FRSA: Free radical scavenging activity; IC50: 50% inhibitory concentration; LC50: Lethal concentration causing 50% mortality; MIC: Minimum inhibitory concentration; ROS: Reactive oxygen species; TAC: Total antioxidant capacity; TFC: Total flavonoid contents; TPC: Total phenolic contents; TRP: Total reducing power; ZOI: Zone of inhibition Conclusion The quantification of important polyphenols and determin- ation of antimicrobial activity in various crude extracts of Q. dilatata is helpful in explaining some of its traditional uses. The use of a wide-ranging solvent system polarity proved crucial to demonstrate phytochemical and biological profiling of the subject plant. The current study proposes DA extract of Q. dilatata as a potential source of phyto- chemicals with substantial antioxidant capability while its M extract has protective activity against various tumori- genic kinases. Similarly, C extract was significantly cytotoxic against brine shrimps, THP-1 human leukemia cells and leishmanial promastigotes. In conclusion, the present study endorses use of polarity dependent extraction as an important factor for the determination of biological spectrum of Q. dilatata and prospects it as substantial source of bioactive metabolites. α-Amylase inhibition assay Therefore, it is crucial to identify and explore the inhibitors of carbohydrate hydrolysing enzymes such as α-amylase from natural sources having fewer side effects. Inhibitors of α-amylase reduce the glucose peaks that can occur after a meal, slowing the speed with which amylase can convert starch to simple sugars until the body can deal with it. This is of particular importance in people with diabetes, where low insulin levels prevent extracellular glucose from being cleared quickly from the blood. Previous studies on α- amylase inhibitors isolated from medicinal plants suggest that several potential inhibitors of this enzyme belong to flavonoid class of phytochemicals [60]. However in the present analysis there was no significant correlation ob- served between TPC and amylase inhibition (R2 = 0.258) or TFC and enzyme inhibition (R2 = 0.258). Table 3 Antifungal activity of Q. dilatata extracts tested against filamentous fungi aZone of inhibition including the diameter of disc (6 mm). In each disc, the sample size was 100 μg per disc (5 μl) in disc diffusion assay. –: no activity. Values are presented as mean ± standard error from triplicate investigation glycosides, unsaturated lactones, sulphur compounds, saponins, cyanogenic glycosides and glucosinolates [61]. Antifungal assay The plant’s antifungal potential was assessed against four strains of filamentous fungi, the results of which are summarized in Table 3. The data indicate that a moder- ate antifungal activity was exhibited by almost all the ex- tracts against the tested strains. In case of A. fumigatus the growth inhibition zones ranged between 7 and 8 mm for all the extracts. A maximum inhibition zone was dis- played by the M, D and E extracts against F. solani (9 ± 1.54 mm), A. niger (9 ± 0.75 mm) and A. flavus (9 ± 0.98 mm) respectively. The absence of growth in- hibition zone around negative control disc confirmed the non-toxic effect of DMSO whereas standard drug Clotrimazole exhibited maximum activity at a concen- tration of 10 μg/disc. It was observed that extraction solvent polarity did not profoundly affect antifungal pro- ficiency of various Q. dilatata extracts. Since, well known plant secondary metabolites exhibiting antifungal activity include flavonoids, phenols and phenolic References 1. Ashraf A, Sarfraz RA, Rashid MA, Shahid M. Antioxidant, antimicrobial, antitumor, and cytotoxic activities of an important medicinal plant (Euphorbia royleana) from Pakistan. J Food Drug Anal. 2015;23(1):109–15. 27. Dent M, Dragovi'-Uzelac V, Peni M, Brn M, Bosiljkov T, Levaj B. The effect of extraction solvents, temperature and time on the composition and mass fraction of polyphenols in dalmatian wild sage (Salvia officinalis L.) extracts. Food Technol Biotechnol. 2013;51(1):84–91. 2. Monzote L, Piñón A, Setzer W. Antileishmanial potential of tropical rainforest plant extracts. Medicines. 2014;1(1):32. 2. Monzote L, Piñón A, Setzer W. Antileishmanial potential of tropical rainforest plant extracts. Medicines. 2014;1(1):32. 3. Ihtisham M, Ihsan-ul-Haq, Sarwar S, Mirza B. HPLC-DAD analysis and free radical scavenging potential of Quercus Dilatata L. Pak J Bot. 2013;45(S1):577–81. 3. Ihtisham M, Ihsan-ul-Haq, Sarwar S, Mirza B. HPLC-DAD analysis and free radical scavenging potential of Quercus Dilatata L. Pak J Bot. 2013;45(S1):577–81. 28. Dudonné S, Vitrac X, Coutière P, Woillez M, Mérillon J-M. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric Food Chem. 2009;57(5):1768–74. 4. Sarwat SZ, Ahmad N. Screening of potential medicinal plants from district sawat specific for controlling women diseases. Pak J Bot. 2012;44(4):1193–8. 29. Santos SAO, Pinto PCRO, Silvestre AJD, Neto CP. Chemical composition and antioxidant activity of phenolic extracts of cork from Quercus suber L. Ind Crop Prod. 2010;31(3):521–6. 5. Dar M. Ethno botanical uses of Plants of Lawat District Muzaffarabad, Azad Jammu and Kashmir. Asian J Plant Sci. 2003;2(9):680–2. 6. Khan MA, Khan MA, Hussain M, Mujtaba G. Medicinal plants used in folk recipes by the inhabitants of himalayan region Poonch Valley Azad Kashmir (Pakistan). J Basic Appl Sci. 2012;8:35–45. 6. Khan MA, Khan MA, Hussain M, Mujtaba G. Medicinal plants used in folk recipes by the inhabitants of himalayan region Poonch Valley Azad Kashmir (Pakistan). J Basic Appl Sci. 2012;8:35–45. 30. Silva N, Fernandes Júnior A. Biological properties of medicinal plants: a review of their antimicrobial activity. J Venomous Anim Toxins Incl Trop Dis. 2010;16:402–13. 7. Qasim M, Aziz I, Rasheed M, Gul B, Khan MA. Effect of extraction solvents on polyphenols and antioxidant activity of medicinal halophytes. Pak J Bot. 2016;48(2):621–7. 7. Qasim M, Aziz I, Rasheed M, Gul B, Khan MA. Effect of extraction solvents on polyphenols and antioxidant activity of medicinal halophytes. Pak J Bot. 2016;48(2):621–7. 31. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 23. Kim J-S, Kwon C-S, Son KH. Inhibition of alpha-glucosidase and amylase by luteolin, a flavonoid. Biosci Biotechnol Biochem. 2000;64(11):2458–61. Competing interests 22. Khan K, Fatima H, Taqi MM, Zia M, ur-Rehman T, Mirza B, Haq I-u. Phytochemical and in vitro biological evaluation of Artemisia scoparia Waldst. & Kit for enhanced extraction of commercially significant bioactive compounds. J Appl Res Med Aromat Plants. 2015;2(3):77–86. Funding k 13. Qasim M, Abideen Z, Adnan M, Gulzar S, Gul B, Rasheed M, Khan M. Antioxidant properties, phenolic composition, bioactive compounds and nutritive value of medicinal halophytes commonly used as herbal teas. S Afr J Bot. 2016;110:240–250. We acknowledge the financial support given by Higher Education Commission Pakistan, under PhD indigenous fellowship program to finance this research project. 14. Araruna MKA, Santos KKA, da Costa JGM, Coutinho HDM, Boligon AA, Stefanello ST, Athayde ML, Saraiva RA, da Rocha JBT, Kerntopf MR, et al. Phenolic composition and in vitro activity of the Brazilian fruit tree Caryocar coriaceum Wittm. Eur J Integr Med. 2013;5(2):178–83. Abbreviations h l Page 15 of 16 Page 15 of 16 Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 Availability of data and materials All the data and materials have been provided in the main manuscript. All the data and materials have been provided in the main manuscript. Author details 1 f 24. Sharma A, Chandraker S, Patel VK, Ramteke P. Antibacterial Activity of Medicinal Plants Against Pathogens causing Complicated Urinary Tract Infections. Indian J Pharm Sci. 2009;71(2):136–9. 1Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan. 2Institute of Sustainable Halophyte Utilization, University of Karachi, Karachi 75270, Pakistan. 25. Celeghini RMS, Vilegas JHY, Lanças FM. Extraction and Quantitative HPLC Analysis of Coumarin in Hydroalcoholic Extracts of Mikania glomerata Spreng: ("guaco") Leaves. J Braz Chem Soc. 2001;12:706–9. Received: 15 November 2016 Accepted: 27 July 2017 Received: 15 November 2016 Accepted: 27 July 2017 26. Hayouni EA, Abedrabba M, Bouix M, Hamdi M. The effects of solvents and extraction method on the phenolic contents and biological activities in vitro of Tunisian Quercus coccifera L. and Juniperus phoenicea L. fruit extracts. Food Chem. 2007;105(3):1126–34. Acknowledgements f 11. Harborne JB. General procedures and measurement of total phenolics. Methods Plant Biochem. 1989;1:1–28. The authors are thankful to Prof. Dr. Rizwana Aleem Qureshi, Department of Plant sciences, Faculty of Biological sciences, Quaid-i-Azam University Islamabad, Pakistan for identifying the plant sample. 12. Proestos C, Boziaris IS, Nychas GJE, Komaitis M. Analysis of flavonoids and phenolic acids in Greek aromatic plants: Investigation of their antioxidant capacity and antimicrobial activity. Food Chem. 2006;95(4):664–71. Authors’ contributions 15. Hogg JS, Lohmann DH, Russell KE. The kinetics of reaction of 2, 2-diphenyl- 1-picrylhydrazyl with phenols. Can J Chem. 1961;39:1588–94. MA executed all experimental work and compiled the data. HF helped in manuscript write up and execution of experiments. MQ and BG were involved in HPLC analysis and interpretation of data. IU contributed in study design, supervised the execution of experiments and revised the manuscript. All authors have read and approved the final manuscript. 16. Krishnaveni M. Antioxidant activity of selected plants. Plant Physiol. 1968;43:193–200. 17. Lea CH. A note on the Chapman and McFarlane method for the estimation of reducing groups in milk powder. Analyst. 1947;72(857):336–40. 17. Lea CH. A note on the Chapman and McFarlane method for the estimation of reducing groups in milk powder. Analyst. 1947;72(857):336–40. 18. Haq IU, Ullah N, Bibi G, Kanwal S, Ahmad MS, Mirza B. Antioxidant and cytotoxic activities and phytochemical analysis of Euphorbia wallichii root extract and its fractions. Iran J Pharm Res (2012). 2010;11(1):241–9. Ethics approval and consent to participate Informed consent was taken from volunteers and protocol was followed according to international ethical guidelines after approval from the ethical committee of the Quaid-i-Azam University (IRB-QAU-116). Informed consent was taken from volunteers and protocol was followed according to international ethical guidelines after approval from the ethical committee of the Quaid-i-Azam University (IRB-QAU-116). 19. Vichai V, Kirtikara K. Sulphorhodaminen B colorimetric assay for cytotoxicity screening. Nat Protoc. 2006;1(3):1112–6. 20. Waseem D, Butt AF, Haq I-u, Bhatti MH, Khan GM. Carboxylate derivatives of tributyltin (IV) complexes as anticancer and antileishmanial agents. DARU J Pharm Sci. 2017;25(8). Consent for publication Not applicable. 21. Yao G, Sebisubi FM, Voo LYC, Ho CC, Tan GT, Chang LC. Citrinin Derivatives from the Soil Filamentous Fungus Penicillium sp. H9318. J Braz Chem Soc. 2011;22(6):1125–9. Competing interests The authors declare that they have no competing interests. References Asifa M, Ansaria SH, Haquea MR, Kalama N. Estimation of total phenolic, flavonoid contents and antioxidant activity in the nut galls of Quercus infectoria olivier. J Pharm Res. 2012;5(7):3855–7. 40. Goli AH, Barzegar M, Sahari MA. Antioxidant activity and total phenolic compounds of pistachio (Pistachia vera) hull extracts. Food Chem. 2005;92(3):521–5. 41. Chavan UD, Shahidi F, Naczk M. Extraction of condensed tannins from beach pea (Lathyrus maritimus L.) as affected by different solvents. Food Chem. 2001;75(4):509–12. 42. Balasundram N, Sundram K, Samman S. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chem. 2006;99(1):191–203. 43. Dai J, Mumper RJ. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules. 2010;15:7313–52. 44. Zhang Y, Seeram NP, Lee R, Feng L, Heber D. Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties. J Agric Food Chem. 2008;56(3):670–5. 45. Huang MT, Xie JG, Wang ZY, Ho CT, Lou YR, Wang CX, Hard GC, Conney AH. Effects of tea, decaffeinated tea, and caffeine on UVB light-induced complete carcinogenesis in SKH-1 mice: demonstration of caffeine as a biologically important constituent of tea. Cancer Res. 1997;57(13):2623–9. 46. Fatima H, Khan K, Zia M, Ur-Rehman T, Mirza B, Haq I-u. Extraction optimization of medicinally important metabolites from Datura innoxia Mill.: an in vitro biological and phytochemical investigation. BMC Complement Altern Med. 2015;15(1):376. 47. Genç Y, Yüzbaşioğlu M, Harput ÜŞ, Kuruüzüm-uz A. Antioxidant activity and total phenolic content of Quercus coccifera L. FABAD J Pharm Sci. 2012;37:17–22. 48. Jain N, Goyal S, Ramawat K. Evaluation of antioxidant properties and total phenolic content of medicinal plants used in diet therapy during postpartum healthcare in Rajasthan. Int J Pharm Pharm Sci. 2011;3(3):248–53. 49. Abdel-Hameed E-SS. Total phenolic contents and free radical scavenging activity of certain Egyptian Ficus species leaf samples. Food Chem. 2009;114(4):1271–7. 50. Wu C. An important player in brine shrimp lethality bioassay: The solvent. J Adv Pharm Technol Res. 2014;5(1):57–8. 51. Nguta JM, Mbaria JM, Gakuya DW, Gathumbi PK, Kabasa JD, Kiama SG. Evaluation of acute toxicity of crude plant extracts from Kenyan biodiversity using brine shrimp, Artemia salina L. (Artemiidae). Open Conf Proc J. 2012;3:30–4. 51. Nguta JM, Mbaria JM, Gakuya DW, Gathumbi PK, Kabasa JD, Kiama SG. Evaluation of acute toxicity of crude plant extracts from Kenyan biodiversity using brine shrimp, Artemia salina L. (Artemiidae). Open Conf Proc J. 2012;3:30–4. 52. Greenlee H. References Chena F, Longa X, Yua M, Liua Z, Liua L, Shao H. Phenolics and antifungal activities analysis in industrial crop Jerusalem artichoke (Helianthus tuberosus L.) leaves. Ind Crop Prod. 2013;47:339–45. 8. Haq IU, Ullah N, Bibi G, Kanwal S, Ahmad MS, Mirza B. Antioxidant and cytotoxic activities and phytochemical analysis of Euphorbia wallichii root extract and its fractions. Iran J Pharm Res. 2012;11(1):241–9. 32. Kubo I, Fujita K, Nihei K, Nihei A. Antibacterial activity of akyl gallates against Bacillus subtilis. J Agric Food Chem. 2004;52(5):1072–6. 33. You BR, Moon HJ, Han YH, Park WH. Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis. Food Chem Toxicol. 2010;48(5):1334–40. 9. Bray HG. Kinetic studies of the metabolism of foreign organic compounds. 3. The conjugation of phenols with glucuronic acid. Biochem J. 1952;3(52):416. 34. Uozaki M, Yamasaki H, Katsuyama Y, Higuchi M, Higuti T, Koyama AH. Antiviral effect of octyl gallate against DNA and RNA viruses. Antivir Res. 2007;73(2):85–91. 34. Uozaki M, Yamasaki H, Katsuyama Y, Higuchi M, Higuti T, Koyama AH. Antiviral effect of octyl gallate against DNA and RNA viruses. Antivir Res. 2007;73(2):85–91. 10. Jafri L, Saleem S, Haq IU, Ullah N, Mirza B: In vitro assessment of antioxidant potential and determination of polyphenolic compounds of Hedera nepalensis K. Koch. Arab J Chem. 2014;10;3699–3702. Page 16 of 16 Page 16 of 16 Page 16 of 16 Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 35. Kumar S, Chashoo G, Saxena AK, Pandey AK. Parthenium hysterophorus: a probable source of anticancer, antioxidant and anti-HIV agents. Biomed Res Int. 2013;2013:11. 36. Ferk F, Chakraborty A, Jager W, Kundi M, Bichler J, Misik M, Wagner KH, Grasl-Kraupp B, Sagmeister S, Haidinger G, et al. Potent protection of gallic acid against DNA oxidation: results of human and animal experiments. Mutat Res. 2011;715(1–2):61–71. 59. Patel DK, Kumar R, Laloo D, Hemalatha S. Diabetes mellitus: An overview on its pharmacological aspects and reported medicinal plants having antidiabetic activity. Asian Pac J Trop Biomed. 2012;2(5):411–20. 37. Brossa R, Casals I, Pintó-Marijuan M, Fleck I. Leaf flavonoid content in Quercus ilex L. resprouts and its seasonal variation. Trees. 2009;23(2):401–8. 38. Popović BM, Štajner D, Ždero R, Orlović S, Gali Z. Antioxidant characterization of Oak extracts combining spectrophotometric assays and chemometrics. Sci World J. 2013;2013:8. 61. Quiroga EN, Sampietro AR, Vattuone MA. Screening antifungal activities of selected medicinal plants. J Ethnopharmacol. 2001;74(1):89–96. 39. 58. Filho VC, Meyre-Silva C, Niero R, Bolda Mariano LN, Gomes do Nascimento F, Vicente Farias I, Gazoni VF, et al.: Evaluation of antileishmanial activity of selected Brazilian plants and identification of the active principles. Evid Based Complement Alternat Med 2013, 2013:7. 59. Patel DK, Kumar R, Laloo D, Hemalatha S. Diabetes mellitus: An overview on its pharmacological aspects and reported medicinal plants having antidiabetic activity. Asian Pac J Trop Biomed. 2012;2(5):411–20. 60. Kyriakis E, Stravodimos GA, Kantsadi AL, Chatzileontiadou DS, Skamnaki VT, Leonidas DD. Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b. FEBS Lett. 2015;589(15):1787–94. 61. Quiroga EN, Sampietro AR, Vattuone MA. Screening antifungal activities of selected medicinal plants. J Ethnopharmacol. 2001;74(1):89–96. 61. Quiroga EN, Sampietro AR, Vattuone MA. Screening antifungal activities of selected medicinal plants. J Ethnopharmacol. 2001;74(1):89–96. 58. Filho VC, Meyre-Silva C, Niero R, Bolda Mariano LN, Gomes do Nascimento F, Vicente Farias I, Gazoni VF, et al.: Evaluation of antileishmanial activity of selected Brazilian plants and identification of the active principles. Evid Based Complement Alternat Med 2013, 2013:7. 59. Patel DK, Kumar R, Laloo D, Hemalatha S. Diabetes mellitus: An overview on its pharmacological aspects and reported medicinal plants having antidiabetic activity. Asian Pac J Trop Biomed. 2012;2(5):411–20. 60. Kyriakis E, Stravodimos GA, Kantsadi AL, Chatzileontiadou DS, Skamnaki VT, Leonidas DD. Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b. FEBS Lett. 2015;589(15):1787–94. Ahmed et al. BMC Complementary and Alternative Medicine (2017) 17:386 References Natural products for cancer prevention. Semin Oncol Nurs. 2012;28(1):29–44. 52. Greenlee H. Natural products for cancer prevention. Semin Oncol Nurs. 2012;28(1):29–44. 53. Jamil M, Mirza B, Yasmeen A, Khan MA. Pharmacological activities of selected plant species and their phytochemical analysis. J Med Plants Res. 2012;6(37):5013–22. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: 54. Rocha-Guzmán NE, Gallegos-Infante JA, González-Laredo RF, Reynoso- Camacho R, Ramos-Gómez M, Garcia-Gasca T, Rodríguez-Muñoz ME, Guzmán-Maldonado SH, Medina-Torres L, Lujan-García BA. Antioxidant activity and genotoxic effect on HeLa cells of phenolic compounds from infusions of Quercus resinosa leaves. Food Chem. 2009;115(4):1320–5. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 55. Machana S, Weerapreeyakul N, Barusrux S. Anticancer effect of the extracts from Polyalthia evecta against human hepatoma cell line (HepG2). Asian Pac J Trop Biomed. 2012;2(5):368–74. 56. Sadeghi-Nejad B, Saki J, Khademvatan S, Nanaei S. In vitro antileishmanial activity of the medicinal plant - Satureja khuzestanica Jamzad. J Med Plant Res. 2011;5(24):5912–5. 56. Sadeghi-Nejad B, Saki J, Khademvatan S, Nanaei S. In vitro antileishmanial activity of the medicinal plant - Satureja khuzestanica Jamzad. J Med Plant Res. 2011;5(24):5912–5. 57. Shah NA, Khan MR, Nadhman A. Antileishmanial, toxicity, and phytochemical evaluation of medicinal plants collected from Pakistan. Biomed Res Int. 2014;2014:7. 57. Shah NA, Khan MR, Nadhman A. Antileishmanial, toxicity, and phytochemical evaluation of medicinal plants collected from Pakistan. Biomed Res Int. 2014;2014:7.
https://openalex.org/W2756868128	https://vtechworks.lib.vt.edu/bitstream/10919/97360/1/cropsci2016.08.0699.pdf	English	null	Comparing Digital and Visual Evaluations for Accuracy and Precision in Estimating Tall Fescue Brown Patch Severity	Crop science	2,017	public-domain	5,675	ABSTRACT Brown patch (Rhizoctonia solani Kuhn), a destructive disease of tall fescue (Festuca arundinacea Schreb.), is typically evaluated visually. The subjectivity of visual evaluations may be reduced using technology like digital image analysis (DIA). This study compared DIA and visual evaluations for accuracy and precision of brown patch ratings of glasshouse grown tall fescue plants. Across four experiments, 112 plants were inoculated with R. solani. Disease was rated visually and using DIA-WP (digital image analysis whole plant canopy). In two experiments, disease evaluations were replicated using three images and three visual evaluations per pot. Absolute error was calculated as the difference between actual disease severity [calculated using an individual leaf DIA method previously quantified as highly predictive of actual brown patch disease severity on tall fescue (r2 = 0.99)] and DIA-WP and visual evaluations, respectively. Standard deviations within repeated measures were also calculated. A mixed-model ANOVA was used to determine differences (P < 0.05) in mean absolute error and mean standard deviation by method, disease range, and method by disease range. Disease ranged from 0 to 100%. Mean absolute error did not differ between methods but did by disease range, exhibiting a bell-shaped curve from 0% to 100% disease severity. Mean standard deviation exhibited significant method by disease range interaction. Mean standard deviation did not differ across the disease range within DIA-WP evaluations but did across the disease range within visual evaluations. The more consistent precision of DIA across the disease range could reduce variability in brown patch evaluations of tall fescue. Abbreviations: DIA, digital image analysis; DIA-IL, digital image analysis of individual leaves, DIA-WP, digital image analysis of whole plant canopy; PDA, potato dextrose agar; PI, plant introduction; V, visual disease evaluation. B B rown patch, caused by the fungus Rhizoctonia solani Kuhn (Piper and Coe, 1919), can be a highly destructive disease on tall fescue (Festuca arundinacea Schreb.) grown in the transition zone. Visual evaluations are commonly used in turfgrass culti var development, both in the initial stages, where breeders may use visual assessment to screen large numbers of individual turf grass genotypes for disease resistance under controlled conditions (Beirn et al., 2015; Curley et al., 2005; Elliot, 1995; Fu et al., 2005), and in the latter stages, where cultivars are evaluated for brown patch resistance under field conditions by multiple evalua tors across many locations. Published September 28, 2017 Published September 28, 2017 RESEARCH Comparing Digital and Visual Evaluations for Accuracy and Precision in Estimating Tall Fescue Brown Patch Severity Virginia R. Sykes,* Brandon J. Horvath, Scott E. Warnke, Shawn D. Askew, Anton B. Baudoin, and James M. Goatley V.R. Sykes, B.J. Horvath, Dep. of Plant Science, Univ. of Tennessee, 252 Ellington, 2431 Joe Johnson Drive, Knoxville, TN 37996. S.E. Warnke, USDA, 10300 Baltimore Avenue, Building 010A BARC- West, Beltsville, MD, 20705. V.R. Sykes, B.J. Horvath, S.D. Askew, A.B. Baudoin, Dep. of Plant Pathology, Physiology, and Weed Science, Virginia Tech, 413 Price Hall, Blacksburg, VA, 24061. J.M. Goatley, Dep. of Crop and Soil Environmental Sciences, Virginia Tech, 330 Smyth Hall, Blacksburg, VA, 24061. Received 23 Aug. 2016. Accepted 28 Mar. 2017. *Corresponding author (vsykes@utk.edu). Assigned to Associate Editor Michael Richardson. © Crop Science Society of America \| 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. ABSTRACT Visual evaluators typically report either the percentage of diseased tissue or use one of several variations of disease rating scales. Visual evaluation is a preferred disease evalu ation method because it is quick, easy, and inexpensive. However, in visual disease evaluations there may be compromises to both precision, defined as “the closeness to each other of repeated mea surements of the same quantity” and accuracy, defined as “the nearness of a measurement to the actual value of the variable being measured” (Horst et al., 1984; Nutter Jr. et al., 1993; Zar, 1999). Published in Crop Sci. 57:3303–3309 (2017). doi: 10.2135/cropsci2016.08.0699 crop science, vol. 57, november–december 2017 www.crops.org 3303 Clear differentiation of disease severity between treatments through both accurate and precise disease evaluations is important in a number of different areas of turfgrass research. These include breeding studies that seek to identify disease resistant genotypes and applied research assessing the impact of inputs and management strategies on disease. A study on the assessment of visual evalua tion techniques done by Horst et al. (1984) showed visual assessment to be inadequate at evaluating turfgrass quality and density. Ten evaluators took quality and density rat ings of ten cultivars of Kentucky bluegrass (Poa pratensis L.) and tall fescue grown in Oregon in 1980 and Texas in 1981. Evaluator ratings and rankings differed signifi cantly. These inconsistencies suggest that the criteria used for evaluation were inconsistent among evaluators. Studies performed by Nutter Jr. et al. (1993) on visual assessment of dollar spot (Sclerotinia homoeocarpa F.T. Benn) severity in creeping bentgrass (Agrostis stolonifera L.) showed similar results. Significant variation was reported for both intra rater repeatability and interrater reliability. The objective of this study was to compare DIA and visual evaluations for accuracy and precision in estimating brown patch disease severity on tall fescue. Inoculation l Fourteen isolates of R. solani were collected from creeping bentgrass putting greens at the Virginia Tech Turfgrass Research Center in Blacksburg, Virginia. These were assessed for virulence on tall fescue. Three of the most virulent isolates (data not shown) were used to create inoculum. Inoculum was created using filter paper cut to 2 cm by 0.5 cm, autoclaved for 1 h prior to use, and placed radially around a 4-mm diam. plug of R. solani on potato dextrose agar (PDA) (Fig. 1). Plates were maintained for 2 wk to allow sufficient colonization of the filter paper. Plants were then inoculated by placing three infected filter paper pieces, one piece from each isolate, within the plant canopy of each cone-tainer. Plants were inoculated approximately 6 wk after planting. Although many studies have looked at disease evalua tion using DIA, very few have examined the accuracy and precision of this method in evaluating leaves that are not perpendicular to the image capture device as is the case in a turfgrass canopy. In turfgrass research, the accuracy of DIA has been determined for evaluating turfgrass color and percent cover (Karcher and Richardson, 2003; Rich ardson et al., 2001). Digital image analysis was also used to successfully measure disease severity of the turfgrass disease dollar spot (Horvath and Vargas, 2005; Steketee et al., 2016). No reports are yet available comparing the pre cision and accuracy of DIA estimates to visual estimates of turfgrass disease severity using a quantified method as a standard of accuracy. Using visual evaluations as a standard of accuracy is common; however, this may have limited efficacy do to the subjectivity of visual evaluations (Zink and Kartunova, 1998). MATERIALS AND METHODS Plant Maintenance Material for Exp. 1 consisted of six seeds of ‘Kentucky 31’ tall fescue planted in 3.8-cm diam. cone-tainers (SC7 stubby cells, Steuwe & Sons, Corvallis, OR) filled with Pro-Mix potting media containing biofungicide (Bacil lus subtilis MBI 600) (Code 0532, Premier Horticulture, Quebec, Canada). Plants were maintained in a glasshouse, watered at a rate of 2.55 cm h-1 for 3 min daily using an overhead irrigation system, and fertilized with three applications per month of a 20:20:20 mix at an N rate of 48.8 kg ha-1. Plants were cut twice a week to a height of 7.6 cm. Material for Exp. 2 consisted of 20 seeds each from 13 plant introductions (PIs) obtained from the USDA germplasm database. Seeds were planted singly in each cone-tainer and maintained in the same manner described for Exp. 1. Digital image analysis (DIA) may reduce both inter- and intrarater variability through increased objectivity and standardization of practices. The accuracy and/or precision of DIA has been shown to be highly effective for a number of different crops with varied leaf shapes and types of pathogen infections (Barbedo, 2014; Bock et al., 2008; Lindow and Webb, 1983; Martin and Rybicki, 1998; Pourreza et al., 2015). However, poor correlations have also been found in DIA estimations of powdery mildew (Erysiphe cichoracearum D.C. Ex Merat) on squash (Cucurbita maxima Dutch) (Moya et al., 2005) and pow dery mildew (Podosphaera clandestine Waller.:Fr.) on sweet cherry [Prunus avium (L.) L.] (Olmstead and Lang, 2001). These mixed results indicate potential variation in DIA estimates due to differences in quantification methodol ogy, image capture methods, and/or the type of disease signs and symptoms evaluated. www.crops.org crop science, vol. 57, november–december 2017 Disease Treatment Disease treatment was set up similarly to other breeding studies screening turfgrass genotypes for disease resistance under controlled conditions (Beirn et al., 2015; Curley et al., 2005; Elliot, 1995; Fu et al., 2005). Plants were placed in a sealed chamber to increase air temperature and pro vide high humidity for disease development. Plants were cut to a height of 7.6 cm prior to inoculation and again prior to disease evaluation. Two methods were used to obtain plants exhibiting a range of disease severity. In Exp. 1, plants were subject to varying days under disease pressure. Plants were placed in a randomized complete block design and six plants were removed and evaluated at 0, 2, 4, 6, 8, and 10 d after inoculation, respectively. After evaluation, plants were not replaced in the disease www.crops.org crop science, vol. 57, november–december 2017 3304 Fig. 1. Inoculum composed of 2-cm by 0.5-cm pieces of sterilized filter paper placed radially around a 4-mm diam. plug of R. solani grown on potato dextrose agar. again after the 24-hr drying period. Leaves exhibit ing natural senescence due to age were identified and removed by hand prior to imaging. Leaves exhibit ing senescence due to severe disease pressure, where the dark edges of coalescing lesions could be seen on the leaf, were not removed. When capturing images, cones were lined up with an index mark placed on the cone-tainer stand and cone prior to inoculation to provide consistent leaf orientation between the initial and final pictures. The stand was set at an angle of approximately 45˚ for the initial and final pictures. The stand was set at an angle of approximately 45˚, and a Canon PowerShot G6 digital camera (Canon, Inc., Tokyo, Japan) was placed on a tripod with the orientation adjusted to take a top down image (Fig. 2). The aperture on the camera was set to f/8 and the shutter speed was manually adjusted for each image until the light meter on the camera read zero. Two Calumet (Calumet Photographic, Inc., Bensenville, IL) light stands, each containing four 40-W compact semispiral, daylight balanced (5500 K) fluorescent bulbs (model 0L2000, Calumet Photographic, Inc. Bensenville, IL) were placed on either side of the stand to provide even illumination. Three images were obtained for each cone with each cone placed in the stand, photographed, and removed from the stand between each image. Fig. 1. Disease Treatment Inoculum composed of 2-cm by 0.5-cm pieces of sterilized filter paper placed radially around a 4-mm diam. plug of R. solani grown on potato dextrose agar. chamber. This method was repeated twice (Exp. 1.1 and 1.2). In Exp. 2, 13 different genotypes were arranged in a randomized complete block design with four replications. These were subject to disease pressure for 12 days. Using the DIA-WP method described below, four plants were chosen from each of the following percent disease ranges: 0 to 20, 20.1 to 40, 40.1 to 60, 60.1 to 80, and 80.1 to 100. This method was repeated twice (Exp. 2.1 and Exp. 2.2). g Pictures were analyzed using APS Assess image analysis software (APS Press, St. Paul, MN). Hue thresholds of 31 (low) and 191 (high) were used to distinguish the plant from the background. The background was then substituted for a solid blue background. An intensity threshold of 66 (low) and 255 (high) was used to select the soil, which was then replaced with a solid blue background. Hue thresholds of 31 (low) and 191 (high) were used to determine the total plant area. Lesion area was determined with hue thresholds of 31 (low) and 101 (high). A ratio of lesion area to total plant area was used by APS Assess to determine percent disease severity. Total percent disease severity was calculated as the difference in disease severity between the pre- inoculation picture and post-inoculation picture. Disease Evaluation Upon removal from the chamber, plants were subirrigated on greenhouse benches for a 24-hr period to allow the leaf canopy to dry prior to disease evaluation. Percent disease was estimated for each plant using visual evaluation (V) and DIA of the whole plant canopy (DIA-WP). A third method, DIA of individual leaves (DIA-IL), was used as a standard of accuracy by which to judge the accuracy of DIA-WP and visual estimations. The DIA-IL method was shown to be highly predictive of actual brown patch severity in previous studies (r2 = 0.99) (Sykes, 2009). Simi lar individual leaf methods using APS assess software have also been quantified as highly accurate (r = 0.97) in other plant–disease combinations (Bock et al., 2008). The V, DIA-WP, and DIA-IL methods are described below. 3. Individual leaf DIA (DIA-IL): All leaves from each plant were cut at the soil surface, placed within a clear plastic cover sheet, and scanned at 300 dpi to produce JPEG images using a CanoScan LiDE 50 scanner (Canon, Inc., Tokyo, Japan). Each set of leaves was scanned three times. The cover sheet was removed and the leaves within it were rearranged between each scan. APS Assess image analysis software was used to evalu ate percent disease in each image. Hue thresholds of 31 (low) and 191 (high) were used to distinguish the plant from the background, which was then replaced by a 3. Individual leaf DIA (DIA-IL): All leaves from each plant were cut at the soil surface, placed within a clear plastic cover sheet, and scanned at 300 dpi to produce JPEG images using a CanoScan LiDE 50 scanner (Canon, Inc., Tokyo, Japan). Each set of leaves was scanned three times. The cover sheet was removed and the leaves within it were rearranged between each scan. APS Assess image analysis software was used to evalu ate percent disease in each image. Hue thresholds of 31 (low) and 191 (high) were used to distinguish the plant from the background, which was then replaced by a 1. Visual analysis: Three evaluators performed visual ratings of disease severity using a percentage scale. All three evaluators were trained in the evaluation of brown patch on tall fescue, though years of experi ence varied. Evaluators had approximately 1, 3, and 14 yr of experience. 2. DIA of whole plant canopy (DIA-WP): Pictures were taken of each plant prior to inoculation and crop science, vol. crop science, vol. 57, november–december 2017 www.crops.org Disease Evaluation 57, november–december 2017 www.crops.org 3305 g. 2. Setup for capturing images using the digital image analysis whole plant (DIA-WP) method. The plant stand was set at an angle o pproximately 45°, and a Canon PowerShot G6 digital camera was placed in a tripod with the orientation adjusted to take a top down mage. Two Calumet light stands, each containing four 40 watt compact semi-spiral, daylight-balanced (5500 K) fluorescent bulbs, were aced on either side of the stand to provide even illumination. Fig. 2. Setup for capturing images using the digital image analysis whole plant (DIA-WP) method. The plant stand was set at an angle of approximately 45°, and a Canon PowerShot G6 digital camera was placed in a tripod with the orientation adjusted to take a top down image. Two Calumet light stands, each containing four 40 watt compact semi-spiral, daylight-balanced (5500 K) fluorescent bulbs, were placed on either side of the stand to provide even illumination. Fig. 2. Setup for capturing images using the digital image analysis whole plant (DIA-WP) method. The plant stand was set at an angle of approximately 45°, and a Canon PowerShot G6 digital camera was placed in a tripod with the orientation adjusted to take a top down image. Two Calumet light stands, each containing four 40 watt compact semi-spiral, daylight-balanced (5500 K) fluorescent bulbs, were placed on either side of the stand to provide even illumination. solid blue background. Hue thresholds of 31 (low) and 191 (high) were used to determine the total plant area and hue thresholds of 31 (low) and 87 (high) were used to determine the lesion area. The ratio of lesion area to total plant area was used by APS Assess to determine percent disease severity. solid blue background. Hue thresholds of 31 (low) and 191 (high) were used to determine the total plant area and hue thresholds of 31 (low) and 87 (high) were used to determine the lesion area. The ratio of lesion area to total plant area was used by APS Assess to determine percent disease severity. solid blue background. Hue thresholds of 31 (low) and 191 (high) were used to determine the total plant area and hue thresholds of 31 (low) and 87 (high) were used to determine the lesion area. The ratio of lesion area to total plant area was used by APS Assess to determine percent disease severity. Disease Evaluation To compare the accuracy (absolute error) and pre cision (standard deviation) of each method, analyses of variance were performed in SAS v. 9.3 (SAS Institute, Inc., Cary, NC) using the GLIMMIX procedure with the following mixed model in which either absolute error or standard deviation were considered the depen dent variable; estimation method and disease range were considered fixed effects and experiment was considered a random effect. In Exp. 2, only one picture, scan, or visual evaluation was performed for each plant. Visual estimation of plant disease was performed by a rater with 3 yr of experience. All other methods followed those described for Exp. 1. Y M D MD E MDE e ijkl i j ij k ijk l ijk = + + + + + + µ ( ) Statistical Analysis where Yijkl is the observed value of absolute error or standard deviation for the lth replication within the ith method by the jth disease range by the kth experiment; µ is the population mean; Mi is the effect of the ith method (i = DIA-WP or Visual); Dj is the effect of the jth dis ease range ( j = 0 to 20, 20.1 to 40, 40.1 to 60, 60.1 to 80, or 80.1 to 100); MDij is the interaction effect of the ith method with the jth disease range; Ek is the effect of where Yijkl is the observed value of absolute error or standard deviation for the lth replication within the ith method by the jth disease range by the kth experiment; µ is the population mean; Mi is the effect of the ith method (i = DIA-WP or Visual); Dj is the effect of the jth dis ease range ( j = 0 to 20, 20.1 to 40, 40.1 to 60, 60.1 to 80, or 80.1 to 100); MDij is the interaction effect of the ith method with the jth disease range; Ek is the effect of The absolute error was calculated for each disease sever ity estimate as the difference between the mean disease severity estimated using the DIA-IL method (standard of accuracy) and the disease severity estimated using either DIA-WP or visual estimation. Standard deviations were calculated within the three images, scans, or visual esti mations performed on each plant. Standard deviation was only calculated for Exp. 1.1 and 1.2. www.crops.org crop science, vol. 57, november–december 2017 3306 3306 Fig. 4. Comparison of mean absolute error by disease range. Only mean absolute error by disease range was significant (P < 0.05), with no significant difference between method or interaction between method and disease range. Absolute error was calculated as the mean absolute difference between estimated disease values (Digital image analysis of whole plants [DIA-WP] or Visual evaluations [V]) and a quantified standard of accuracy (Digital image analysis of individual leaves [DIA-IL]). Mean absolute errors were compared using ANOVA with means differentiated using Tukey’s HSD. Means not connected by the same letter are significantly different (P < 0.05). Error bars indicate the standard error of the mean. RESULTS AND DISCUSSION The mean and distribution of disease severity differed among experiments (Fig. 3). Blocking by experiment helped control this variation and allowed for compari son of methods across the full range of potential disease values (0% to 100%). Across the four experiments, the total number of observations per disease group were n = 226 (0 to 20% disease), n = 126 (20.1 to 40% disease), n = 66 (40.1 to 60% disease), n = 40 (60.1 to 80% disease), and n = 46 (80.1 to 100% disease). There was no interaction between experiment and method for either absolute error or standard deviation analyses. Fig. 4. Comparison of mean absolute error by disease range. Only mean absolute error by disease range was significant (P < 0.05), with no significant difference between method or interaction between method and disease range. Absolute error was calculated as the mean absolute difference between estimated disease values (Digital image analysis of whole plants [DIA-WP] or Visual evaluations [V]) and a quantified standard of accuracy (Digital image analysis of individual leaves [DIA-IL]). Mean absolute errors were compared using ANOVA with means differentiated using Tukey’s HSD. Means not connected by the same letter are significantly different (P < 0.05). Error bars indicate the standard error of the mean. Fig. 4. Comparison of mean absolute error by disease range. Only mean absolute error by disease range was significant (P < 0.05), with no significant difference between method or interaction between method and disease range. Absolute error was calculated as the mean absolute difference between estimated disease values (Digital image analysis of whole plants [DIA-WP] or Visual evaluations [V]) and a quantified standard of accuracy (Digital image analysis of individual leaves [DIA-IL]). Mean absolute errors were compared using ANOVA with means differentiated using Tukey’s HSD. Means not connected by the same letter are significantly different (P < 0.05). Error bars indicate the standard error of the mean. Fig. 3. Scatterplot matrix showing the distribution of disease severity in experiments 1.1, 1.2, 2.1, and 2.2. Each dot represents the actual disease severity of an individual cone as measured using the digital image analysis of individual leaves (DIA-IL) method. observed. This indicates the differences in standard devia tion by disease range differed by method. Therefore, each method was considered separately. Within the DIA-WP method, standard deviation did not differ by disease range (Fig. 5). Statistical Analysis the kth experiment (k = 1.1, 1.2, 2.1, or 2.2); MDEijk is interaction effect of the ith method with the jth disease range and kth experiment; el(ijk) is experimental error or residual. Means were separated using Tukey’s HSD. Statistical significance was determined using a P-value of less than 0.05 for all tests. RESULTS AND DISCUSSION However, within the visual estimation method, the standard deviation did differ by disease range, with a significant increase in standard deviation being observed Fig. 5. Comparison of mean standard deviation between digital image analysis of whole plants (DIA-WP) and visual evaluation (V) by disease range. Mean standard deviation exhibited a significant method by disease range interaction (P < 0.05), indicating the differences in mean standard deviation by disease range differed by method. Standard deviations were calculated within the three replications per evaluation method performed on each plant. Mean standard deviations were compared using ANOVA with means differentiated using Tukey’s HSD. Means not connected by the same letter are significantly different (P < 0.05). Error bars indicate the standard error of the mean. Fig. 3. Scatterplot matrix showing the distribution of disease severity in experiments 1.1, 1.2, 2.1, and 2.2. Each dot represents the actual disease severity of an individual cone as measured using the digital image analysis of individual leaves (DIA-IL) method. Mean absolute error values between DIA-WP and visual estimations did not differ and the interaction effect between evaluation method and disease range was not significant. Because methods did not differ, data were combined across methods to assess differences in mean absolute error by disease range. Mean absolute error did differ by disease range, exhibiting a bell-shaped curve across the range of disease severity (Fig. 4). The highest mean absolute error occurred at the 40.1 to 60% range while the lowest mean absolute errors occurred at the 0 to 20% and 80.1 to 100% ranges. Fig. 5. Comparison of mean standard deviation between digital image analysis of whole plants (DIA-WP) and visual evaluation (V) by disease range. Mean standard deviation exhibited a significant method by disease range interaction (P < 0.05), indicating the differences in mean standard deviation by disease range differed by method. Standard deviations were calculated within the three replications per evaluation method performed on each plant. Mean standard deviations were compared using ANOVA with means differentiated using Tukey’s HSD. Means not connected by the same letter are significantly different (P < 0.05). Error bars indicate the standard error of the mean. Fig. 5. Comparison of mean standard deviation between digital image analysis of whole plants (DIA-WP) and visual evaluation (V) by disease range. crop science, vol. 57, november–december 2017 www.crops.org RESULTS AND DISCUSSION Mean standard deviation exhibited a significant method by disease range interaction (P < 0.05), indicating the differences in mean standard deviation by disease range differed by method. Standard deviations were calculated within the three replications per evaluation method performed on each plant. Mean standard deviations were compared using ANOVA with means differentiated using Tukey’s HSD. Means not connected by the same letter are significantly different (P < 0.05). Error bars indicate the standard error of the mean. In the model examining precision, standard deviation differed by method and disease range and a signifi cant interaction between method and disease range was crop science, vol. 57, november–december 2017 www.crops.org 3307 3307 between the 0% to 20%, 20.1% to 40%, and 40.1% to 60% ranges. Standard deviation for DIA-WP was significantly lower than that of visual estimations within the 40.1% to 60% disease range. Within the remaining disease ranges, these two methods did not differ. by using a portable light box or drone technology to assess field plots could allow these analysis methods to be used for field scale disease evaluation. Continued research would need to assess whether the image analysis methods described would translate to larger areas of turfgrass cover under less controlled conditions where additional abiotic and biotic stressors could potentially influence efficacy. Although DIA-WP was more consistently pre cise across the range of disease severity, it did not show improved accuracy compared with visual evaluations. Potential improvements in image capture were identi fied which might provide improved accuracy in future DIA evaluations. In all DIA-WP evaluations, a purple cloth was placed around the cone to mask the stand and provide a uniform background for the image. Although the same cloth was used for each image capture, the color appears different in certain images. Light stands were used to provide even illumination; however, this did not entirely negate the influence of light from outside sources. Better control of outside light sources could help to further reduce variability and potentially improve accuracy. Additional concerns revolve around the time and expense of this technology. One of the distinct advan tages of visual evaluation is that it requires no expensive equipment and analysis is very rapid. RESULTS AND DISCUSSION Although the cost of equipment required for digital image analysis has dropped significantly in the past decade, the time required to capture and process an image still exceeds the time required to visually assess a plant for a single trait. With increased levels of automation and the use of macros to assess multiple parameters such as color, percent cover, and disease simultaneously, the time requirement of digital image analysis might become more comparable to visual evaluation. Hue variance may also have been caused by the camera settings used when capturing images. The aperture was set to f/8 and the shutter speed adjusted to balance the light meter to zero for each image. This resulted in slight overexposure of images with greater disease severity. This minor overexposure did not affect the ability of DIA to detect disease when parameters were defined on a single image basis. However, when evaluating disease using a DIA macro, where the parameters are set prior to batch evaluation of images, this could impact the ability of DIA to consistently define diseased areas in each image due to differences in exposure. Further reducing variability by using a closed light source and balancing the camera set tings to a neutral image prior to image capture could aid in increasing the accuracy of DIA methods. CONCLUSION The DIA-WP method evaluated in this study was sig nificantly more precise than visual evaluation within the 40.1% to 60% disease range. Additionally, while no differ ences in precision were observed among the disease ranges within the DIA-WP method, precision differed between the 0 to 20%, 20.1 to 40%, and 40.1 to 60% range within visual evaluations. The DIA-WP method demonstrated improved precision; however, it did not show improved accuracy compared with visual evaluation. An increase in absolute error under either evaluation method as disease values approached the 40.1 to 60% disease range indicates a strong need for continued efforts to improve the accu racy of disease evaluation methods. Reduction in image variability introduced by differences in light quality and camera settings may result in further improvements to both the accuracy and precision of DIA in evaluating brown patch disease severity on tall fescue. These results are important for turfgrass evaluation since leaf orienta tion differs from many of the previous research studies examining the use of DIA to evaluate plant disease. This technology offers improved phenotyping capability that is critical to the early stages of cultivar improvement, when large numbers of individual genotypes are screened for disease resistance under controlled conditions. Continued research focused on improving accuracy and translat ing this methodology to field-based turfgrass evaluations would further expand the utility of this technology in turfgrass research. The DIA method exhibited improved precision, and potential improvements to accuracy were identified. However, other factors may compromise overall efficacy in adoption of this technology. Our disease inoculations were performed in a glasshouse setting with highly con trolled conditions that limited the introduction of abiotic or biotic stresses other than the pathogen of interest. Images were also captured in a highly controlled setting with uniform plant placement and lighting. This type of methodology would work very well for disease resistance screenings in the initial stages of cultivar development, where hundreds of plant introductions could be rapidly assessed for resistance to specific pathogens. This type of controlled environment is common in the initial stages of breeding studies, as it allows for screening of larger num bers of genotypes and reduces the potential for escapes being identified as resistant. In later stages of cultivar development under field conditions, these methods may not be as effective. Modification of image capture methods References Barbedo, J.G. 2014. An automatic method to detect and measure leaf disease symptoms using digital image processing. Plant Dis. 98:1709–1716. doi:10.1094/PDIS-03-14-0290-RE 3308 www.crops.org crop science, vol. 57, november–december 2017 Beirn, L.A., W.A. Meyer, B.B. Clarke, and J.A. Crouch. 2015. A greenhouse-based inoculation protocol for fungi causing crown rust and stem rust diseases of Kentucky bluegrass turf. HortScience 50:1509–1513. Moya, E.A., L.R. Barrales, and G.E. Apablaza. 2005. Assessment of the disease severity of squash powdery mildew through visual analysis, digital image analysis and validation of these methodologies. Crop Prot. 24:785–789. doi:10.1016/j. cropro.2005.01.003 Bock, C.H., P.E. Parker, A.Z. Cook, and T.R. Gottwald. 2008. Visual rating and the use of image analysis for assessing differ ent symptoms of citrus canker on grapefruit leaves. Plant Dis. 92:530–541. doi:10.1094/PDIS-92-4-0530 Nutter, F.W., Jr., M.L. Gleason, J.H. Jenco, and N.C. Christians. 1993. Assessing the accuracy, intra-rater repeatability, and inter-rater reliability of disease assessment systems. Phytopa thology 83:806–812. doi:10.1094/Phyto-83-806 Curley, J., S.C. Sim, S. Warnke, S. Leong, R. Barker, and G. Jung. 2005. QTL mapping of resistance to gray leaf spot in ryegrass. Theor. Appl. Genet. 111:1107–1117. doi:10.1007/s00122-005- 0036-x thology 83:806–812. doi:10.1094/Phyto-83-806 Olmstead, J.W., and G.A. Lang. 2001. Assessment of severity of powdery mildew infection of sweet cherry leaves by digital image analysis. HortScience 36(1):107–111. Elliott, M.L. 1995. Disease response of Bermudagrasses to Gaeu mannomyces graminis var. graminis. Plant Dis. 79:699–702. doi:10.1094/PD-79-0699 Piper, C.V., and H.S. Coe. 1919. Rhizoctonia in lawns and pastures. Phytopathology 9(2): 89-92. Pourreza, A., W.S. Lee, E. Etxeberria, and A. Banarjee. 2015. An evaluation of a vision-based sensor performance in Huan glongbing disease identification. Biosystems Eng. 130:13–22. doi:10.1016/j.biosystemseng.2014.11.013 Fu, D., N.A. Tisserate, Y. Xiao, D. Settle, S. Muthukrishnan, and G.H. Liang. 2005. Overexpression of rice TLPD34 enhances dollar-spot resistance in transgenic bentgrass. Plant Sci. 168:671–680. doi:10.1016/j.plantsci.2004.09.032 Richardson, M.D., D.E. Karcher, and L.C. Purcell. 2001. Quan tifying turfgrass cover using digital image analysis. Crop Sci. 41:1884–1888. doi:10.2135/cropsci2001.1884 Horst, G.L., M.C. Engelkle, and W. Meyers. 1984. Assessment of visual evaluation techniques. Agron. J. 76:619–622. doi:10.2134/agronj1984.00021962007600040027x Steketee, C.J., A.D. Martinez-Espinoza, K.R. Harris-Shultz, G.M. Henry, and P.L. Raymer. 2016. Effects of genotype and isolate on expression of dollar spot in seashore paspalum. HortScience 51(1):67–73. Horvath, B.J., and J.M. Vargas. 2005. Analysis of dollar spot dis ease severity using digital image analysis. Int. Turfgrass Soc. Res. J. 10:196–201. Karcher, D.E., and M.D. Richardson. 2003. Quantifying turf grass color using digital image analysis. Crop Sci. 43:943–951. doi:10.2135/cropsci2003.9430 Sykes, V.R. 2009. crop science, vol. 57, november–december 2017 www.crops.org References Use of digital image analysis to identify Rhizoc tonia solani and Rhizoctonia zeae resistance in Festuca arundinacea plant introductions. Virginia Tech. Zar, J.H. 1999. Biostatistical analysis. 4th ed. Prentice Hall, Inc., Upper Saddle River, NJ. Lindow, S.E., and R.R. Webb. 1983. Quantification of foliar plant disease symptoms by microcomputer-digitized video image analysis. Phytopathology 73:520–524. doi:10.1094/Phyto-73- 520 Zink, A.G., and E. Kartunova. 1998. Wood failure in plywood shear samples measured with image analysis. For. Prod. J. 48:69–74. Martin, D.P., and E.P. Rybicki. 1998. Microcomputer-based quan tification of maize streak virus symptoms in Zea mays. Phyto pathology 88:422–427. doi:10.1094/PHYTO.1998.88.5.422 3309 crop science, vol. 57, november–december 2017 www.crops.org
https://openalex.org/W2921352792	https://www.matec-conferences.org/10.1051/matecconf/201927602014/pdf	English	null	Risk management system model to improve the reputation of oil and gas companies in the Java island - Indonesia	MATEC web of conferences	2,019	cc-by	3,829	Risk management system model to improve the reputation of oil and gas companies in the Java island - Indonesia Nova Nevila Rodhi1,2,, I Putu Artama Wiguna2, and Nadjadji Anwar 2 Nova Nevila Rodhi1,2,, I Putu Artama Wiguna2, and Nadjadji Anwar 2 1Departement of Civil Engineering, Universitas Bojonegoro, Bojonegoro, Indonesia 2Departement of Civil Engineering, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia 1Departement of Civil Engineering, Universitas Bojonegoro, Bojonegoro, Indonesia 2Departement of Civil Engineering, Institut Teknologi Sepuluh Nopember, Surabaya, Indo Abstract. Risk management has been widely studied and applied in oil and gas pipeline projects, but the reality is that the impacts still occur. This is due to the ineffectiveness of existing risk management applications, so of course in this case an effective risk management system is needed, in which risk management must pay attention to all aspects that exist, both internal aspects, external aspects, and other aspects that can affect and influenced by existing risks and risk management not only can be used as a preventive method. But it can also support sustainable development targets. This paper presents the application of risk management by oil and gas companies in Indonesia that has been adapted to ISO 31000:2009 as a framework that can integrate various other management processes, including the management of HSE (Health, Safety, and Environment) risk in the hope of that sustainable development can be achieved . But in the reality it can not be denied that the activities that continue to this day still cause negative impact, especially for the environment. International oil and gas companies nowadays place more emphasis on preventive measures than the methods of mitigation. Thus it shows that the concept of sustainable development has not been fully considered in risk management applications. The existence of a continuous negative impact would greatly affect the credibility of oil and gas companies. Based on the results of research conducted with the hybrid method and analyzed with the help of System Dynamics it can be concluded that there are 3 (three) factors that can affect reputation risk, these factors are social, environmental, and economic and the model built in this study shows that it will can help the oil and gas company players to predict and improve the company’s reputation. https://doi.org/10.1051/matecconf /201927602014 https://doi.org/10.1051/matecconf /201927602014 , 0 (2019) MATEC Web of Conferences 276 ICAnCEE 2018 2014 1 Introduction The oil and gas projects have many risks that potentially become hazardous to life, property, and the environment if those activities are not properly controlled and regulated [1]. One of the activities at risk is the pipeline project. If there is a leak in the oil and gas * Corresponding author: nova.nevila@gmail.com * Corresponding author: nova.nevila@gmail.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/). * Corresponding author: nova.nevila@gmail.com * Corresponding author: nova.nevila@gmail.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/). , 0 (2019) MATEC Web of Conferences 276 ICAnCEE 2018 2014 https://doi.org/10.1051/matecconf /201927602014 pipeline and often occurs in the negative impact, it can lead to social conflict and casualties. [2]. The existence of an impact due to the risk that is not managed correctly [3]. A risk is an internal or external situation that can affect and change the initial state and affect time, cost and security [4]. According to Nielsen (2006), the oil and gas pipeline project has very complex risk [5]. Ogwu (2011), states that the oil and gas pipeline project has enormous environmental, social, and economic impacts [6]. pipeline and often occurs in the negative impact, it can lead to social conflict and casualties. [2]. The existence of an impact due to the risk that is not managed correctly [3]. A risk is an internal or external situation that can affect and change the initial state and affect time, cost and security [4]. According to Nielsen (2006), the oil and gas pipeline project has very complex risk [5]. Ogwu (2011), states that the oil and gas pipeline project has enormous environmental, social, and economic impacts [6]. As explained by Hasan (2016) in his paper, that although the failure of the pipeline project is rare, but if the failure can have a significant impact on the social environment, it is because, in addition to many areas damaged by the incident, the failure of oil and gas pipeline also took a massive loss of life [4]. The impacts caused by the project in the oil and gas industry can affect the credibility of oil and gas companies, thus automatically affecting the sustainability of existing projects [7]. The risks contained in the oil and gas pipeline project can certainly be anticipated with risk management applications, but if risk management has been applied and still has a negative impact, it is one of them because the risk management application is not systematic and ineffective. Therefore, it is necessary to develop a concept of risk management that can build an effective risk management system [3]. Currently, oil and gas companies in Indonesia certainly have applied risk management. * Corresponding author: nova.nevila@gmail.com In general, risk management applied by some oil and gas companies is more emphasized in managing the risk of HSE (Health, Safety, and Environment) [8]. In ISO 31000:2009, it is mentioned that the effectiveness of risk management can be achieved by taking into account 11 (eleven) risk management principles. The risk management principle shows that it must be done concerning all aspects of the project management field so that risk management can help companies achieve their goals. For risk management applications following the corporate characteristic certainly needed an effective method. Dynamics systems can be used to understand complex behavior and system dynamics over time [9]. Shin et al. (2013) state that dynamic systems can analyze feedback mechanisms. For example, an event X will affect the occurrence of Z and vice versa, in which case, of course, the event must be analyzed thoroughly and cannot be analyzed separately, where the analysis is only done on the effect of X to Z only or vice versa. The concept of feedback loop system analysis or causal loops is an advantage possessed by System Dynamics [10]. Based on the description above. This paper builds a model of the risk management system to improve the reputation of oil and gas companies in the Indonesian island of Java by using the method of System Dynamics. 2 Literature review In Indonesia’s oil and gas industries there are 2 (two) main activities, namely core business which consist of upstream business which includes exploration and exploitation. In the upstream business, there is also a downstream business which provides processing, transportation, storage, and trading. In addition to the core business, in the oil and gas industry, there are also supporting businesses which include construction services and non- construction services. One of the much needed supporting infrastructure in the project is a gas pipeline that serves to support the transmission and distribution of natural gas. Currently, the Indonesian government is targeting the construction of a transmission network with a length of 7558.3 km and a distribution network of 2733.15 km spread throughout the working area can be realized in 2018. The distribution pipeline network in Java is planned to be longer than in other areas, along 1244.15 km [11]. In an effort to achieve the target of pipeline development, oil and gas industry players will face various challenges and obstacles, whether from the risk of internal factors or external ones have a negative impact on the sustainability of the oil and gas pipeline project 2 2 , 0 (2019) MATEC Web of Conferences 276 ICAnCEE 2018 2014 https://doi.org/10.1051/matecconf /201927602014 itself also to the surrounding environment. This can be seen from some of the harmful events that are caused by the oil and gas pipeline project, as well as the theft by punching holes in pipes which are very dangerous. The existence of a leakage pipe causes fire until the explosion of oil and gas pipelines in Rig Pumpindo Majalengka that claimed casualties and caused burns to residents [12]. The tragic events caused by pipeline projects also occur in some other countries. As explained by Hasan (2016) in his paper, that although the failure of the pipeline project is rare, but if the failure can have a massive impact on the social environment, it is because, in addition to many areas damaged by the incident, the failure of oil and gas pipeline also took a massive loss of life. The impacts caused by the project in the oil and gas industry can affect the credibility of oil and gas companies, thus automatically affecting the sustainability of existing projects [7]. 2 Literature review Currently, in addition to the principles that must be adhered to also there is a concept that must be considered and taken into account in every activity. It is a concept of sustainable development [13]. Sustainable development is a development concept that aims to provide a better quality of life for all people, both at present and in future generations [14]. Thus, construction project risk management must be taking into consideration the concept of sustainable development so that the planned project can achieve a sustainable construction project and can support sustainable development targets [15]. Sustainable Construction Project is a project that is based on balance, whether it be a financial balance, environment and operational considerations on all existing processes [16]. The concept can inspire the engineer of construction services and other project actors regarding determining methods and planning to achieve project implementation efficiency. Among them is done by integrating the preparation of mapping design and project process to be undertaken [17], as well as by project scheduling [18]. In order to achieve a sustainable construction project requires an effective and acceptable method by all parties. This can be done by security risk management considering at the design and planning stage, building efficient designer and constructor communication, and effective planning and safety management during construction. [19] According to Bakhtiari, risk management is an essential tool for achieving sustainable development, as risk management is a crucial key to maximizing sustainable development goals [20]. To achieve sustainable development, construction project risk management must be carried out concerning the objectives of Sustainable Development Goals, which achieve sustainable development and lift people out of poverty. In the effort to alleviate poverty, it must be accompanied by economic growth, so that all kinds of risks that could adversely affect economic growth must be well managed, including those related to disaster risk and vulnerability in the development plan. This is because disaster can be a significant threat in achieving and sustaining development plans and objectives [20]. Dynamics systems can be used to understand complex behavior and system dynamics over time [9]. 2 Literature review Yames mentions the model built with systems approach which has 4 (four) virtues [21], namely 1) enable conducting cross-sectoral research with broad scope, 2) can experiment with the system without disturbing (giving treatment) pertain to the system, 3) able to determine the purpose of management activities and improvements to the system under study, and 4) can be used to predict (predict) behavior and state of the system in the future. 3 Research methods In this research is done by using a hybrid method, where preliminary data obtained based on the identification of literature review which then continued with validation based on 3 3 3 , 0 (2019) MATEC Web of Conferences 276 ICAnCEE 2018 2014 https://doi.org/10.1051/matecconf /201927602014 respondent perspective. In this study, related respondents are employees of oil and gas companies in the island of Java Indonesia. respondent perspective. In this study, related respondents are employees of oil and gas companies in the island of Java Indonesia. System modeling starts from the system conceptualization stage which is done by building a conceptual model illustrated with the Causal Loop Diagram (CLD). It aims to describe the simulation of System Dynamics that will be carried out in general, where this diagram is formed based on the variables that already exist. This conceptualization phase will produce a causal diagram which will then be combined into a complete system The design of respondents used in this study is the design of respondents remain because the respondents formed following specific rules and do not change during the process of withdrawal of respondents take place. The design of fixed respondent chosen in this research is the cluster sampling method, which is a technique to select a respondent from a small group of clusters or clusters. 4 Results and discussion From the literature, there are three variables can affect the reputation risk of oil and gas companies. Namely, social, economic, and environmental. Furthermore, it can be explained that each variable has parameters that can affect each other. The identification results are validated with the actual situation by distributing questionnaires and assessing the risks to each variable. The variables are presented in Table 1. From Table 1, risk analysis is performed to obtain the most dominant variables and will be used in the preparation of this research model. The initial stage analysis results can be seen in Table 2. Table 2 presents seven parameters affect social risk, while for economic risk the origin has five parameters into two parameters, as well as for the environment, which initially has eight parameters, after analyzing there are only two parameters. To shorten the parameter names in the model, in this case, a code is created for each parameter. Referred to in Table 2 then, in this case, can be built model premises to describe the causal loop diagram first, as for the basic model CLD can be seen in Fig. 1 below: sosial economy environment quality of life institution and technology health and safety skills negative impact social benefits environmental compensation sustainable policy contractor with env performance conservation historical damage - - -+ -- - - - - - - - - reputation + + + - - - - Fig. 1. Causal Loop Diagram (CLD) of the basic model. Fig. 1. Causal Loop Diagram (CLD) of the basic model. , 0 (2019) MATEC Web of Conferences 276 ICAnCEE 2018 2014 https://doi.org/10.1051/matecconf /201927602014 Table 1. Risk variables of oil and gas pipeline project in Indonesia. 4 Results and discussion Variables Parameters Resources Social Improving the quality of human life with poverty alleviation [13], [18], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31] Planning that can be adapted by local human institutions and technologies Health and safety The skills of the people who participated in the project Avoids the negative social impact of construction works Social benefits during construction (eg, employment opportunities) Equal compensation for environmental change Economy financial affordability Employment Opportunity Full cost accounting balance and real costing related to efficiency in the process Improve market competitiveness by adopting policies and practices that promote sustainability issues Selects environmentally-responsible suppliers and contractors that can demonstrate environmental performance Environment Reduce the use of four common sources used in construction, energy, water, materials, and land, at each stage of the project life cycle (REDUCE) Maximizing resource reuse, and/ or recycling to reduce waste (REUSE) Recycling (reduction of raw materials used in new products) (RECYCLE) Use renewable resources to choose non-renewable resources (RENEWABLE) Minimize air, soil, and water pollution Create and use environmentally friendly products in construction, on human health and safety and minimize environmental damage. Conservation efforts by conserving life support systems, conserving plant biodiversity, animals, and other organisms Minimizes damage to sensitive landscapes, including valuable areas from a landscape, cultural, historical, or architectural point of view, and minimizes disturbance to the wilderness area Table 1. Risk variables of oil and gas pipeline project in Indonesia. 5 , 0 (2019) MATEC Web of Conferences 276 ICAnCEE 2018 2014 https://doi.org/10.1051/matecconf /201927602014 Table 2. Variables used in the model. 4 Results and discussion Variables Parameters Code Social Improving the quality of human life with poverty alleviation Quality of life Planning that can be adapted by local human institutions and technologies Institutions and technology Health and safety Health and safety The skills of the people who participated in the project Skills in the project Avoids the negative social impact of construction works Negative impact Social benefits during construction (eg, employment opportunities) Social benefits Equal compensation for environmental change Environmental compensation Economy Improve market competitiveness by adopting policies and practices that promote sustainability issues Sustainable policy Selects environmentally-responsible suppliers and contractors that can demonstrate environmental performance Contractor with environmental performance Environment Conservation efforts by conserving life support systems, conserving plant biodiversity, animals and other organisms Conservation efforts Minimizes damage to sensitive landscapes, including valuable areas from a landscape, cultural, historical, or architectural point of view, and minimizes disturbance to the wilderness area Historical damage Variables Parameters Code Social Improving the quality of human life with poverty alleviation Quality of life Planning that can be adapted by local human institutions and technologies Institutions and technology Health and safety Health and safety The skills of the people who participated in the project Skills in the project Avoids the negative social impact of construction works Negative impact Social benefits during construction (eg, employment opportunities) Social benefits Equal compensation for environmental change Environmental compensation Economy Improve market competitiveness by adopting policies and practices that promote sustainability issues Sustainable policy Selects environmentally-responsible suppliers and contractors that can demonstrate environmental performance Contractor with environmental performance Environment Conservation efforts by conserving life support systems, conserving plant biodiversity, animals and other organisms Conservation efforts Minimizes damage to sensitive landscapes, including valuable areas from a landscape, cultural, historical, or architectural point of view, and minimizes disturbance to the wilderness area Historical damage social economy environment social risk rate rate of env risk avoid soc. negative impact Health and safety Institution and technology Quality of life Social benefits Env compentation reputation sust. policy contractor with EP conservation historical demage rate of ec risk Fig. 2. Stock and Flow Diagram (SFD) of the basic model. Environment Minimizes damage to sensitive landscapes, including valuable areas from a landscape, cultural, historical, or architectural point of view, and minimizes disturbance to the wilderness area social economy environment social risk rate rate of env risk avoid soc. 5 Conclusions Based on the results of the discussion on point 4 Fig. 2 can be concluded that there are 3 (three) factors that can affect the reputation risk, namely social, environment, and economy and the model built System Dynamics in this research shows can help the perpetrators oil and gas companies to predict and enhance the company's reputation. It is based on a causal relationship between the variables. As for the relationships that are built, among others, the relationship of loop balancing and reinforcing loop. 4 Results and discussion negative impact Health and safety Institution and technology Quality of life Social benefits Env compentation reputation sust. policy contractor with EP conservation historical demage rate of ec risk Fig. 2. Stock and Flow Diagram (SFD) of the basic model. social economy environment social risk rate rate of env risk avoid soc. negative impact Health and safety Institution and technology Quality of life Social benefits Env compentation reputation sust. policy contractor with EP conservation historical demage rate of ec risk Fig. 2. Stock and Flow Diagram (SFD) of the basic model. Fig. 2. Stock and Flow Diagram (SFD) of the basic model. 6 , 0 (2019) MATEC Web of Conferences 276 ICAnCEE 2018 2014 https://doi.org/10.1051/matecconf /201927602014 Fig. 2 is the based-model reputation of oil and gas companies. The model consists of 3 sub-based models, namely social sub-models, economic sub-models, and environmental sub-models. In the CLD explained that there are three main variables which can affect the reputation of oil and gas companies, these variables are social, economic, and environmental. Each variable is influenced by several parameters, and it can affect as an amplifier and also as a counterweight to the existing variables. Besides that, among parameters can also influence each other. After the relationship between variables is known, then the CLD can be developed into stock and flow diagrams. 6 References 1. O.W. Achaw, E.D. Boateng, Int. J. of Development and Sustainability 1, 2 (2012) 2. D. Ambarsari, Minyak kami tanggung jawab kami-transparansi migas untuk pembangunan berkelanjutan: belajar dari Blora dan Bojonegoro (2009) 3. N.V. Thuyet, S.O. Ogunlana, P.K. Dey, Int. J. of Energy Sector Man. 1, 2 (2007) 4. A. Hasan, J. of Pipeline Systems Eng. and Practice 7, 3 (2016) 5. S. Nasution, Y. Arkeman, K. Soewardi, T. Djatna, J. of Industrial Res. 8, 2 (2016) 6. F.A. Ogwu, FORUM Ejournal 10 (2011) 7. H. Conner, Managing environmental risk in the oil and gas industry (Thesis, Claremont Graduate University, California, 2015) 8. Y.A. Cintya, Implementasi Standar Internasional ISO 31000: 2009 risk management -principles and guidelines di sektor energi-minyak dan gas. Available at: http://crmsindonesia.org/www2/knowledge/crms-articles/implementasi-standar- internasional-iso-31000-2009-risk-management-%E2%80%93 (2014) 9. Y.M Goh, P.E.D. Love, Safety Science 50 (2012) 10. M. Shin, H.S. Lee, M. Park, M. Moon, M, S. Han, Accident Analysis and Prevention 68 (2013) 11. Kementerian Energi dan Sumber Daya Mineral. Peluang investasi sektor ESDM (Menteri Energi dan Sumber Daya Mineral, Jakarta, 2011) 12. T. Purwati, Pipa gas dan minyak Pertamina meledak, 1 tewas. Available at: www.pikiran-rakyat.com/jawa-barat/2016/02/08/360129/pipa-gas-dan-minyak- pertamina-meledak-1-tewas (2016) 13. G. Nyirenda, C.C. Ngwakwe, J. of Env. Economics 5, 1 (2014) 14. M. Abduh, Seminar Nasional Sustainability dalam Bidang Material, Rekayasa, dan Konstruksi Beton (2007) 15. C.S. Goh, S. Rowlinson, J. of Legal Affairs and Dispute Resolution in Engineering and Construction 5, 4 (2013) 7 7 , 0 (2019) MATEC Web of Conferences 276 ICAnCEE 2018 2014 https://doi.org/10.1051/matecconf /201927602014 16. L.Kh.M. Suliman., dan A. Omran, Journal of Economic, Social, Political, and Cultural Problems of the Future Society 10 (2009) 17. S. Asad, M.M.A. Khalfan, Emirates J. for Eng. Research 12, 2 (2007) 18. J. Irizarry, S. Zolfagharian, M. Nourbakhsh, Zin, M. Rosli, K. Jusoff, R Zakariya, J. of Information Technology in Construction 17 (2012) 19. K.S. Dewlaney, M.R. Hallowell, B.R. Fortunato, J. of Construction Engineering And Management 138, 8 (2012) 20. S. Bakhtiari, OIDA Int. Journal of Sustainable Development (2014) 21. Y. Barlas, European J. of Operational Research 42, 1 (1996) 22. U. Rosentröm, The J. of Transdisciplinary Environmental Studies 5, 1-2 (2006) 23. B. Masood, Postmodernism and architecture (Khak publication, Iran, 2007) 24. E.S. Bakhoum, D.C. Brown, J. of Construction Eng. and Man. 138, 1 (2012) 25. El Attar, W. Khattab, Proc. of the Int. Maritime Transport & Logistics Conf. (2013) 26. N. Wang, K. Wei, H. Sun, J. 6 References of Man. in Eng. 30, 2 (2014) 27. D. Paton, D. Johnston, L. Mamula-Seadon, C.M. Kenney, Disaster & development: examining global issues and cases (Springer, New York, 2014) 28. H. Zabihi, F. Habib, L. Mirsaeedi, Int. J. of Emerging Sciences 2, 4 (2012) 29. W.N.B. Osman, Z.M. Udin, D. Salleh, J. of Southeast Asian Res. 2012 (2012) 30. J. Morelli, J. of Environmental Sustainability 1, 1 (2011) 31. S. Zolfagharian, M. Nourbakhsh, J. Irizarry, A. Ressang, M. Gheisari, Construction Research Congress 2012 (2012) 8 8
https://openalex.org/W4321638496	https://uet.edu.al/polis/wp-content/uploads/2023/01/The-Approach-of-the-Albanian-Reality-to-Autism-Spectrum-Disorder.pdf	English	null	The Approach of the Albanian Reality to Autism Spectrum Disorder	Polis	2,022	cc-by	9,617	g p 2 Stela Kapo graduated in psychology from the European University of Tirana. Her areas of interest are mainly related to counseling psychology, social psychology, organizational psychology and scientific research. He has been part of several projects related to mental health before and has conducted various courses and trainings in this field.i 1 Lorela Dema has graduated from European University of Tirana with a Bachelor’s degree in Psychology, Mental Health profile. Her fields of interest include clinical psychology, personality psychology, abnormal psychology and counseling. She has volunteered in different organizations which focus on the rights of different minorities, thus she is dedicated to further contribute in the study fields that relate to these groups. gi 3 Jonad Kosova recently finished his second year of his Bachelors in Psychology with a focus on mental health, in European University of Tirana. His areas of interest include psychotherapy, clinical psychology, psychosocial and psychiatric rehabilitation. As someone who seeks to understand mental illnesses and mental health issues better and is on the path of training to provide quality and socially conscious mental health services, he was very excited to partake in this study. 4 Eneda Sala finished the second year of Bachelor in Psychology, at European University of Tirana. Areas such as social psychology, personality psychology, engineering psychology and psychodynamics are of great interests to her. Her future perspective consists to operate as a research psychologist. The acknowledgment of the necessity for more research and thoughtfulness, towards autistic individuals added to the personal motivation to be part of the study. Abstract Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder associated with persistent deficits in social communication and social interaction across multiple contexts and restricted, repetitive patterns of behaviour, interests, or activities. The ways in which autism, culture and the education system interact have not yet been explored sufficiently, even more so when it comes to Albania. Through literature reviews, interviews, and case studies we aim to further investigate how autism is assessed and treated within the Albanian school system and how culture can affect the parent-teacher-child triangle. We also investigate the implementation of laws and policies regarding inclusive education, and its benefits to autistic individuals, specifically in the development of social skills that further help them to integrate into society. We also try to figure out the difficulties autistic individuals face within the school system and in their day-to-day life in Albania, as well as how Autism Spectrum Disorder affects the families of these individuals. This qualitative study found that the reality in Albania is not supportive enough towards autistic individuals. With further research into the field, we might be able to back this statement up with empirical data as well as figure out how this condition can be improved. Keywords: autism spectrum disorder, special education, cultural influence, inclusivity Eneda SALA4 Department of Education, Psychology and Sports European University of Tirana esala2@uet.edu.al Eneda SALA4 Department of Education, Psychology and Sports European University of Tirana esala2@uet.edu.al The Approach of the Albanian Reality to Autism Spectrum Disorder Lorela DEMA1 Department of Psychology European University of Tirana ldema5@uet.edu.al Stela KAPO2 Department of Psychology European University of Tirana skapo2@uet.edu.al Stela KAPO2 Department of Psychology European University of Tirana skapo2@uet.edu.al Jonad KOSOVA3 Department of Psychology European University of Tirana jkosova@uet.edu.al 1 Lorela Dema has graduated from European University of Tirana with a Bachelor’s degree in Psychology, Mental Health profile. Her fields of interest include clinical psychology, personality psychology, abnormal psychology and counseling. She has volunteered in different organizations which focus on the rights of different minorities, thus she is dedicated to further contribute in the study fields that relate to these groups. g p 2 Stela Kapo graduated in psychology from the European University of Tirana. Her areas of interest are mainly related to counseling psychology, social psychology, organizational psychology and scientific research. He has been part of several projects related to mental health before and has conducted various courses and trainings in this field. gi 3 Jonad Kosova recently finished his second year of his Bachelors in Psychology with a focus on mental health, in European University of Tirana. His areas of interest include psychotherapy, clinical psychology, psychosocial and psychiatric rehabilitation. As someone who seeks to understand mental illnesses and mental health issues better and is on the path of training to provide quality and socially conscious mental health services, he was very excited to partake in this study. 107 1. Introduction Living in a society where prejudice is something that almost everyone holds within themselves, makes people of special target groups more aware of how others approach them. This is because, very often, it affects their quality of life and their psychological state. Autistic individuals for example, can be categorized 4 Eneda Sala finished the second year of Bachelor in Psychology, at European University of Tirana. Areas such as social psychology, personality psychology, engineering psychology and psychodynamics are of great interests to her. Her future perspective consists to operate as a research psychologist. The acknowledgment of the necessity for more research and thoughtfulness, towards autistic individuals added to the personal motivation to be part of the study. POLIS / No. 21, 2022 108 as a vulnerable group that can be affected in considerable levels by the way the others approach them, including here not only the general public, but also the systems, laws, institutions, schools, etc., that are in place. Autistic is the term referring people diagnosed with Autism Spectrum Disorder, which is defined by American Psychological Association as any one of a group of disorders with an onset typically occurring during the preschool years and characterized by varying but often marked difficulties in communication and social interaction. Autism Spectrum Disorder was formerly said to include such disorders as the prototype autism, Asperger’s disorder, childhood disintegrative disorder, and Rett syndrome; it was synonymous with pervasive developmental disorder but more commonly used, given its reflection of symptom overlap among the disorders. It is now the official term used in DSM–5, where it encompasses and subsumes these disorders: Autism, Asperger’s disorder, and childhood disintegrative disorder are no longer considered distinct diagnoses, and medical or genetic disorders that may be associated with Autism Spectrum Disorder, such as Rett’s syndrome, are identified only as specifiers of the disorder. (Vandenbos) h This disorder caught our attention and inspired us to conduct this study as it is a very delicate and important matter of discussion. Another factor in deciding our study subject were the rising rates of Autism Spectrum Disorder diagnosis. Autism Spectrum Disorder has firstly been tracked by researchers in 2000 and had a prevalence of 1 in 150 children in United States, while in 2016 the prevalence was 1 in 54 children and the in 2017 was 1 in 44 children. Description of chapters Chapter I: This chapter provides a brief introduction to the topic to present its purpose, importance and perspectives.h Chapter II: The second chapter includes a review of the literature, in which various sources are reviewed to provide an explanatory framework regarding autism, Albanian law in support of it, and gender differences in treatment.h f Chapter III: The third chapter explains step by step the study methodology, including the hypothesis, the research question, the objectives, the purpose, the relevance, the dependent and independent variables, the instruments used, the sample, and the restrictions. Chapter IV: In this chapter the analysis of the data is done and in the case of our qualitative study it is organized in the form of a summary of the interviews and the main data provided by them that will help us to examine the hypothesis of the study. Chapter V: In the last chapter, the conclusions of the study are given, considering the results obtained and verified hypothesis. 1. Introduction (Wright, 2017) With the growing number of autistic individuals, we thought it would be worthwhile to conduct a study which would focus on this category, to provide information so that we can come to their aid as much as possible. We added the frameworks of the Albanian reality to our study, to make the field of study more specific and tangible. Given the history, this disorder is considered new in Albania, so we find it more valuable to focus research on this country. This would provide more scientific information on this topic which is almost lacking for Albania. i In this study we aim to reach results regarding the way the Albanian reality approaches autistic individuals. When we say the Albanian reality, we are talking about the legal aspect, the institutional aspect and the human aspect and the way these work towards autistic individuals. We have tried to achieve this through a qualitative study method based on literature review and analysis of interviews conducted with competent people who can confidently talk about this field. ii What is worth noting is the need that these individuals have for support and inclusion. They should be involved in life activities just like everyone else, but conditions should be created for them to cope with these activities on their own terms. This would help them to coexist better with their disorder and take steps towards development and improvement. POLIS / No. 21, 2022 109 5 From here on after, Autism Spectrum Disorder and ASD will be used interchangeably POLIS / No 21 2022 110 5 From here on after, Autism Spectrum Disorder and ASD will be used interchangeably The importance of the role gender plays in the perception of ASD Parallel to the research that strives to explain the effect of how a culture overall affects the way autism is viewed, there is also an increasing number of research that points to the importance of addressing and explaining the specific role that the social construct of gender plays in the perception of autism symptom severity. (Geelhand et al., 2019) As pointed out by a few studies measuring cognitive differences in boys and girls conducted by the Journal of American Academy of Child & Adolescent Psychiatry there do seem to exist a few differences between them, specifically in the area of adaptive skills, problem-solving and problem externalization, overall cognitive ability and intellectual level. (Dworzinsky et al., 2012; Frazier et al., 2013)f Other studies have concluded that there are few to none gender differences when it comes to autism symptom severity and the areas where there do seem to be slight differences, it was mostly a matter of differences in IQ and age. (Rivet & Matson 2011) The most widely reported male–female ratio for autism prevalence is 4–5:1, lower in individuals with intellectual disability and higher at the high-functioning end. (Fombonne E., Quirke S., Hagen A. 2011) The overall nature of the studies who have taken such subjects into consideration is one of contradiction, leading to the relationship between gender differences and autism symptoms to remain unclear and complicated. 2. Literature review Cultural influences on Autism Spectrum Disorder Cultural influences on Autism Spectrum Disorder As it stands today, the diagnosis of Autism Spectrum Disorder5 is one that not only affects the individual who receives it, but one that affects them along with their families, communities that they live in, the ways in which they interact with the healthcare system and the school system as a whole. Research on the impact of cultural beliefs specific to autism is very limited, although studies focusing on other developmental disorders suggest that it is influential. (Herbert & Koulouglioti, 2010). Even though there is recent evidence that suggests that autism symptomatology is culturally influenced (Norbury & Sparks, 2013; Zaroff& Uhm, 2012), as it stands today, the ways in which culture specifically impacts people with ASD and their families is insufficiently researched. Cultural context has been shown to influence the expression, recognition, interpretation, and reporting of autism symptoms. (Leeuw et al., 2020) Despite the lack of research, a number of studies have also pointed out the importance of further researching the ways in which the families respective culture influences the way they view autism, how they understand and interpret autism symptoms and finally the treatment options they choose to move forward with, if at all. POLIS / No. 21, 2022 110 On the other hand, the same factors must be pointed out when it comes to the way culture influences the professionals’ point of view and how they choose to utilize different diagnostic instruments for ASD and offer treatment options. It is important to focus on both macrolevel cultural factors—factors at the dominant culture level that affect the people in that society and microlevel factors—factors at the family level that affect response to diagnosis or treatment choice. (Bernier et al., 2010) There is a consensus between practitioners that there exists a need to conduct further research on how autism may present in middle- and low-income countries, typically non-western ones. Parent – teacher-child triangle The lack of studies on the topic of cultural influence and gender differences in autism, also seems to bleed into a lack of studies in the area of parent-teacher-child interaction and cooperation when the child is diagnosed with ASD, especially when it comes to families that are part of a cultural minority. There seem to be two main ways through which this triangle is maintained, one is through the co-development of teaching strategies and the other is through parental POLIS / No. 21, 2022 111 training facilitated by special education teachers and the school system. These have not only shown positive results in the management of autism symptoms of the child, but also perceived as very beneficial by the parent. (Kashinath Sh., Woods J., Goldstein H., 2006) As such, the utilization of such strategies and guidelines such as the 2nd Edition of Kids in the Syndrome Mix of ADHD, LD, ASD, Tourette’s, Anxiety and More. h The main conclusion that is presented in studies around this topic comes down to the importance of sharing knowledge with parents, communities, and individuals. Something that would increase public awareness of available services and resources for culturally and linguistically diverse children with ASD and their families, and thus help them access usual sources of care. (Lin, Yu & Harwood, 2012) Parent-implemented PECS training showed that through such an intervention parents can effectively implement improvisation training and a clear relation between parent-implemented training and improvisation of mands by children with autism. (Chaabane et al., 2009) As for minorities, parent-focused interventions do not seem to address their issues as effectively. Rather, it was shown that ASD education programs that address informational and cultural needs may better promote ASD adjustment among ethnic minority families. (Gordillo M., Chu A., Long K., 2020) Another very important point to mention is the importance of providing documentation and resources for families in their native language. This would help practitioner understand and respect cultural difference, and thus contribute to the delivery of family-centered service. (Barrio et al., 2018) Studies clearly point to the importance of combining research-based interventions with the understanding and respect needed to provide useful resources to parents and families that are a part of a cultural minority. Parent – teacher-child triangle Doing so will ensure the well-management and functionality of the parent-teacher-child triangle,resulting in a better education experience for the child diagnosed with ASD.hi There are a lot of factors to be considered while examining the significance of education for children with ASD; this can vary as of: class formation, teacher’s attitude, assistant teacher ease of access, parent–school–child triangle (Brooker, 2010), the role of school psychologist, inclusive or exclusive education style, infrastructure and physical environment, practices challenges&IEP. “Due to legal and therapeutic reasons, children with autism spectrum disorders (ASD) are often educated in general education settings. As such, it is important to understand the variables that might affect a student’s placement in inclusive education settings, simultaneously considering student variables (e.g., disability label) and teacher variables (e.g., knowledge of autism).” (Matthew J.Segalla, 2014) Teaching children with ASD can be quite challenging for teaching staff. ASD can take unique forms in different individuals therefore, not every child with POLIS / No. 21, 2022 112 ASD will necessitate additional staffing and specialist placement. Nevertheless, all children can benefit from staff that has professional knowledge of ASD, with diverse teaching style; and from an adjusted education environment. “First, they need to help the child in managing the classroom and school environment. This may involve reorganizing the furniture; labelling areas and equipment; providing a dedicated work area; or teaching the children strategies with which to interpret and respond to demands as they arise” In example using Social Stories and visual timetables. (Gray 1994; Smith 2001) & (Mesibov& Howley 2003) Autism Spectrum Disorder in Albania Autism Spectrum Disorder in Albania The law for inclusive education in Albania The law for inclusive education in Albania Albanian law that discusses special education for children with disabilities is included in the 11th section of law number 69 published in 2012 concerning the pre-university education system, containing three articles in total, namely Article 63, Article 64 and Article 65.Along with the three articles included under the aforementioned section, there is also Article 19 under section number 2 “EDUCATIONAL INSTITUTIONS AND FORMS OF EDUCATION” of the same law. Under Albanian law, services for students with special needs are ensured by the state (Article 19,Law nr.69,2012).The ways in which these services will be delivered and the criteria needed to be able to receive such services is decided by The Council of Ministers, as explained by Article 19 of Law number 69,2012.Section 11 titled “EDUCATION FOR CHILDREN WITH DISABILITIES”, as stated above, contains three articles, each containing three, six and four main points respectively. The first one, Article 63, states the three main principles around education for children with disabilities, SD being a diagnosis that puts a child in that category. It explains that the goal of special education for these children should be to help them achieve their full physical and mental potential, while also improving their quality of life. Furthermore, it states that the inclusion of children with disabilities in ordinary schools is of primary importance and also the guarantees the right to communicate through sign language and the usage of Braille writing. Article 64,titled “THE ATTENDING OF EDUCATIONAL INSTITUTIONS BY CHILDREN WITH DISABILITIES”, consists of six main points and describes the role of specialized institutions in the process of attending educational institutions by children with disabilities and also the role that the parents play in deciding whether or not their child will or will not attend such institutions. Article 65,titled “ORGANIZATION OF EDUCATION FOR CHILDREN WITH DISABILITIES”, consists of four main points. It describes the ways in which children with disabilities may follow the course of a certain subject, whether it be alongside other students via the POLIS / No. 21, 2022 113 ordinary teaching plan, a teaching plan adjusted to their needs or one that is made specifically for them. The role of the parent in this entire process is also included, stating that the development of the personalized education program is done in collaboration with the child’s parents. Autism Spectrum Disorder in Albania (Article 65,Law nr.69, 2012) Even though such law acknowledges the importance of special education services, the inclusion of the child’s parents in the process of making decisions regarding their child’s education and the role specialized institutions play in the education of children with disabilities, it still lacks important points regarding the support that should be offered for the needs of children with disabilities, while also excluding children who show signs of social maladaptation from receiving special services. Overall it is characterized as a legal document that offers limited information and guidelines as to how children with disabilities fit into the education system. The Albanian education system in relation to ASD The Albanian education system in relation to ASD h y A study conducted in the Albanian education system indicated a lot of tasks and issues regarding the quality and services offered in schools. “Related to the barriers of children with learning difficulties in compulsory education. Autistic children, similarly, to all children with learning difficulties, face and are hindered by a considerable number of barriers. These barriers can be structural, pedagogical, conceptual, professional, financial, and behavioral. The group of autistic children faces specific barriers at a different level, when compared to other children with learning difficulties.”(Gjedia, 2015) Correspondingly in parents’ voices these obstacles in the Albanian education environment are regarded as fundamental in their children’s optimal development. “In terms of what parents feel is most needed for the provision of an appropriate education for their children, almost all studies examining this question specifically mention the need for specialized teacher training and knowledge of autism, effective collaboration and communication, staff being able to manage the child’s behavior, the child’s progress in terms of social skills, and the need for structure.” (Foy, 2012) An additional question that originated during the conduction of the research was: “If provided mainstream education or special school education which would be the best way to support ASD children optimal development? “National Autistic Society Northern Ireland (2012) survey identified that more than one in four children do not feel happy at school with almost one-third of parents indicating that the education their child gets is inadequate to meet their needs.” (Marshall, 2015) Additionally, less than half of the parents repeatedly have to take their child out of school because of the difficulties that staff has to go through to handle aggressive behaviors. This situation in an Albanian context can get more problematic due to parents’ approach. Mainstream schools and POLIS / No. 21, 2022 114 neurodiversity classrooms can be challenging for all components, then again: “The benefits of mainstream inclusion for children with ASD include displaying more social behavior, having more advanced education goals” (Elder et al. 2010) and increased social skills (Reiter and Vitani 2007). Certainly, benefits of general/inclusive education setting can vary from improved social skills due to modeling of typical behaviors, lower stigmatization and improved self-concept, advanced academic expectations. (Mesibov & Shea, 1996) Although teachers, psychosocial experts and REA representatives recommend that for the Albanian context and conditions it would be a better choice not to include all children in regular schools. The Albanian education system in relation to ASD “Some extreme cases that need a caregiver, besides an assistant teacher, recommend being put in special education institutions until they make progress. They mainly mention cases of children that are not able to care for their hygiene (e.g., they urinate in class or take their clothes off) or others that have aggressive behaviour towards other children or teachers.” (Duci, Ndrio, Dragoti, (Nasufi), & Ismaili, 2016) Literature indicates the importance of mainstream inclusive education, nonetheless specifies the importance of IEP (Gartin, 2005) & assistant teacher and staff. IEP functionality interconnects with teacher’s behavior and child engagement & response. Studies have pointed out that the success of IEP is equally dependent on a child’s reaction to the setting and its quality. (Ruble, 2013) Referring back to the Albanian setting, IEP and assistant teacher & staff seem challenging. “In most cases, their individual plans do not meet the real needs of students with autism. Individual Education Plans, in most cases are written by the teacher and are not built by a specialized team that is capable of such task.”(Gjedia, 2015) Another prominent element in in the revised literature is the importance of teachers’ attitude towards autistic children. “Positive teacher attitudes are an important predictor of the successful education of children with disabilities, including those with autism spectrum disorders.” (Rodríguez, 2012) Parents’ Perspective on ASD Parents’ Perspective on ASD There’s a lack of literature on the parents’ perspective in the Albanian setting therefore we have referred to cross cultural studies to thoroughly link to the perspective conveyed by the interviews conducted. Parent’s perspective is important, regarding the amount of time they spend with their children. Parents can express moreover what the children may not be able to express analogically. Their perspective is not only their experience but also the children’s experience. In a survey conducted in 2012 by Elizabeth M. Starr and Janis B. Foy: “Parents stated that what is needed are “fully trained experts of any profession, so I need not be the only expert (really, I do all of the programming). These quotations also highlight the commonly expressed frustration related to the parents ‘perceived POLIS / No. 21, 2022 115 need to educate their child’s teacher each year.” Some of the most common needs that parents mention in surveys are increased educational assistant time, the incorporation of technology, increased assistant time in education, social skills training, and sensory integration. Various literatures point toward parents commonly wanting their autistic children to reach their full potential in life. y g p Another valuable component of education for autistic children is the psychologist. The psychologist assists not only the children but also serves as care coordinator for the child- school-parent triangle. School psychologists are positioned to facilitate care coordination because they possess the required training. In domains such as: child development and developmental disabilities, constructing data- based resolution and offering consultation, professional academic and mental health mediations, psychological assessment, affiliating with families and other disciplines and structures. “The concept of care coordination assumes a central role within the National Association of School Psychologists’(2010) Model for Comprehensive and Integrated School Psychological Services” (NASP Practice Model) (Shahidullah, 2020) 3.1. Study purpose and research question 3.1. Study purpose and research question The purpose of this study is to analyze the reality and the culture that individuals diagnosed with Autism Spectrum Disorder face in Albania. By gathering data on the day-to-day activities of autistic individuals as well as their interactions with others, we may be able to understand what the general approach to autistic people in the Albanian society is, whether it be cultural or legislative. Variables: In this study we have two variables from which one is dependent and one independent. Respectively the dependent variable of this study is the approach of the Albanian reality and the independent variable are the autistic individuals. Study analysis: In this study the main analysis is based on a methodology of a qualitative study model. Qualitative research is the process of collecting, analyzing, and interpreting non-numerical data, such as language. Qualitative research can be used to understand how an individual subjectively perceives and gives meaning to their social reality. (McLeod, 2019) To collect data about our study we chose to interview some individuals that are in contact with this reality on a daily basis and are competent to give opinions and evidence about the situation of individuals diagnosed with Autism Spectrum Disorder in Albania. We believe that this model is the most appropriate one for our study thesis as it allows for gathering more extensive information as well as specific details. We have used the semi-structured type of the interview, with pre-prepared questions as well as additional questions that were asked based on the course of the interview. Hypothesis: In this study we aim to confirm these hypotheses: Hypothesis 1: The reality in Albania is not supportive enough for individuals diagnosed with Autism spectrum disorder. Hypothesis 0: The reality in Albania is quite supportive for individuals diagnosed with Autism spectrum disorder. Variables: Objectives: Objectives: • In this study we aim to reach these main objectives:f • Understand how the law in Albania affects the life of autistic individuals • Understand how the law for these individuals is implemented, especially in schools, as it is one of the main institutions in which autistic individuals are part of and affected by. fi f • Understand more about the school system and its’ efficiency for these individualsfi f • Understand more about the school system and its’ efficiency for these individualsfi Understand the difficulties that these individuals face in Albanian reality • Understand the difficulties that these individuals face in Albanian reality • Understand the approach of other people toward these individuals Understand the approach of other people toward these individuals Understand how these individuals are seen by the society and cultural aspec • Understand how these individuals are seen by the society and cultural aspects POLIS / No. 21, 2022 116 Research question: What is the reality of the approach towards individuals diagnosed with Autism Spectrum Disorder in Albania? Participants As mentioned before, in this study we tried to seek information from individuals who have significant contact with autistic individuals, including here psychologists, parents of autistic children diagnosed, and special needs learning support assistants. Respectively we have interviewed two parents of two autistic boys, one psychologist for children and adolescents and two special needs learning support assistants. For parents, as it is clear that they spent most of the time of their day taking care of their children diagnosed with Autism Spectrum Disorder, they have a lot of information about how their children are seen by others, how the law supports them, how they are involved in school lessons and how efficient is this process for them and all other objectives that answer our research question. About the special needs learning support assistants, their everyday work is to assist these individuals in schools, so they know what the difficulties they face during the learning process are. The psychologist also works with autistic individuals, and has a wide experience with them, therefore she was able to share more formal and organized information about all our questions. Why is this study important? This study has considerable importance especially in Albania. By gathering information on the ways in which autistic individuals interact with the social environment, in their day-to-day life as well as within establishments such as schools, we may be able to highlight the difficulties these individuals are faced with, and where these difficulties may stem from. By talking to professionals, we can figure out what needs to be done to get the best outcome for autistic individuals. By compiling both, the law for education and firsthand experiences of the people who are constantly in contact with autistic individuals, we will be 117 POLIS / No. 21, 2022 able to check how well these laws are implemented. This study can also help in opening a path to further research, both qualitative and quantitative, in order to get a full picture on the reality that autistic people face in Albania. With further research that can provide empirical data for the field of studying ASD, we may be able to bring forth positive change in the lives of autistic people, whether it be by bringing awareness, reducing stigma or even influencing the legislation. Methodology The process of conducting this study has passed through some phases. We initially decided on the individuals we should collect information from and realized that there are three key figures that have a significant role in the life of autistic individuals of all ages: parents of these individuals, psychologists and special needs learning support assistants. We proceeded to find our contacts and reached out to them for an interview. As mentioned above, we found two parents, a psychologist and two special needs learning support assistants that accepted to share with us their information anonymously except for the psychologist and one of the special needs learning support assistants. They agreed to share the evidence they have witnessed with sincerity and objectivity based on facts and situations. We prepared the questions for each group that would be interviewed, resulting in a different set of questions for parents, a different set of questions for the psychologist and a different set of questions for the special needs learning support assistants. We set the dates for the interviews, POLIS / No. 21, 2022 118 which were held on different days and from different people. Every interview was recorded as evidence for this study. After completing the interviews, we made the transcriptions and organized the data collected from the answers of the people who were interviewed. We proceeded to prepare a detailed analysis of the data so we could reach the conclusions of this study. In the end of the process, we controlled the hypothesis and understood which one was proved. Instruments Instruments used for this study consist of the interviews we had prepared to gather as much information as needed to collect the data we wanted. The interviews were semi-structured and created by us, so not based on a standardized inventory or questionnaire. The interview of the psychologist as well as the interviews of the special needs learning support assistants consisted on a different number or questions based on the natural flow of the conversation and information they were willing to share. The interview of the parents consisted of a different number of questions from each other because of the age of the children diagnosed with Autism Spectrum Disorder, so we decided to focus the attention of our interview in different aspects for these two individuals. The questions of the interviews on each group where mostly open-ended questions, based on their experiences with autistic individuals, their evidence about the life of these individuals, the way they think and approach with these individuals, their understanding of others culture and approach to autistic individuals, etc. We have adjusted by adding new questions and not using some that we have pre-prepared on the process of interviewing because of the dynamics of the interviews and the information we aimed to collect the most. First interview The first interview was conducted with S. A., special needs learning support assistant. She expressed that inclusive education is crucial when it comes to these Children. She reported that the biggest challenge when it comes to special needs education is the introduction process, especially when it comes to children on the autism spectrum. She linked establishing a good relationship with the child to a form of testing, both on the teacher’s end and on the child’s end. The child through their behaviors creates a block, which makes it very hard for the teacher to connect to them. However, she expressed that if these children were allowed to be themselves it would create a better atmosphere in the class. It is also worth noting that this would allow both parties to experience very positive emotions. For Ms. A. the main issue with special needs education was related to the adults, who allow their own opinions and mentality to affect the quality of education. Both, the teacher, and the special needs learning support assistant, need to focus on inclusivity to help create harmony between all the children in the class, Ms. A. stated. She regurgitated that it is very important to allow autistic children to be open and try to interact with others, as well as the fact that most issues arise from the adults in these settings. She put emphasis on the fact that each behavior that autistic children exhibit has the function of trying to communicate something specific to the teacher and it is up to the teacher to decode it. To conclude, Ms. A. distinguished between successful and unsuccessful cases when it comes to autistic children. Success cases for her were the ones in which nonverbal children become able to speak and express themselves. Restrictions This study, because of its nature, has some restrictions which we are going to list below. As it is a qualitative study, there is a small number of interviews to take into consideration and this stops us from being able to generalize on our conclusions. We give conclusions based on the detailed information we collected by our interviews, but we cannot say that these interviews express all the Albanian reality about autistic individuals. Another restriction for this study is also the instrument used, that is not standardized but self-created. However, the questions are adapted to the purpose of the study, and we tried to choose the best ones that could help us collect the information we needed. Even with these restrictions we have respected all ethical rules and a strictly objective process, from which we could build our data base and reach reliable conclusions which others can take into consideration for further quantitative studies. 119 POLIS / No. 21, 2022 Third interview The third interviewee was the parent of an autistic child who shared her struggles with us. She stated that the hardest part of this journey was accepting that she had an autistic child. As she was not informed on autism spectrum disorder, when the child was very young, she believed that he would speak very soon, and it was no cause of concern. She claimed that accepting that her son needed help was the first step in helping him. Afterwards, her son was assessed by a multidisciplinary team and was diagnosed with autism spectrum disorder, and they started the process of taking him to therapy. She states that though it is a hard process, both emotionally and financially, her son is now better. In the end she expressed again how important it is to accept autistic children, be it acceptance from the parents or other children in inclusive classrooms. Second interview The second interviewee was also a special needs learning support assistant who shared her experiences with us. She believed that integrating autistic children in inclusive classrooms helps them interact better with others. She stated that during her experience she has mainly worked with the other children in the classroom. She considered this work very important as by explaining and familiarizing other children with the concept of autism spectrum disorder she had been able to help them cooperate with the autistic children in the class. She had also observed that other children who interact with autistic children had become more empathic and more willing to cooperate, as well as more accepting to the idea that everyone is unique. She recalled her most successful moment POLIS / No. 21, 2022 120 of her career as a case in which the child was unable to stay in class for the full length of the lesson, however by the end of the year, the same child actively participated in a celebration the class held. of her career as a case in which the child was unable to stay in class for the full length of the lesson, however by the end of the year, the same child actively participated in a celebration the class held. Fourth interview The fourth person to be interviews was Ms. K. Sh. who works as a psychologist for children and adolescents. She stated that the policies to create inclusive classrooms have been revolutionary in autism awareness as well as in integrating autistic children in society. However, she noted that due to the high number of students in a class, managing them becomes difficult, therefore it is necessary to have a special needs learning support assistant that caters to only one or two autistic children. She then talked about the difficulties of implementing inclusive education in Albania. Ms. Sh. said that the infrastructure was the first difficulty, bringing as an example the classroom décor that can sensory problems for autistic children who deal with hypersensitivity. The second point she brought up was getting to know the child and recognizing specific behaviors they exhibit that might need intervention, something that teacher often have troubles with. For this exact reason, Ms. Sh. believed that the best help can be offered through a team consisting of the teacher, special needs learning support assistant psychologist, social worker. Another topic that came up was the curriculum, which needs to be adapted to autistic children through the usage of individual education plans and specific classed that offer sources for children with special needs. The latter is very limited as these classes are not available in every school. When asked about the most effective method of helping these children, Ms. Sh. said that it goes on a case-by-case basis, but mostly focuses on teaching social skills as well as integration into social activities organized by the class and interaction 121 POLIS / No. 21, 2022 with peers. When asked about the most important thing to keep in mind in this field of work, she noted three things: being sensitive to the child’s needs and emotions, listening to the child, group collaboration. When asked about her most successful case, Ms. Sh. brought into attention the fact that improvement in autistic children is slow, extended in time and with very small steps, but she mentioned a case where a child had managed to be integrated into groups. Fourth interview When asked about the importance of inclusive education, she mentioned that not all autistic children should be part of inclusive classrooms because some might require more specialized treatment and only after these treatments become part of inclusive classrooms, however it is important for everyone to recognized and accept people who are different from our own self. 5. Discussion As stated previously, the purpose of this study is to reveal the reality and the culture that individuals diagnosed with Autism Spectrum Disorder, face in Albania. The research question which this study aims to give an answer to is: “How does the reality in Albania approach individuals diagnosed with Autism spectrum disorder?”. From the interviews conducted, we aimed to gather information on the point of view of both professionals and parents of autistic individuals to paint a fuller picture of the realities that autistic individual face. We also reviewed the laws in place and the support they offer for autistic individuals. h f Hypothesis 1: The reality in Albania is not supportive enough for individuals diagnosed with Autism Spectrum Disorder.i As it stands, the law does not have a specific section that caters to autistic individuals’ needs. It only refers to what is offered to special needs students, within all categories, to facilitate their learning processes. In article 63, section XI of the “Law on Pre-University Education” it is clearly stated that inclusive education is the end goal of special needs education in Albania. This aligns with the findings of the interviews conducted with special needs learning support assistants and a psychologist, in specific interviews 1,2 and 4. Throughout all three of these interviews, it is noted that, for autistic children, taking part in inclusive classrooms is crucial to their development. This is because it creates the tools and develops the skills necessary to be integrated into society as they grow up. The second interview emphasized the positive impact inclusive education has on other children as well, as it allows them to become more accepting and empathic. These findings are further backed up by studies reviewed previously such as Mesibov& Shea (1996), which speak to the benefits inclusive education can offer. if In the meantime, though inclusive education is the end goal for Albanian education, there are a lot of difficulties that come with implementing it into the existing education system. These difficulties were brough to our attention by the interviewees in the first, second and fourth interview as well. Specifically, in the fourth interview it was noted that the infrastructure is a big problem as it can cause reactivity in autistic children. Another issue is the curriculum, which is not fully adapted to autistic children. Fifth interview The fifth interviewee is the mother of a 20-year-old autistic man. She explains that originally, her son was being helped by professional and offered ABA therapy, up until he was 14 at the Regional Autism Centre. Since he left, he has been taking medication, which the mother states do not help him. She also regretted not having given him a sedative medication she was suggested when he was a child to calm his anger. She recalled a certain incident in which her son had become violent, hurting both her and her husband. They brought him to a hospital and isolated him in order to have him calm down as they were unable to restrain him. She claimed to often feel hopeless as the violent outbursts of her older son have become a threat to their youngest son. When asked about her opinion on the current treatments offered in Albania, she again recalled a specific incident. When her son was following treatment at the above-mentioned facility, he had gone to the gym where he had been hit by the professionals or the therapists supposed to help him. When she had gotten in contact with the higher ups, she was told that there was nothing they could do and had put the blame on her son’s behavior. Though the situation was later resolved, it led her to believe that there is no facility that can help her son. She stated that to improve the quality of help offered to autistic children, the main thing that needs to change is for people, specifically professionals, to be more open-hearted and compassionate towards these children. She described life with ASD in the family as an ongoing struggle that affects every single member of the family. She also opened about financial struggles that come with having to pay for therapy and medication as well as worsening mental health due to the situation she was currently in. She concluded the interview by stating that the most important thing when it comes to dealing with ASD in the family is accepting that you have an autistic child and helping them follow are the necessary therapies. POLIS / No. 21, 2022 122 5. Discussion The studies reviewed also note the importance of Individual Learning Plans, plans that according to Gjedja (2005), do not meet the needs of the students as they are not compiled by the professionals necessary in this process. It was pointed out, in interviews and in studies, that the approach teachers and professionals have towards autistic individuals may pose an issue as well. More 123 POLIS / No. 21, 2022 specifically, on our first interview, it was stated that adults often allow their own mentality and opinions to affect the quality of education offered. From the fourth interview we learn that teacher may have difficulties recognizing certain behaviors in autistic children, and thus are not able to offer the help needed. As Rodriguez (2012) stated that positive teacher attitudes are crucial predictors of the success of special needs education, it is clear to see that Albanian special needs educations lacks in this regard. All three interviewees who are involved in the education process as professionals (interview 1,2,4) defined success for autistic children in similar ways. For them, a case would be considered successful the moment that autistic children are able to interact, integrate, and cooperate with other children in groups. From the interview conducted with a psychologist we learned that though this progress is achievable, it is slow and in very small steps. This approach to success seems to also be in line with what one of the parents we interviewed said, in our third interview. We also examined what autistic individuals face outside of a classroom setting. For this, the interviews conducted with children of autistic individuals come to our aid. In both interviews we found one common problem; the parents’ unwillingness to accept that their child has autism and will need special help. In the third interview we conducted, the mother of an autistic child states that she herself had trouble with this notion when the child was very young. This becomes an issue as in the “Law on Pre-University Education”, section XI, article 64, it is stated that the parent can decide which educational institution their child will be part of, as well as deciding to pull their child from school at any moment. If the parent is unwilling to accept that their child needs special help and force them to go through an educational path that does not cater their needs, it can hinder the child’s progress. Conclusions and recommendations Will all the information we gathered from reviewing the existing literature, comparison to previous studies conducted in Albania, as well as the firsthand experiences of special needs learning support assistants, a psychologist, and parents of autistic children, we conclude that our hypothesis is proven as correct. Thus, the reality in Albania is not supportive enough for individuals diagnosed with Autism Spectrum Disorder. We also reached our goals of analyzing how the law, education system and culture affect autistic people in Albania. However, as the study we conducted if of a qualitative nature, we cannot generalize our conclusions as pertaining to the whole Albanian reality. We may say that these conclusions reveal a part of how the reality in Albania is for autistic individuals, based on the limited information we were able to gather through interviews. It is necessary for further quantitative studies to be conducted to generalize. This study still has weight in the field as it provides firsthand accounts of people affected by as autism as well as people who support those affected. Furthermore, it can offer recommendations for future studies, such as studies on the perception of parents of autistic children on the special education system in Albania. Quantitative studies in the future can also shed light on the discrimination autistic individuals face in our society. 5. Discussion As there is a lack of literature regarding parent’s perceptions, these conclusions are only pulled by our own research. However, there is evidence that the implement of parent training can improve the conditions of an autistic child (Chaabane et al., 2009). From the last interview conducted we also learned of the discrimination autistic individuals face in their everyday life. One instance was retold by the mother of an autistic man where he was physically abused in an establishment that was supposed to help him. This incident was later blamed on her son. This aligns with that the first interviewee stated when it came to the fact that professionals allow their own opinions and perceptions to affect the way they offer help. This negative biases towards autistic individual may be what Bernier et al. (2010) and Leeuw et al. (2020) were referring to when they spoke about cultural influence in the expression, perception and help offered to autistic individuals. Furthermore, it may be one of the barriers that autistic individuals face, as Gjedja (2005) stated. POLIS / No. 21, 2022 124 Both parents of autistic children interviewed spoke of the financial difficulties they were faced with once their child was diagnosed. The third interviewee only mentioned that the treatment for her child was a financially taxing process. Meanwhile the fifth interview went more in detail on this regard. While she stated that she her son was given help up util the age of 14 at the Regional Autism Centre, she also talked about the numerous medication that he had to be on. Thus, continuous therapy, medication, emergency situations and further intervention were all very costly processes that worsened their financial state. Furthermore, as autism does not only affect the autistic individual, but also their family, she mentioned that she herself had gone to therapy. Thus, more costs were added as mental health help is also necessary for the other members of the family. Barrio, B. L., Hsiao, Y. J., Prishker, N., & Terry, C. (2018). The Impact of Culture on Parental Perceptions about Autism Spectrum Disorders: Striving for Culturally Competent Practices. Multicultural Learning and Teaching, 14(1). https://doi.org/10.1515/mlt-2016-0010 Bernier, R., Mao, A., & Yen, J. (2010). Psychopathology, Families, and Culture: Autism. Child and Adolescent Psychiatric Clinics of North America, 19(4), 855–867. https://doi.org/10.1016/j. chc.2010.07.005 Barrio, B. L., Hsiao, Y. J., Prishker, N., & Terry, C. (2018). The Impact of Culture on Parental Perceptions about Autism Spectrum Disorders: Striving for Culturally Competent Practices. Multicultural Learning and Teaching, 14(1). https://doi.org/10.1515/mlt-2016-0010 Bernier, R., Mao, A., & Yen, J. (2010). Psychopathology, Families, and Culture: Autism. Child and Adolescent Psychiatric Clinics of North America, 19(4), 855–867. https://doi.org/10.1016/j. chc.2010.07.005 References Barrio, B. L., Hsiao, Y. J., Prishker, N., & Terry, C. (2018). The Impact of Culture on Parental Perceptions about Autism Spectrum Disorders: Striving for Culturally Competent Practices. Multicultural Learning and Teaching, 14(1). https://doi.org/10.1515/mlt-2016-0010 125 POLIS / No. 21, 2022 Brooker, L. (2010). Constructing the triangle of care: Power and professionalism in practitioner/ parent relationships. British Journal of Educational Studies, 58(2), 181-196. Chaabane, D. B. B., Alber-Morgan, S. R., &DeBar, R. M. (2009). THE EFFECTS OF PARENT- IMPLEMENTED PECS TRAINING ON IMPROVISATION OF MANDS BY CHILDREN WITH AUTISM. Journal of Applied Behavior Analysis, 42(3), 671–677. https://doi. org/10.1901/jaba.2009.42-671i Duci, P. V., Ndrio, P. M., Dragoti, P. P., (Nasufi), P. A., & Ismaili, P. E. (2016). FACING THE CHALLENGES OF INCLUSIVE EDUCATION IN ALBANIA. Tirana: ISOP. Fombonne E., Quirke S., Hagen A. Epidemiology of pervasive developmental disorders. In: Amaral D.G., Dawson G., Geschwind D.H., editors.Autism Spectrum Disorders.Oxford University Press; New York: 2011. pp. 90–111. y pp Foy, E. M. (2012). In Parents’ Voices: The Education of Children With Autism Spectrum Disorders. Remedial and Special Education, 33 (4) 207-216. Frederickson, N., Jones, A. P., & Lang, J. (2010). Inclusive provision options for pupils on the autistic spectrum. Journal of Research in Special Educational Needs, 10(2), 63-73.h Gartin, B. C., &Murdick, N. L. (2005). Idea 2004: The IEP. Remedial and Special Education, 26(6), 327-331.h Geelhand, P., Bernard, P., Klein, O., van Tiel, B., &Kissine, M. (2019). The role of gender in the perception of autism symptom severity and future behavioral development. Molecular Autism, 10(1). https://doi.org/10.1186/s13229-019-0266-4 Gjedia, F. K. (2015). The Reality of Education of Children with Autism in mainstream 9-year schools in Albania. Academic Journal of Interdisciplinary Studies, 4 No 2 S2. Gordillo, M. L., Chu, A., & Long, K. (2020). Mothers’ Adjustment to Autism: Exploring the Roles of Autism Knowledge and Culture. Journal of Pediatric Psychology, 45(8), 877–886. https://doi.org/10.1093/jpepsy/jsaa044 Gordillo, M. L., Chu, A., & Long, K. (2020b). Mothers’ Adjustment to Autism: Exploring the Roles of Autism Knowledge and Culture. Journal of Pediatric Psychology, 45(8), 877–886. https://doi.org/10.1093/jpepsy/jsaa044 Howley, M. (2015). Outcomes of structured teaching for children on the autism spectrum: does the research evidence neglect the bigger picture? Journal of Research in Special Educational Needs, 15(2), 106-119. Howley, M., &Kime, S. (2003). Policies and practice for the management of individual learning needs. In Strategies to promote inclusive practice (pp. 34-49). Routledge. Isabel R. Rodríguez, D. S. (2012). References Attitudes toward the Education of Students with Autism Spectrum Disorders. Autism Research and Treatment. Jones, G. (2006). Department for Education and Skills/Department of Health Good Practice Guidance on the education of children with autistic spectrum disorder. Child: care, health and development, 32(5), 543-552. Kałużna-Czaplińska, J., Żurawicz, E., &Jóźwik-Pruska, J. (2018). Focus on the Social Aspect of Autism.Journal of autism and developmental disorders,48(5), 1861–1867. https://doi. org/10.1007/s10803-017-3407-7 Kashinath, S., Woods, J., & Goldstein, H. (2006). Enhancing Generalized Teaching Strategy Use in Daily Routines by Parents of Children With Autism. Journal of Speech, Language, and Hearing Research, 49(3), 466–485. https://doi.org/10.1044/1092-4388(2006/036) Lai, M. C., Lombardo, M. V., Auyeung, B., Chakrabarti, B., & Baron-Cohen, S. (2015). Sex/ gender differences and autism: setting the scene for future research.Journal of the American Academy of Child and Adolescent Psychiatry,54(1), 11–24. https://doi.org/10.1016/j. POLIS / No. 21, 2022 126 jaac.2014.10.003 j Leeuw, A., Happé, F., & Hoekstra, R. A. (2020). A Conceptual Framework for Understanding the Cultural and Contextual Factors on Autism Across the Globe. Autism Research, 13(7), 1029–1050. https://doi.org/10.1002/aur.2276 Ligj për Sistemin Arsimor Parauniversitar në Republikën e Shqipërisë, nr.69 (2012),Tiranë:Qendra e Publikimeve Zyrtare Lin, S. C., Yu, S. M., & Harwood, R. L. (2012). Autism spectrum disorders and developmental disabilities in children from immigrant families in the United States. Pediatrics,130, S191– 197 Mandell, D. S., & Novak, M. (2005). The role of culture in families’ treatment decisions for children with autism spectrum disorders. Mental Retardation and Developmental Disabilities Research Reviews, 11(2), 110–115. https://doi.org/10.1002/mrdd.20061 Marshall, D., & Goodall, C. (2015). The Right to Appropriate Appropriate and Meaningful Education for Children with ASD. Journal of Autism and Developmental Disorders , 3159- 3167. Matheis, M., Matson, J. L., Hong, E., & Cervantes, P. E. (2018). Gender Differences and Similarities: Autism Symptomatology and Developmental Functioning in Young Children. Journal of Autism and Developmental Disorders, 49(3), 1219–1231. https://doi.org/10.1007/ s10803-018-3819-z Matthew J.Segalla, J. M. (2014). Factors influencing the educational placement of students with autism spectrum disorders. Research in Autism Spectrum Disorders, 8, 31-43.f Norbury, C. F., & Sparks, A. (2013). Difference or disorder? Cultural issues in understanding neurodevelopmental disorders. Developmental Psychology, 49(1), 45–58.l Ravindran, N., & Myers, B. J. (2011). Cultural Influences on Perceptions of Health, Illness, and Disability: A Review and Focus on Autism. Journal of Child and Family Studies, 21(2), 311–319. https://doi.org/10.1007/s10826-011-9477-9 Rodríguez, I. R., Saldana, D., & Moreno, F. J. (2012). Support, inclusion, and special education teachers’ attitudes toward the education of students with autism spectrum disorders. Autism research and treatment, 2012. Ruble, J. H. (2013). Teacher and Child Predictors of Achieving IEP Goals of Children with Autism. Journal of autism and developmental disorders, 43(12) 2748-2763. Sansosti, F. J., Powell-Smith, K. A., & Kincaid, D. (2004). A research synthesis of social story interventions for children with autism spectrum disorders. Focus on autism and other developmental disabilities, 19(4), 194-204. p Shahidullah, J. D. (2020). Coordinating Autism Care Across Schools and Medical Settings. Intervention in School and Clinic, 1-8. Shahidullah, J. D. (2020). Coordinating Autism Care Across Schools and Medical Settings. Intervention in School and Clinic, 1-8. Vandenbos, G. (n.d.). Autism spectrum disorder (ASD). Retrieved June 27, 2022, from APA Dictionary: https://dictionary.apa.org/autism-spectrum-disorderf Vandenbos, G. (n.d.). Autism spectrum disorder (ASD). Retrieved June 27, 2022, from APA Dictionary: https://dictionary.apa.org/autism-spectrum-disorderf Werling, D. M., &Geschwind, D. H. (2013). jaac.2014.10.003 Sex differences in autism spectrum disorders. Current Opinion in Neurology, 26(2), 146–153. https://doi.org/10.1097/wco.0b013e32835ee548h Werling, D. M., &Geschwind, D. H. (2013). Sex differences in autism spectrum disorders. Current Opinion in Neurology, 26(2), 146–153. https://doi.org/10.1097/wco.0b013e32835ee548h Wright, J. (2017, March 3). The Real Reasons Autism Rates Are Up in the U.S. Retrieved June 27, 2022, from Scientific American: https://www.scientificamerican.com/article/the-real- reasons-autism-rates-are-up-in-the-u-s/ Wright, J. (2017, March 3). The Real Reasons Autism Rates Are Up in the U.S. Retrieved June 27, 2022, from Scientific American: https://www.scientificamerican.com/article/the-real- reasons-autism-rates-are-up-in-the-u-s/ POLIS / No. 21, 2022 127
https://openalex.org/W2190265773	https://www.matec-conferences.org/10.1051/matecconf/20153303011/pdf	English	null	Phase Transformations and Fatigue of NiTi	MATEC web of conferences	2,015	cc-by	4,238	DOI: 10.1051/ / 0 0 ( 2015) 201 conf Web of Conferences 5 MATEC atec m 3 , 3 0 0 3 3 3 3 1 1 1 1 DOI: 10.1051/ / 0 0 ( 2015) 201 conf Web of Conferences 5 MATEC atec m 3 , 3 0 0 3 3 3 3 1 1 1 1 DOI: 10.1051/ / 0 0 ( 2015) 201 conf Web of Conferences 5 MATEC atec m 3 , 3 0 0 3 3 3 3 1 1 1 1 DOI: 10.1051/ / 0 0 ( 2015) 201 conf Web of Conferences 5 MATEC atec m 3 , 3 0 0 3 3 3 3 1 1 1 1 / C ⃝Owned by the authors, published by EDP Sciences, 2015 / C ⃝Owned by the authors, published by EDP Sciences, 2015 Eduardo Alarcon1,2,a, Ludek Heller2, Shabnam Arbab Chirani1, Petr Sittner2, Luc Saint-Sulpice1 and Sylcain Calloch3 1LBMS, ENIB, Technopôle Brest-Iroise, CS73862, 29238 Brest, France 2Institute of Physics, Academy of Sciences of the Czech Republic, 182 00 Prague, Czech Republic 3LBMS, ENSTA Bretagne, 29200 Brest, France 1LBMS, ENIB, Technopôle Brest-Iroise, CS73862, 29238 Brest, France 2Institute of Physics, Academy of Sciences of the Czech Republic, 182 00 Prague, Czech Republic 3LBMS, ENSTA Bretagne, 29200 Brest, France Abstract. Tensile fatigue of superelastic medical graded NiTi wires was analysed in stress control regime with special attention paid to the deformation/transformation processes involved. Concave shaped samples were machined from NiTi wires allowing us to confine deformation processes into the centre of the sample. The localization of phase transformations within the centre was confirmed using in-situ infrared imaging. Characteristic stress-displacement responses for amplitudes inducing either R-phase only or both R-phase and martensite were identified for these samples. A limited number of pull-pull fatigue tests in force-control regime were performed. The obtained S-N curve and associated stress-displacement responses allowed for identification of three fatigue regimes differing in the fatigue life-time by orders of magnitude. Particularly, fatigue life-time deteriorating effect of R-phase transformation was identified. As in other engineering materials, two different approaches can be adopted to characterize the mechanical fatigue of Nitinol: defect-tolerant (crack propagation analysis) and total-life (characterization of total fatigue life-time to failure in terms on cyclic stress or strain range) [1]. Melton and Mercier [4] were the first to investigate the fatigue behaviour of Nitinol under the two aforementioned approaches. Since then, several fatigue studies have been carried out using different testing procedures. For medical devices, the Bending-rotation fatigue (BRF) testing is widely used [6][7][8]. Fatigue of laser-cut struts, the building units of medical stents, are often tested in cyclic tension-compression [9][10]. Other studies present results of the behaviour of Nitinol tubes under cyclic tension-tension (pull-pull) [11]. Classical pull-pull fatigue tests have been performed on Nitinol wires and sheets as well. Dog bone samples, cut from wire electro discharge machining, were tested in [12] in order to investigate the pull-pull fatigue behaviour of Nitinol sheets. In the case of pull-pull fatigue of Nitinol wires, most of the results in literature are from straight non-machined samples [13][14]. However, from our experience, these samples are not suitable for HCF tests since they tend to fail within the jaws of the fatigue device, which puts the obtained number of cycles in doubts. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits distribution, and reproduction in any medium, provided the original work is properly cited. Phase Transformations and Fatigue of NiTi Eduardo Alarcon1,2,a, Ludek Heller2, Shabnam Arbab Chirani1, Petr Sittner2, Luc Saint-Sulpice1 and Sylcain Calloch3 Article available at http://www.matec-conferences.org or http://dx.doi.org/10.1051/matecconf/20153303011 3 Experimental conditions The concave geometry acts as stress raiser, ideal for pull-pull fatigue tests of NiTi wires since there is no shear band localization of strain. Besides, the volume which transforms during the forward and reverse MT is strongly confined in the centre of the sample. Local changes in sample’s temperature due to latent heat absorption/release might be effectively suppressed by heat conduction into surrounding non-transforming material. Therefore, the wire stress-strain response might be less rate dependent. Concave geometry is also more suitable for microscopic observations because all the deformation processes are localized into a well-defined spot. On the other hand, material strains and stresses vary along the length of the sample. Static tensile and pull-pull fatigue tests were carried out using an Instron 8872 servohydraulic fatigue testing device in a thermal chamber at 40°C. Testing temperature is thus well above the Af temperature (see Table 1 for identified transformation temperatures). The temperature of the environment was monitored by two thermocouples placed near the sample and at the inner chamber casing. In order to estimate the strain, a 10 mm gauge-length clip-on extensometer was placed at the centre of the concave section of samples. Extensometer controlled tensile tests at a rate of 2.4x10-4mm.s-1 were performed to characterize the tensile response of NiTi samples. Fatigue tests were performed in a force-controlled regime at 25Hz and stress ratio = equal to 0.1. All tensile responses are presented in terms of the engineering stress at the mid-cross-section (applied load divided by the initial mid-cross-section area) plotted against the displacement recorded between the extensometer arms surrounding the concave part. Motivated by fatigue endurance of NiTi wires used in medical application, we selected a medical graded superelastic NiTi wire of diameter 1.78 mm provided in as drawn condition by Fort Wayne Metals Ltd. The provider specified the composition of the raw material as 50.92 at. % Ni and cold-work of about 40%. The as drawn wires were machined into concave shaped samples as explained above. Afterwards, electropolishing was performed in order to remove surface defects and oxide. Finally, samples were heat treated at 500°C for 30min in constrained straight shape and water quenched. We also include results of additional experiments characterizing deformation processes in concave shaped samples using stress-displacement-temperature evolutions performed in force controlled tension on Bose Electroforce 3300 with in-situ infrared imaging using fast Flir SC7000 (max. 200 Hz). 1 Introduction Mechanical fatigue may be thought as a change in material properties due to mere repeated application of stresses or strains which can finally lead to cracking or failure. Origins of fatigue cracks of polycrystalline solids are often associated with irreversible shear displacement along slip bands [1]. However there are many material factors affecting the fatigue properties such as stress and strain state, grain size, precipitates, impurities, inclusions, texture, surface finish as well as phase transitions including martensitic transformation, which gives rise to functional properties of shape memory alloys. From the early days of Nitinol research, it has been known that thermoelastic martensitic transformation is accompanied by dislocation slip and accumulation of residual martensite [2]. These irreversible processes are often blamed for the functional fatigue as well as the drop in fatigue performance systematically reported whenever the loading enters the martensitic transformation (MT) regime [3]. This low cycle fatigue (LCF) is contrasted with high cycle fatigue (HCF) observed at lower strain/stress amplitudes within the Austenite elasticity (ratio between maximal stress applied and martensite transformation stress between 0.3 and 0.7) [4]. Moreover, heat treated cold worked wires used in applications exhibit R-phase transformation preceding the MT [5]. This transformation has to be also considered in fatigue analysis of NiTi wires as a distinct deformation process although it shows much lower transformation strains and negligible hysteresis compared to MT. Surprisingly, the role of R-phase transformation in the fatigue of Nitinol has not been extensively studied. In this paper, we present first results in the context of complex fatigue investigations being carried out on concave shaped superelastic Nitinol wires. First, we introduce investigated NiTi material and particularly the concave geometry of NiTi samples tested in fatigue. Second, we describe typical tensile responses of concave shaped samples including data from in-situ infrared imaging showing the localization of transformation a Corresponding author: eduardo.alarcon@ensta-bretagne.org a Corresponding author: eduardo.alarcon@ensta-bretagne.org This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits distribution, and reproduction in any medium, provided the original work is properly cited. MATEC Web of Conferences processes. Finally, we present fatigue data gathered so far from “pull-pull” tests. Namely, the S-N curve interpreted using tensile fatigue responses and their evolutions during fatigue cycling. Figure 2 DSC curve of NiTi concave shape samples for fatigue testing. 1 Introduction The samples were prepared from raw NiTi material with composition of 50.92 at. % Ni that was cold worked down to 1.78 mm wires with final cold work of about 40%. Then the concave shape was machined and, afterwards, the samples were heat treated at 500°C for 30 min. and water quenched. 2 Material and samples geometry In order to confine the stress-induced phase transformation and other deformation processes proceeding in NiTi, concave samples were produced as shown in Figure 1. The grinding was applied to machine a concave 5 mm long profile with radius of 10 mm within the central section of 40 mm long NiTi wires of diameter 1.78 mm. The diameter at mid-cross-section was decreased by grinding down to 1.14 mm. Figure 2 DSC curve of NiTi concave shape samples for fatigue testing. The samples were prepared from raw NiTi material with composition of 50.92 at. % Ni that was cold worked down to 1.78 mm wires with final cold work of about 40%. Then the concave shape was machined and, afterwards, the samples were heat treated at 500°C for 30 min. and water quenched. Figure 1 Geometry of concave shaped samples made from 1.78mm as drawn NiTi wires by grinding followed by electropolishing. Table 1 Phase transformation temperatures identified from the DSC curve. °C As 21.45 Af 34.52 Rs 26.48 Rf 12.15 Ms -46.16 Mf -105.9 Table 1 Phase transformation temperatures identified from the DSC curve. °C As 21.45 Af 34.52 Rs 26.48 Rf 12.15 Ms -46.16 Mf -105.9 Table 1 Phase transformation temperatures identified from the DSC curve. °C As 21.45 Af 34.52 Rs 26.48 Rf 12.15 Ms -46.16 Mf -105.9 Figure 1 Geometry of concave shaped samples made from 1.78mm as drawn NiTi wires by grinding followed by electropolishing. 4 Phase transformations in concave shaped NiTi samples rate 4 N/s) and annealed samples (loading rate 4.9 N/s), respectively. We suppose that the increase in temperature measured on the annealed sample between loading stages 2 and 3 (see Figure 4a and related temperature profiles 2 and 3 in 4b) is associated with the austenite-R-phase transformation temperature as the solutionized sampled doesn‘t present such a temperature increase at the corresponding loading range denoted 1 and 2 in Figure 3a (see also related temperature profiles in Figure 3b). The following transformations from R-phase to martensite as well as from austenite to martensite are marked with pronounced peaks in temperature-displacement evolutions as seen in Figure 4a and 3a, respectively. Moreover, the related temperature increases is well localized around the mid-cross-sections (see temperature profiles 3, 4 in Figure 3b and 4, 5 in Figure 4b). The increase in temperature and localization of temperature peaks suggest that the martensitic transformation clearly proceeds from the early stage of the plateau appearance in stress-displacement evolutions (loading stages denoted 3 in Figure 3a for the solutionized sample and 4 in Figure 4a for annealed sample). As introduced above, degradation of fatigue performance of superelastic nitinol correlates with the appearance of MT. Hence, before any meaningful tensile fatigue testing on concaved shaped samples we have to understand how the transformations proceed in such shaped samples upon tensile loading. Therefore, we performed in-situ infrared imaging to trace the onset of transformation processes in concave shaped NiTi samples subjected to force controlled tensile loadings. Note that this study was performed on samples that received different heat treatment from the one used for the fatigue analysis. We studied two concave shaped samples that received different thermal treatments and analysed the responses of a solution heat treated sample undergoing directed austenite-martensite transformation path and annealed sample undergoing sequential austentite-R-phase- martensite transformation path. The solutionized sample was prepared by solution heat treatment at 800°C for 2 h followed by water quenching. The annealed sample was prepared by annealing at 450°C for 30 min. The transformation paths were verified using DSC measurements the results of which are not shown here for the sake of brevity. Figure 4 a Stress-displacement-temperature evolutions as obtained during force controlled tensile loading (4 N/s) of the annealed sample. b Temperature profiles related to loading stages numbered in a. c Infrared images related to denoted loading stages. 3 Experimental conditions Note that the clip-on extensometer was not used here and the grip displacement were considered instead. Phase transformation temperatures of the heat treated sample were measured by Differential Scanning Calorimetry (DSC). From the DSC curve shown in Figure 2, phase transformation temperatures listed in Table 1 were identified. Note that the R-phase peak is located near the room temperature denoted in Figure 2 by the vertical line. 03011-p.2 ESOMAT 2015 4 Phase transformations in concave shaped NiTi samples Figure 3 a Stress-displacement-temperature evolutions obtained during force controlled tensile loading (4 N/s) of the solutionized sample. b Temperature profiles related to loading stages numbered in a. c Infrared images related to denoted loading stages. Figure 4 a Stress-displacement-temperature evolutions as obtained during force controlled tensile loading (4 N/s) of the annealed sample. b Temperature profiles related to loading stages numbered in a. c Infrared images related to denoted loading stages. Figure 3 a Stress-displacement-temperature evolutions obtained during force controlled tensile loading (4 N/s) of the solutionized sample. b Temperature profiles related to loading stages numbered in a. c Infrared images related to denoted loading stages. This in-situ infrared imaging study confirmed that the concave shaped samples, indeed, confine the transformation processes to the concave section of the sample. In addition, at the onset of the plateau in stress- displacement evolution the MT is effectively triggered right at the mid-cross-section of the concave section. Figures 3 and 4 summarize stress-displacement- temperature evolutions as recorded during force controlled tensile loading on the solutionized (loading 03011-p.3 MATEC Web of Conferences mechanism. The presumable R-phase onset at ~ 150 MPa is denoted by red bullet. 5 Static tensile characterization To identify the onset of MT and R-phase transformations in samples for fatigue testing we measured static loading- unloading tensile responses of virgin samples. The tests were done at temperatures 40°C at which the fatigue testing was performed in clip-on extensometer controlled loading regime at a rate of 2.4x10-4mm.s-1. Figure 5 shows responses measured at amplitudes below the onset of MT (1), at the onset of MT (2) and above the onset of MT (3). The onset of MT was identified at = 607 . Although the response (1) looks like a linear elastic response of austenite, it deviates from linearity at ~150MPa as clearly seen in Figure 6. We suppose that this is due to the onset of austenite-R-phase transformation as Rs temperature is near the room temperature (see Table 1) and, moreover, the corresponding response at 80°C is perfectly linear (see Figure 7) as expected from the Clausius-Clapeyron temperature dependence of R-phase transformation stresses. Figure 7 Comparison of tensile responses at a fixed stress amplitude below the onset of MT measured at 40°C showing nonlinear shape probably due to R-phase transformation and at 80°C showing linear response suggesting that only Austenite elasticity takes place. Figure 7 Comparison of tensile responses at a fixed stress amplitude below the onset of MT measured at 40°C showing nonlinear shape probably due to R-phase transformation and at 80°C showing linear response suggesting that only Austenite elasticity takes place. 1 2 3 Figure 5 Tensile responses of concave shaped samples at 40°C at three particular displacement amplitudes – 1 below onset of MT, 2 at the onset of MT, 3 beyond the onset of MT 1 2 3 High cycle fatigue R-phase regime at moderate displacements. 6 Fatigue testing Fatigue tests were performed in a force-controlled regime at frequency of 25 Hz and stress ratio = equal to 0.1. A fatigue runout test was defined at 1.5x106 cycles. All failures occurred in mid- cross-section of the concave zone. Only one test was performed for each applied amplitude, so no statistical measures could be evaluated. Nevertheless, the measured S-N curve shown in Figure 8b shows rather continuous decrease in number of cycles before fracture with increasing applied stress amplitude. The fatigue limit of the material () was identified at 397 MPa using the staircase method [15]. Beyond this limit the fatigue life- time drops to 105 cycles and rather continuously decreases down to few 104 cycles. The fatigue life-time drops down again to few thousands of cycles at stress amplitude of 650 MPa being above the onset of MT ( = 607 ). To associate deformation processes with different stages of fatigue performance, stress- displacement responses recorded in 100th cycle of three particular fatigue tests are plotted in Figure 8a. Runout tests show nonlinear reversible responses suggesting that the austenite-R-phase transformation takes place. Similarly, tests at stress amplitudes ranging from 450 MPa to 500 MPa with reduced fatigue life-time by two orders of magnitude feature nonlinear reversible responses, however, in this R-phase fatigue regime, samples underwent larger displacements. Finally, the test at stress amplitude of 650 MPa with reduced fatigue life- time by 3 orders of magnitude, shows nonlinear hysteretic response suggesting that MT operates. Therefore we may classify the fatigue performance of the wire into 3 regimes: Figure 5 Tensile responses of concave shaped samples at 40°C at three particular displacement amplitudes – 1 below onset of MT, 2 at the onset of MT, 3 beyond the onset of MT. Figure 5 Tensile responses of concave shaped samples at 40°C at three particular displacement amplitudes – 1 below onset of MT, 2 at the onset of MT, 3 beyond the onset of MT. Figure 6 Detail view of the tensile response of concave shaped samples at 40°C, at an amplitude below the onset of MT. The tensile response feature non-linear non-hysteretic shape suggesting that the R-phase is involved in deformation High cycle fatigue R-phase regime at moderate displacements. 03011-p.4 ESOMAT 2015 during a low cycle MT fatigue regime test at stress amplitude of 650 MPa and temperature of 40°C. 6 Fatigue testing Low cycle fatigue R-phase regime at larger displacements featuring amplitude dependent life-time ranging from 105 to 104 cycles To investigate the R-phase transformation in fatigue of NiTi wires, we compared fatigue performance at a fixed stress amplitude of 450 MPa and different temperatures: 40°C, 80°C, 150°C. Although the stress amplitudes were equal, tests at 80°C and 150°C ran out at maximum number of cycles, whereas test at 40°C finished by a failure after just ~30,000 cycles. When comparing the responses at respective temperatures (see Figure 10) we notice that the response at the temperature of 40°C is nonlinear suggesting that austenite-R-phase is involved as operating deformation mechanism i.e. low cycle fatigue R-phase regime is in place. At two higher temperatures of 80°C and 150°C, the sample responses feature rather linear slopes suggesting that austenite elasticity takes place or the moderate displacement high cycle fatigue R-phase regime is entered at 80°C as its slope is lower than at 150°C i.e. high cycle fatigue Austenite regime (150°C) and R-phase regime with moderate amplitude (80°C) take place. Low cycle fatigue MT regime with life-times in the range of 103 cycles This classification is based on rather low number of fatigue tests, therefore, further tests are needed to confirm this conclusion. this conclusion. Figure 8 a- Three typical tensile responses within the stress amplitude range covered during fatigue, the responses were measured at the 100th b- S-N curve resulting from pull-pull fatigue tests performed on concave shaped samples at 40°C. a b a b Figure 10 Fatigue tensile responses in cycle 100 of three fatigue tests performed at a fixed stress amplitude of 450 MPa and three different temperatures. Figure 8 a- Three typical tensile responses within the stress amplitude range covered during fatigue, the responses were measured at the 100th b- S-N curve resulting from pull-pull fatigue tests performed on concave shaped samples at 40°C. In accordance with this classification, we propose a “Fatigue failure advanced warning” in terms of pronounced hysteresis appearing on stress-displacement curve before failure. In case of R-phase fatigue regime, hysteresis appears and continuously growths within approximately 100 cycles before failure occurs. In case of MT fatigue regime showing inherent hysteresis from the first cycle, hysteresis starts increasing within approximately 100 cycles before failure occurs as illustrated in Figure 9. 6 Fatigue testing Note that the hysteresis may change continuously even before the final 100 cycles, however, the change become noticeable right before the failure. Figure 10 Fatigue tensile responses in cycle 100 of three fatigue tests performed at a fixed stress amplitude of 450 MPa and three different temperatures. 7 Conclusions We have investigated tensile pull-pull fatigue of superelastic NiTi wires in view of the involved deformation/transformation processes. Fatigue-life approach and concave shaped samples were purposely machined from NiTi wires in order to confine the transformation processes into a small and well localized material volume. failure. In-situ infrared imaging during tensile loading of concave shaped samples confirmed the localization of deformation/transformation processes into the centre of the sample and linked the onset of the plateau in stress- displacement evolution with the onset of martensitic transformation. A limited number of fatigue tests were performed on concave shaped samples showing rather regular decrease of material life-time with increasing stress-amplitude. The obtained S-N curve and stress-displacement Figure 9 Continuous enlargement of hysteresis that appears as advanced failure warning within 100 cycles before failure Figure 9 Continuous enlargement of hysteresis that appears as advanced failure warning within 100 cycles before failure 03011-p.5 MATEC Web of Conferences “Microstructure and structural fatigue of ultra-fine grained NiTi-stents,” Mater. Sci. Eng. A, vol. 503, no. 1–2, pp. 96–98, Mar. 2009. responses allowed for identification of three distinct fatigue regimes as follows: High cycle fatigue R-phase regime at moderate displacements. A life-time larger than runout set at 1.5x106 cycles. 10. 10. M. Frotscher, K. Neuking, R. Böckmann, K.-D. Wolff, and G. Eggeler, “In situ scanning electron microscopic study of structural fatigue of struts, the characteristic elementary building units of medical stents,” Mater. Sci. Eng. A, vol. 481–482, pp. 160–165, May 2008. Low cycle fatigue R-phase regime at larger displacements featuring amplitude dependent life time ranging from 105 to 104 cycles Low cycle fatigue MT regime with life-times in the range of 103 cycles 11. S. W. Robertson, Stankiewicz, Gong, and R. O. Ritchie, “Cyclic Fatigue of Nitinol,” Proc. Int. Conf. Shape Mem. Superelastic Technol. SMST- 2003, 2006. The activity of the austenite-R-phase transformation was confirmed by fatigue tests at a constant stress- amplitude and different temperatures. Fatigue life-time exceeding runout was confirmed for higher temperature tests (80°C and 150°C) where the R-phase transformation did not occur whereas the wire broke at much lower number of cycles till failure at lower temperature where the R-phase transformation did occur. 12. C. Maletta, E. Sgambitterra, F. Furgiuele, R. Casati, and a. Tuissi, “Fatigue properties of a pseudoelastic NiTi alloy: Strain ratcheting and hysteresis under cyclic tensile loading,” Int. J. Fatigue, vol. 66, pp. 78–85, 2014. 13. C. M. 7 Conclusions Otsuka, K; Wayman, Shape Memory Materials. Cambridge: Cambridge University Press, 1998. Finally, a “Fatigue failure advanced warning” in terms of pronounced hysteresis appearing on stress- displacement curve in final 100 cycles before the failure is proposed. Finally, a “Fatigue failure advanced warning” in terms of pronounced hysteresis appearing on stress- displacement curve in final 100 cycles before the failure is proposed. 14. G. Eggeler, E. Hornbogen, a Yawny, a Heckmann, and M. Wagner, “Structural and functional fatigue of NiTi shape memory alloys,” Mater. Sci. Eng. A, vol. 378, no. 1–2, pp. 24–33, Jul. 2004. pp 15. W. Dixon and A. Mood, “A method for obtaining and analyzing sensitivity data,” J. Am. Stat. …, vol. 43, no. 241, pp. 109–126, 1948. References 1. S. Subresh, Fatigue of Materials. 2004. 2. S. Eucken and T. Duerig, “The effects of pseudoelastic prestraining on the tensile behaviour and two-way shape memory effect in aged NiTi,” Acta Metall., vol. 37, no. 8, pp. 2245–2252, 1989. pp 3. P. Sedmák, P. Šittner, J. Pilch, and C. Curfs, “Instability of cyclic superelastic deformation of NiTi investigated by synchrotron X-ray diffraction,” Acta Mater., vol. 94, pp. 257–270, 2015. 4. K. . Melton and O. Mercier, “Fatigue of NITI thermoelastic martensites,” Acta Metall., vol. 27, no. 1, pp. 137–144, 1979. 5. P. Šittner, M. Landa, P. Lukáš, and V. Novák, “R- phase transformation phenomena in thermomechanically loaded NiTi polycrystals,” Mech. Mater., vol. 38, no. 5–6, pp. 475–492, 2006. 6. S. Miyazaki, K. Mizukoshi, T. Ueki, T. Sakuma, and Y. Liu, “Fatigue life of Ti–50 at.% Ni and Ti– 40Ni–10Cu (at.%) shape memory alloy wires,” Mater. Sci. Eng. A, vol. 273–275, pp. 658–663, Dec. 1999. 7. M. F.-X. Wagner and G. Eggeler, “Stress and strain states in a pseudoelastic wire subjected to bending rotation,” Mech. Mater., vol. 38, no. 11, pp. 1012–1025, Nov. 2006. pp 8. A. R. Pelton, J. Fino-Decker, L. Vien, C. Bonsignore, P. Saffari, M. Launey, and M. R. Mitchell, “Rotary-bending fatigue characteristics of medical-grade Nitinol wire.,” J. Mech. Behav. Biomed. Mater., vol. 27, pp. 19–32, Nov. 2013. 9. M. Frotscher, P. Nörtershäuser, C. Somsen, K. Neuking, R. Böckmann, and G. Eggeler, 03011-p.6
https://openalex.org/W1566543514	https://www.intechopen.com/citation-pdf-url/34675	English	null	A framework for VoIP testability and functionality extension with interactive content delivery	InTech eBooks	2,012	cc-by	10,603	A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery Janez Stergar, Janez Klanjšek and Sibila Vadlja University of Maribor, Faculty of Electrical Engineering and Computer Science Slovenia Janez Stergar, Janez Klanjšek and Sibila Vadlja University of Maribor, Faculty of Electrical Engineering and Computer Science Slovenia Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 12.2% 191,000 210M TOP 1% 154 7,200 8 www.intechopen.com 1. Introduction Also a router is used as a gateway with IP telephony services operation system to simulate the WAN network environment. The framework is designed for IP phone applications development and testing. Therefore a discussion will be included where critical implementation parameters in a real-time environment are evaluated and tested (jitter, frame dropping, priority queuing, etc.). The extra value of IP telephony is the support for applications extending the voice capability of IP phones with interactive content delivery. These are the so called IP phone services. These enable representation of multimedia content from a server locally or anywhere in the Internet cloud. Applications are delivered from a server and rendered on IP phones using HTTP protocol. The deployment of a typical application regarding the XML capabilities will be discussed and the workflow of an application realization presented. Also limits for image streaming will be evaluated. 2. IP Telephony components When deploying the IP Telephony solutions of almost any prospering IPT solutions provider on the market the key areas to emphasize are (Kaza & Asadullah, 2005): - Network Infrastructure, - Call Processing, - Call Manager Directory Services, - IP Telephony endpoints, - Call Admission Control, - Legacy Fax Messages, - Media Resources and - Applications. 1. Introduction The telephony as we know it has changed when VoIP emerged in 2004. In the third quarter of 2000 the second generation of IP enabled phones came to the market with full QoS capability. Performance was quadrupled and the basic voice functionality was extended with the addition of a large screen with HTTP/XML driven capability. That was a major market breakthrough extending VoIP phones with the possibilities of interactive content delivery. Classic telephony systems are being rapidly replaced by IP Telephony (IPT) in corporate and home environments. Especially IPT has gained wide acceptance in the industry offering new ways to exchange information with rich media communication capabilities. Therefore stationary telephony is migrating into the internet. Voice over packet switching networks can significantly reduce the per-minute cost, resulting in reduced long distance costs. Therefore many dial-around-calling schemes already relay on VoIP backbones to transfer voice. There is even more potential extending VoIP with interactive content using tailored applications for the end user. Successful deployment of any new technology solution requires thorough understanding of the function of various components involved and the interaction among them. The architects and engineers who are tasked with implementing the IPT solution must ensure that the proposed architecture meets all the requirements and is also scalable in the future (Kaza & Asadullah, 2005). Therefore an IP Telephony framework will be introduced with the goal to demonstrate typical limitations and an IP Telephony delivery platform for interactive content applications. The VoIP framework can deliver IP Telephony services and internet access at the same time based on one core device. We implemented VoIP technology with content delivery support using a common unified communication device in combination with typical networking devices such as a router and switch. The platform is capable of carrying voice data and multimedia traffic with QoS management. The system consists of diverse IP phones with dissimilar capabilities and a PoE entry access switch (delivering power for the www.intechopen.com 168 New Technologies – Trends, Innovations and Research IP phones). Also a router is used as a gateway with IP telephony services operation system to simulate the WAN network environment. The framework is designed for IP phone applications development and testing. Therefore a discussion will be included where critical implementation parameters in a real-time environment are evaluated and tested (jitter, frame dropping, priority queuing, etc.). IP phones). 2.1 Network infrastructure Network infrastructure plays a key role in building multiservice networks e.g. Cisco AVVID. Integration of data and voice traffic puts strong requirements on packet loss, delay and jitter (variable delay of VoIP packets). LAN/WAN components with QoS mechanisms support are indispensable when designing IPT networks as well faster convergence in case of network failures to avoid destructive delay, jitter and frame dropping. Voice traffic in addition to the existing data traffic increases the bandwidth a critical issue because of the high-speed LAN switching technologies availability. However, when transporting the voice traffic across the WANs, one has to ensure that adequate bandwidth is available to support the additional bandwidth required to transport voice calls. If the WAN links do not have adequate bandwidth their bandwidth has to be increased to support the additional voice traffic. After the bandwidth is assured, QoS mechanisms have to be properly configured on operating systems of LAN/WAN networking devices to prioritize voice traffic with adequately bandwidth allocation. Network infrastructure plays a key role in building multiservice networks e.g. Cisco AVVID (Architecture for Voice, Video and Integrated Data) is the foundation of converged enterprise communication networks. www.intechopen.com 169 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery 2.4 IP Telephony endpoints In an IPT network, endpoints are the devices that accept or initiate a VoIP session. Typical endpoints that are used are: IP Phones, Soft Phones e.g. Cisco IP Communicator, Wireless IP Phones, Voice gateways which connect the IPT network to the PSTN or a PBX, Survivable Remote Site Telephony (SRST) which provides the fallback support for the IP phones that are connected behind a router running a suitable operating system software version that supports SRST and in a specific Cisco environment the so called Call Manager Express - CME which delivers key system functionality for small and midsize branch offices using Cisco IP stationary, wireless and software phones. 2.2 Call processing The core of an IP Telephony solution is usually a software call manager. For Cisco devices this software component is called Cisco CallManager. The specially designed IOS software with embedded CallManager handles all the call-processing requests received from various clients in the IP Telephony network. For the Cisco IP Telephony AVVID solution CallManager software runs on a compatible router or the Microsoft Windows Server operating systems. Call manager is installed on the Cisco Media Convergence Server (MCS) for medium to large-scale networks but can be also operated from a router (CallManager Express) or a specific device for smaller unified networks e.g. the Cisco UC 500. The selection of the hardware platforms depends on the size of the network in which IP Telephony is going to be deployed, including its high-availability and performance requirements (typically 300-7500 devices per dedicated server for medium to large-scale systems). For large-scale systems a clusterization of servers is inevitable. Call Manager servers are grouped to form clusters to support more devices (IP phones, gateways, etc.). For the current Cisco CallManager version, a Call Manager cluster can have up to eight Call Manager servers running the call manager service. 2.3 Call Manager directory services Call Manager stores system and device configurations in a Microsoft SQL database. The application scripts and the subsequent information are stored in a Lightweight Directory Access Protocol (LDAP) compliant directory the so-called DC Directory (DCD): - User authentication and authorization - Extension Mobility profiles - Personal Assistant profiles - Internationalization information - Personal Address Book (PAB) - Spoken name - Call Forward All information The DCD process replicates the information among the members of the cluster. This process is similar to Microsoft SQL replication. 2.8 Applications There is a wide range of applications that can be deployed in an IPT network. These applications are optional, and their deployment adds more features and capabilities to the overall IPT network. Design and deployment of the applications, such as Customer Response Solution and IP Phone services is a very important topic for new converged IPT networks. Cisco offers many proprietary services e.g. IVR, IPCC Express, Cisco Unity, Cisco Emergency Responder, Cisco Conference Connection and so on. In the following we will try to emphasize the non-proprietary solutions and therefore focus on IP Phone Services. 2.7 Media resources The function of media resource devices is to mix the multiple streams into a single output stream, converting the data stream from one compression type to another, and so forth. The media resources can be hardware or software. The limitation of software media resources is that they can't combine the streams that use different compression techniques. Hardware media resources have the same features as software media resources with an additional advantage of mixing the streams that use different compression types. Characteristic media resources are conferencing, transcoding, and MoH (Music on Hold) which provides music or announcements when the users are put on hold. 2.6 Legacy FAX messages There is still a large portion of long-distance minutes based on legacy fax traffic. One of the most important functionalities in the transition to converged networks is therefore support for fax communications. As network implementations increasingly provide for e-mail attachments and web-downloadable documents, fax communication nonetheless is still a significant method of immediate document delivery worldwide. Three methods to transmit legacy fax traffic across the IP network are common: the Pass-Through mode, where the gateways do not distinguish a fax call from a voice call, the Cisco proprietary Fax Relay mode, where the gateways terminate the T.30 fax signalling and the plain old T.38 Fax Relay mode. 2.5 Call admission control In VoIP networks, Call Admission Control (CAC) does the bandwidth management. CAC ensures that enough bandwidth is available before granting permission to a gateway for www.intechopen.com 170 New Technologies – Trends, Innovations and Research placing the call across the IP WAN. When deploying IPT solutions with multiple locations, there are two choices for implementing the CAC: Call Manager locations-based CAC and Gatekeeper CAC. Call Manager locations-based CAC is one mechanism to limit the calls sent across an IP WAN in a single Call Manager cluster deployment, whereas the Gatekeeper CAC provides call admission and call routing between the Call Manager clusters in distributed call processing deployments. 3. IP Telephony deployment architectures By using the Call Manager software it is possible to bypass the plain old PBX and replace it with IP Telephony over a next generation converged network. The Call Manager application software provides call-control functionality and, when used in conjunction with IP hardware/software phones, can provide PBX functionality in a distributed and scalable manner. The deployment solution models of Cisco IPT can be categorized into one of the following architectures (Kaza & Asadullah, 2005): www.intechopen.com 171 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery - Single-site deployment g y Centralized call processing with remote branc p g - Distributed call-processing deployment - Clustering over the IP WAN Selection of the deployment model depends on implementation requirements, such as the size of the network, features, and availability of the WAN bandwidth. Fig. 1. Single - Site IPT Model Fig. 1. Single - Site IPT Model 3.1 Single-site model In this deployment model, Call Manager applications such as voice mail, IP-IVR, AutoAttendant (AA), Transcoding, and conferencing resources are located at the same physical location (Fig. 1). All the IP phones are located within this single site. The PSTN is used to route the off-net calls. 3.2 Centralized call processing model In this deployment model all the call processing is done at the central site. This is suitable for implementations in which the majority of the workforce is concentrated at a single site and small numbers of employees work at the remote branches (Fig. 2). Fig. 2. Centralized Call-Processing Model Fig. 2. Centralized Call-Processing Model www.intechopen.com 172 New Technologies – Trends, Innovations and Research At each remote branch, SRST (Survivable Remote Site Telephony) routers ensure that call processing is preserved in case of WAN link failure. The voice traffic travels via the IP WAN and falls back to the PSTN if not enough bandwidth is available across the WAN link, by using the Automated Alternate Routing (AAR) feature available in the Call Manager software application. This deployment model is cost effective and provides many benefits, such as a unified dial plan, less administrative overhead, and potential savings on communications costs as the remote branches calls use the IP WAN as first choice. The only limitation is that the remote sites will have limited features available in the case of a WAN failure. 3.3 Distributed call processing model In the distributed call-processing deployment architecture, Call Manager software and applications are located at each site. Device weights and dial plan weight calculations determine the number of IP phones supported at each site. In the figure (Fig. 3) a distributed call-processing model is depicted in which headquarters and branch Y IP phones are served by separate Call Manager clusters and branch X is served by the Cisco CallManager Express (CME) feature that is enabled on the router. CME solution is suitable for a small branch. 3.4 Large scale architecture – Clustering over the IP WAN The Cisco IPT solution allows organizations to build disaster recovery sites by separating the single Call Manager cluster across the WAN. Call Manager servers in a cluster update the configuration information via the Microsoft SQL replication process. To ensure successful SQL replication and propagation of other critical information in real time, the round-trip time (RTT) between any Call Manager servers in the cluster should not exceed 40 ms. Many other requirements have to be satisfied before selecting this deployment model. Fig. 3. Distributed Call Processing Deployment Fig. 3. Distributed Call Processing Deployment www.intechopen.com 173 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery When using the clustering over the IP WAN deployment model, voice gateways, media resources, and voice mail have to be deployed locally at each site. Essential services such as DHCP, DNS, and TFTP that are critical for the functioning of IP phones and other IPT endpoints also require local implementation. This configuration avoids dependency on a single site for crucial resources. Clustering over the WAN can support two types of deployments: - Local failover deployment model where each site contains a primary Call Manager subscriber and at least one backup subscriber. All the servers are part of the same Call Manager cluster. - Local failover deployment model where each site contains a primary Call Manager subscriber and at least one backup subscriber. All the servers are part of the same Call Manager cluster. - Remote failover deployment model where each site contains at least one primary Call Manager subscriber and might or might not have a backup subscriber. Branch X and branch Y as depicted on the previous models have only primary subscribers, and the backup subscriber is not located in each site. - Remote failover deployment model where each site contains at least one primary Call Manager subscriber and might or might not have a backup subscriber. Branch X and branch Y as depicted on the previous models have only primary subscribers, and the backup subscriber is not located in each site. 4. The framework for VoIP testability and functionality extension In this chapter we will introduce the educationally tailored IP Telephony platform based on the Centralized Call Processing Model (Fig. 4). The IPT platform is intended as a test framework for QoS parameters evaluation, configuration, critical examination of the VoIP network parameters as well as delivery and multimedia media applications platform for the Cisco IP Phones. 1 2 ABC 3 DEF 4 5 JKL 6 MNO GHI 7 8 TUV 9 WXYZ PQRS * 0 OPER # 7 960 CISCO IP PHONE i me ssages directories settings services 1 2 ABC 3 DEF 4 5 JKL 6 MNO GHI 7 8 TUV 9 WXYZ PQRS * 0 OPER # 7960 CISCO IP PHONE i messages di rectories settings services Fig. 4. The VoIP test framework The introduced VoIP framework represents a development environment for IP phone applications using XML to add the extra value with additional interactive content on phones with touch screen display option. The architecture model is applicable in small and mid- sized business environments. It supports up to 100 IP phones and includes all the Fig. 4. The VoIP test framework Fig. 4. The VoIP test framework The introduced VoIP framework represents a development environment for IP phone applications using XML to add the extra value with additional interactive content on phones with touch screen display option. The architecture model is applicable in small and mid- sized business environments. It supports up to 100 IP phones and includes all the www.intechopen.com 174 New Technologies – Trends, Innovations and Research functionalities that small businesses need; voicemail, auto-attendant, e-mail integration, and call attainability functions. Voice mail provides a voice messaging system for cases when called persons are not available, auto-attendant provides voice narrated guide system and greeting if required. Call attainability functions support the user call seeking service (Cisco Systems, 2011). The core of the system is a unified communication (UC) appliance and a VoIP services enabled router with a customized operating system (VoIP software extensions with CME). The UC appliance and router support the convergence of voice communication, data communication, mobile phone support and video (multimedia) support. Additionally the UC appliance offers a wireless module supporting connection of wireless IP phones. It runs special software for VoIP control, the so called CallManager. The call manager handles processes and routes the incoming calls comparable to a PBX system. It is actually an IP-PBX system. 4. The framework for VoIP testability and functionality extension The UC appliance functions as a router and a switch for all connected devices e.g. IP phones, computers. The Call manager software offers centralized/distributed control of the calls and routes the calls to the intended users (Cisco Systems, 2011). In the presented platform a switch is used to connect and power IP phones in combination with a router. The installed Call manager software works as an IP-PBX and simulates two separate VoIP networks connected through a WAN via the H.323 trunk. The H.323 trunk encapsulates the calls appropriately. Actually our platform is build of two separate local VoIP networks. The ports that are used are all FastEthernet 100Mbps ports. The IP phones represent the end devices of the presented network platform. These are entry to high-end IP phones intended for home and business environments. Some models have a HiRes color display and support up to eight call lines which can be configured with different numbers and speed dial functions. Supported are traditionally soft keys which are programmable with functionalities change based on the configuration. Templates can be used to apply the same configuration to multiple phones. All the configurations are centralized. The Call manager software is used for central point phone management with complete control over the IP phones in the system. The IP phones load the configuration settings using the Call manager software integrated trivial file transfer protocol (TFTP) server (Cisco Press, 2006). Similar UC management software as on the router is implemented on the UC appliance. It has a graphical user interface and supports the control and creation of user IP telephony system. Users can be incorporated into the voicemail system and the creation of voice messages mailbox and e-mail notification of messages are also supported for every individual user mailbox. Thus users can access voice messages from anywhere (Au et al., 2005, Cisco Press, 2011c). The system is configured on network devices in console line in text mode with exception of the entry level access switch which is managed through a GUI. The call manager software takes care of network operation in VoIP network while unity express software takes care of the users that are connected to the system. 4.1 VoIP communication and protocols All main VoIP protocols can be used and analyzed in our system. The data examination access to forwarding connections is implemented with port mappings. The network is flexible so it can support various Session initiation protocol (SIP) IP phones from different www.intechopen.com 175 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery vendors as well as soft phones that run on computers. It can offer a SIP trunk or a H.323 trunk for WAN connection to a telephony service provider (Cisco Press, 2007; Hatting, S. et al., 2010; H.323 Implementation, 2011). Students can observe operations in VoIP network of SIP, H323 and other proprietary protocols (e.g. SKINNY protocol) with Wireshark network analyzer (Wireshark, 2011). For voice transfer and direct communication between two users Real time protocol (RTP) protocol is used. IP phones and attached computers use Virtual local area networks (VLAN) and Dynamic host configuration protocol (DHCP). VLANs are used to provide basic security and Quality of service (QoS). Actually data traffic is separated from voice traffic applying two VLANs, thus voice traffic does not interfere with data traffic. Interference of the two kinds of traffic could cause degradation of voice communication service. With basic security implementation we mean that any computer in the network cannot see the IP phones on OSI L2 and compromise its identity. DHCP is used to automatically configure network parameters such as IP address, network masks, and DNS address for both the IP phones and computers connected to the network. That optimizes time necessary for adding new users and reduces administrators work - there is no need to manually enter device configuration. Every configuration for any device in the network can be done from a single point. That is one of the tasks performed by the Call Manager software which runs on UC appliance (Deel, D. & Nelson, M., 2004; Cisco Resources, 2011). There is a trunk connection between the UC appliance and the router. A trunk is used to carry multiple calls or data transfers over a single Ethernet link. The educational platform supports SIP and H.323 trunks. We used a H.323 trunk. Using this kind of connection the UC appliance and router act as two dial peers. The right parameters have to be configured in the dial peer list to correctly route the incoming and outgoing calls. 4.1 VoIP communication and protocols The dial peer list presents similar information to the Call Manager software devices as a routing table does to forwarding router. A dial plan is also implemented. It defines mapping of local phone numbers into global phone numbers and translation of local phone numbers into global phone numbers for and outgoing call and vice versa for an incoming call (similar to network address translation). The system can also be designed that the UC appliance has only one global telephone number which is used for registration at the telephony service provider. The UC appliance then routes calls appropriately inside the local IP telephony network (H.323 Implementation). 5.1 Basic concepts Here we present the introduction to building blocks for development of applications on IP phones. The HTTP integrated client on the IP phone enables the capability to deliver content on the display of an IP phone. The content is gathered from a Web server with the HTTP protocol. It has to be emphasized that applications are not part of a call service but actually are data applications separated from VoIP communication. All they have in common with VoIP is the shared IP network infrastructure (Fig. 4). Applications are displayed on the IP phone on user demand. They are loaded from the Web servers on which they are resident. On the Web server applications are developed, executed and changed. The interface for communication between the Web server and the phone is the TCP/IP stack. Applications use TCP for reliable transfer which is invoked by the HTTP and the proprietary SKINNY protocol. The SKINNY protocol relays on top of the TCP stack intended for signalization and control of the IP phone with the IP phone central unit on CME (Call Manager Express). The connection with the central unit (CME, IP-PBX) is necessary because this is how the IP phone acquires its IP address. The logical address is necessary for the connectivity between the IP phone and the server. The SKINNY protocol uses the IP phone firmware for data link layer communication with the central unit. The firmware is also responsible for the initiation of the HTTP request calling the application on the server. Basically the data travels between the IP phone, IP PBX (which serves as a proxy) and the Web server (Deel, D. & Nelson, M., 2004). Let us examine the data flow more in detail: The firmware on the IP phone initiates a HTTP request to the server (HTTP GET message). The request includes a URL (Uniform Resource Locator) containing the address of the server and a file, script or program ID resident on that server. Data that is requested is embedded in an XML object and sent to the Web server. The Web server responds and also embeds data in XML objects. Then the data is sent back to the IP phone with the HTTP protocol. The IP phone understands and is capable of parsing only supported XML objects which are intended for displaying the dynamic or static content (Deel, D. 5.1 Basic concepts & Nelson, M., 2004, Cisco Developer Community – Resources, 2011). 5. IP Phone services application development Applications on stationery IP phones are an added value to IP telephony especially in the business environment. A phone actually transforms to a tool displaying business information, multimedia applications or entertainment applications that serve the user needs. IP network enables that functionality. Applications also called IP phone services are interactive services which relay on the IP phones keyboard or touch screen capability. First we have to introduce how the IP phone actually invokes a service. The phone is drawn into a service by virtue of a URL attached to a button on the phone the so called services buttons. If the phone is equipped with a touch screen, the button functionalities are taken over with hot spot fields on the screen of the IP phone. The button assignment is done in one of the Cisco CallManager Administration screens. The services button by default is assigned www.intechopen.com 176 New Technologies – Trends, Innovations and Research a URL that points to a Web page on the CallManager server (GetServicesMenu.asp). It simply presents the user with a menu of services that have been configured for that particular phone. Each of the different menu items is connected to another URL. It is important to remember that the Services Menu usually seen on the local CallManager system is only one of many possible ways to invoke services. The Cisco IP Phones can have a URL attached to the directories button and the messages button. Most system administrators will confine the use of those buttons to specialized services. 5.1.2 Web server usage options Application can also communicate with distributed Web services on the internet from the local Web server. A Web server can request content from the server on the internet. That server responds with the requested data. The Web server then processes the received information and sends it in XML form to the IP phones. Examples of such applications are stock market information, weather information and even Google maps (Cisco Developer Community – Resources, 2011). The Web server is the core unit of applications development but also involves many distributed processes. These processes can be database queries, other server queries or even a connection to other users and multimedia devices – the so called backend processing on the server. Only the simplest applications communicate solely with the development Web server (Deel, D. & Nelson, M., 2004). 5.2 The IP phone and application server interaction There are three major topics to emphasize in communications between the server and the IP phone: The HTTP Protocol, the customized XML language and the applications on the IP PBX. 5.1.1 Choosing a Web server and the programming language There is an open choice of the Web server and Web programming languages. IP phone understands only XML and the applications are processed on the server. This is why we need to select a Web server and a compatible programming language. If we decide for a Microsoft IIS then the optimal programming languages to use are C# and ASP. If we decided for a JAVA server, the JAVA language is the most suitable. If we decided for the Apache, then scripting languages e.g. PHP, Perl, Javascript are the advised programming tools (Deel, D. & Nelson, M., 2004). www.intechopen.com 177 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery For the introduced test-bed we decided to use the Apache Web server with the PHP scripting language. PHP enables a programmer to implement much work with small amount of code making the writing of applications faster. In addition with implementation of MySQL database, one has the capability of saving data from IP phones into a database or displaying data from the database on the IP phones (Deel, D. & Nelson, M., 2004, Gilmore, W. J., 2010). That enables far broader spectra of applications that can be designed. Apache is the most popular Web server and it’s also open source. It represents approximately 60% of all the active Web servers implemented on the internet (Apache Usage Statistics, 2011). 5.2.1 The HTTP protocol IP phones use a HTTP client, which communicates with the HTTP server. Nevertheless the IP phone does not operate the same as a Web browser, because it is not capable of processing demanding complex Web pages built with HTML. This is also the reason that it does not understand the HTML language. IP phone is limited because of its lack of memory and processor resources which is common for all embedded systems. The IP phone also has an embedded HTTP server for sending information about the configuration, firmware, name and status of the device. For more advanced functions it uses CGI, which enables external program to change the configuration on the IP phone. Both client and the server embedded in the IP phone use HTTP version 1.1 for communication with other entities in the network (Cisco Systems, 2011a). The communication between the IP phone and the server is accomplished with HTTP messages. HTTP requests are sent by the phone. The request includes a header, a method, a body of request and status messages about the capabilities of the phone. HTTP response is sent back to the phone with similar content messages. The IP phone supports only a few from the huge variety of HTTP headers because it is not capable of processing them all. It www.intechopen.com 178 New Technologies – Trends, Innovations and Research would be too difficult to implement that variety into the embedded system. IP phone uses solely the HTTP GET method. This method has an URI identifier, which is a path to the server folder containing the data, a process which returns static or dynamic content as depicted in Fig. 5. Fig. 5. HTTP message exchange between the server and IP phone Fig. 5. HTTP message exchange between the server and IP phone Fig. 5. HTTP message exchange between the server and IP phone We also have to mention the headers that the IP phone can read: We also have to mention the headers that the IP phone can read: - Content-type: MIME type of data, so the phone knows what to process and parse. This has to be set to Text/Xml so the phone starts to parse XML objects and correctly displays them. p y - Refresh: Page refresh in seconds. Enables the phone to refresh the application. Manually or automatically. - Expire: Expiration of the requested URL. 5.2.1 The HTTP protocol With this header one determines how long certain content is available. IP phone also has a history stack, which can store up to ten pages. With this header the history can also be erased. - Location: Intended for redirection. - Location: Intended for redirection. - Set-Cookie: Saved information about the session. When the phone uses the application again, the Web server can recognize that particular IP phone. - Accept: Informs the Web server about the capabilities of the IP phone, its language and charset. One can define or read header parameters in the Web programming language code (Deel, D. & N l M 2004) One can define or read header parameters in the Web programming language code (Deel, D. & Nelson, M., 2004). www.intechopen.com 179 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery 5.2.2 The IP phone customized XML language XML language is used to display the content on IP phones. It is a hierarchical language, it has one main element and many sub elements with different attributes. An XML object is predefined and IP phone proprietary. Inside of the XML object there is dynamic content which is processed on the server and sent to the phone via the IP network. The Web server does all the operational work related to the application. The contents of the application is packed into XML objects and sent to the phone which parses and displays the contents. One can only use predefined proprietary XML objects, tags and attributes. Those predefined elements are the only elements the IP phone can parse. They are defined in the XML schema, which is integrated with the phone through its firmware. This schema is defined by the vendor (Cisco) and cannot be changed by the user. The schema is being changed and updated regularly by the vendor. New functionalities are added and new ways of checking the XML with phone firmware updates are possible. Rules and a dictionary of behaviours and display of the objects are defined in XML schema. Therefore an overall knowledge of XML for the development of applications on Cisco IP phones is not necessary. Only the rules and Cisco custom XML objects knowledge is needed. XML objects are an envelope for the content of applications (Web based programming language). One is limited with the usage of objects with different phone models. Some models do not accept all the available objects; they have a limited scope of functionality (Cisco Systems, 2011a). The server must have the “text/xml” MIME extension enabled otherwise the IP phone will display just plain text. For the Web server files navigation URLs and URIs are used. The IP phone also has soft keys with which we normally navigate through application. They use proprietary XML tags to describe their functionality. With the properly designed soft keys we enhance the user experience. We can set the order, function and the text they show. Every Cisco XML object also has its own default soft key defined which can be arbitrary altered. Internal URI can be used on soft keys. Those URIs use various functions already built in the phone, for example Dial or Transfer Call. External URI is actually a URL for a Web server. Soft keys are defined inside Cisco XML objects. 5.2.2 The IP phone customized XML language All Cisco XML objects, elements, tags and attributes, their definition and usage, are described in references (Deel, D. & Nelson, 2004; Cisco Systems, 2011a). It is not our intention to describe them explicitly as that would exceed the topics of the article. 5.3 The service architecture As already mentioned Cisco IP phone service is not running on the IP phone locally despite it could seem so. The phone is actually reduced to an I/O device, being used to render screens of data and retrieve input from the user. The main processing usually takes place on a Web server where the applications reside. The location of such a server can be locally but can be also anywhere in the Internet. Most IP Phone services are centralized on a Web server, but generally involve many distributed processes. The “locally positioned” Web server receives the request from the IP phone and generally does some local parsing. Nevertheless it relies on additional components, programs, and processes to do the back- end work (such as database access, scripting, security, and interfacing with scripts and CGI). Only the simplest services do all their processing on just one server. 5.2.3 Enabling applications on the IP PBX To make the application available to the IP phones we have to configure the IP PBX correctly. IP-PBX serves as a proxy between the IP phone and the server and also sends all parameters to the phone. The phone cannot be directly configured. IP-PBX is managed with the proprietary Call manager software package. All one has to do is determine the path to the Web server where the application resides. URL services parameter has to be changed in the Call manager configuration. The Call manager then sends this parameter to the IP phone which then knows where to find the developed applications. IP-PBX is also important because IP phones and its users are registered to it. By registering they get an IP address and consecutively a connection to the IP network. With the determination of the URL one is not www.intechopen.com 180 New Technologies – Trends, Innovations and Research limited to the local network (the introduced network schema) but can choose any Web server on the internet that hosts appropriate applications. The only condition is that the IP- PBX is connected to the internet. That means that one can share the locally resident applications with any Cisco IP-PBX and Cisco IP phones on the internet. In the URL services parameter one can just write a single URL. Therefore the phone has limited access to that particular application. To solve this problem we created a menu with Cisco XML tag IPPhoneMenu where we defined many URLs that point to different applications on different locations. Therefore the user can choose between different applications (UC500.com, 2011; Cisco Systems, 2011). 5.4 Developing a phone service To develop IP phone services for a Cisco IP Telephony environment, one needs access to the proprietary CallManager application on the suitable networking device (usually a Cisco router or a unified communication device). With XML over HTTP, one can provide targeted functionality designed specifically for the device, while still keeping with the desire to maintain but expand the core phone functionality. XML provides the horsepower through Cisco IP Phone objects and tags to push the content. Additionally XML is user-readable, machine-readable, and low-cost, plus it expands the use and value of the phone without diminishing the phone functionality. Instead it enhances the phone to the extent that it can be considered munch more than a legacy phone. The key to the concept is XML, a user- and machine-readable way of structuring and encapsulating data; this simply provides the data to the phone to use with its existing menu structure. An application can send simple text to the IP phone, but when developing real services for the IP phone system, the odds are that XML objects have to be created that the phone understands. The extra flexibility and power we get with these objects make them almost mandatory. XML enables the design engineer to provide data to the native functionality of the device. With tailored sophisticated XML objects including the CiscoIPPhoneGraphicMenu and CiscoIPPhoneImage enhanced services in IP phones have been enabled. The main purpose of the introduced VoIP framework (Fig. 4) is the development of IP phone applications which add extra value and usability. A user does not need a desktop computer to browse the internet or read local intranet information. Even stock markets and www.intechopen.com 181 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery other dynamic changing data can be checked from an IP phone. Various applications can be implemented with the use of XML, PHP and JavaScript and can then be tested for responsiveness and usability. All applications are served from an http server which is directly or indirectly connected to the UC appliance. One has to configure the IP address and server folder location on the UC appliance and the applications are ready to be accessed on IP phones. There are various solutions for server implementation e.g. Microsoft IIS or Apache most frequently but also other free PHP servers. 5.4 Developing a phone service As already mentioned services on IP Phones are applications that run rather on a dedicated WEB server than on IP Phones and can be very useful in a home environment, branch offices, faculties, etc. The range of possible application domain use is very large, from straightforward applications e.g. unit conversion, messaging systems, opinion polls etc. to more entertaining applications as quizzes, games, bulletin boards, photo albums. Nevertheless severe applications for smoke detection or other alarms can be deployed as well as support and control of other applications. IP Phones have a HTTP compliant client that is used for services and directories functionality. The HTTP client enables the phone to use a simple standard mechanism for retrieving data and providing output to and from standard Web servers. Therefore the method for providing content to the phones is straightforward. All IP Phones services used in our platform use the client functionality for every request; the client is used to request URLs and provides the HTTP GET functionality. The main framework we have used for designing such applications is based on use of XML i.e. IP Phone XML objects and tags, scripting language PHP and SQL for data management. The proprietary IP Phone XML objects can be: - ProprietaryIPPhoneMenu, - ProprietaryIPPhoneText, - ProprietaryIPPhoneInput, - ProprietaryIPPhoneImage, - ProprietaryIPPhoneError, - ProprietaryIPPhoneResponse, - ProprietaryIPPhoneDirectory, - ProprietaryIPPhoneGraphicMenu, - ProprietaryIPPhoneIconMenu, - ProprietaryIPPhoneExecute, etc. The listed proprietary objects are providing functionality to soft keys. When pressed, the soft key invokes the associated action, with exceptions to ProprietaryIPPhoneResponse and ProprietaryIPPhone-Execute (UC500.com, 2011). We have to emphasize what each of these objects is capable of and what kind of functionality it can provide, e.g. how many characters can be in a single ANSI string, how many instances a menu can have or maximum of pixels in a bitmap. We decided to use PHP (Still, 2005; Holzner, 2007). PHP is necessary for more advanced applications development. Most of dynamic applications need some data managing system to deal with different data, for that we used an open source data base management system (Gilmore, 2010). Under the services button on the IP Phone we can install more than one application at the same time using menus. www.intechopen.com 182 New Technologies – Trends, Innovations and Research 6. Designing practical applications When designing applications special care must be taken to adapt the application to the right phone model. This can be done with decisive structures in the application code. Also the phone firmware can be helpful for adapting the content to the physical capabilities of the IP phone. For demonstrational purposes we will present a multimedia application of improvised video surveillance. Fig. 6. The test environment for video surveillance application Fig. 6. The test environment for video surveillance application www.intechopen.com 6.1 The video surveillance prototype The settings of the program enabled us to take the picture at certain time intervals. The picture is saved locally on the disk and is overwritten with every new snapshot. The format of the picture snapshot is JPEG (Lundie 2011) Then the Web server comes into play p p ( ) p y – it converts the picture and sends it to the phone. Server is an Apache type with PHP and MySQL (Apache Server, 2011). PHP script does the work on converting pictures to PNG format and correctly altering the picture to be displayed on the phone (display capabilities). Then its sends the picture in XML objects to the IP phone. MySQL database adds functionality to the users with the option of saving certain pictures for a later review (Gilmore, W. J., 2010, UC500.com, 2011). y – it converts the picture and sends it to the phone. Server is an Apache type with PHP and MySQL (Apache Server, 2011). PHP script does the work on converting pictures to PNG format and correctly altering the picture to be displayed on the phone (display capabilities). Then its sends the picture in XML objects to the IP phone. MySQL database adds functionality to the users with the option of saving certain pictures for a later review (Gilmore, W. J., 2010, UC500.com, 2011). The main goal of the server is converting the picture to the expected format. For that PHP language is used. PHP needs an extension for a successful conversion. This extension enables, that new objects are added to enhance the capabilities of writing applications. Therefore we used the ImageMagick software suite. The package enables creation, editing and conversion of bitmap pictures. It can read, write and convert in 100 different picture formats (Still, 2005). In PHP ImageMagick is used as an extension through ImageMagick API. We included the extension for the PHP and also for the ImageMagick software suite (Powers, 2008; Holzner, 2007). With the extension selection we have to be careful using the correct DLL library version for the used PHP installation version. With the appropriate implementation the conversion of the picture can be done with a few lines of code. That is the positive part of PHP language. It gives us an opportunity to write applications quickly and do a lot of processing with a few lines of code. 6.1 The video surveillance prototype The video surveillance is improvised because the IP phone is not capable of directly video streaming via display. We have to take snapshots from the Web camera and send them to the IP phone. Some models of IP phones have video call functionality built in but not for application development purposes (Cisco Systems, 2011b). Many distributed process are involved in the operation of the application. Here included are IP phone, IP-PBX, Web server, database and an IP/Web camera (Fig. 6). For the application we used a Cisco IP phone with color display, which has a 298x168 pix space for displaying the picture. This is the biggest area display available on Cisco IP phone models. We have to consider that the phone only displays pictures in PNG format. The picture can only have 12 bits of color depth which means we have to resize and correctly alter the picture to display it correctly. Because video cannot be streamed to the phone we have to take picture snapshots at certain intervals. For that purpose we need a designated program that will be able to snapshot the picture from the IP/Web camera, resize it and send in on request to the IP phone. We used two programs for that purpose. The picture can then be automatically or manually refreshed locally on the phone. www.intechopen.com 183 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery For acquiring snapshot pictures from the Web camera we used an open source program Fwink. The settings of the program enabled us to take the picture at certain time intervals. The picture is saved locally on the disk and is overwritten with every new snapshot. The format of the picture snapshot is JPEG (Lundie, 2011). Then the Web server comes into play – it converts the picture and sends it to the phone. Server is an Apache type with PHP and MySQL (Apache Server, 2011). PHP script does the work on converting pictures to PNG format and correctly altering the picture to be displayed on the phone (display capabilities). Then its sends the picture in XML objects to the IP phone. MySQL database adds functionality to the users with the option of saving certain pictures for a later review (Gilmore, W. J., 2010, UC500.com, 2011). For acquiring snapshot pictures from the Web camera we used an open source program Fwink. header("Content-type: text/xml"); We use a “Title” tag for the name of the picture that will be displayed on the top of the screen and “Prompt” for displaying information about the picture. With the tag “Location” we determined the position of the picture on the phone display. With the “URL” tag we specify where the application resides on the IP phone and where the picture is located. Inside of our CiscoIPPhoneImageFile XML object we also define softkeys. As already mentioned, softkeys are created for user application interaction; updates, application closing, etc. (UC500.com, 2011). <CiscoIPPhoneImageFile> <Title>LDIS-G215</Title> <Prompt>Laboratorij LDIS</Prompt> <LocationX>0</LocationX> <LocationY>0</LocationY> <URL>http://192.168.10.11/image.png</URL> <SoftKeyItem> <Name>UPDATE</Name> <URL>http://192.168.10.11/cam1.php</URL> <Position>2</Position> </SoftKeyItem> <SoftKeyItem> <Name>BACK</Name> <URL>SoftKey:Exit</URL> <Position>5</Position> </SoftKeyItem> </CiscoIPPhoneImageFile> <CiscoIPPhoneImageFile> <Title>LDIS-G215</Title> <Prompt>Laboratorij LDIS</Prompt> <LocationX>0</LocationX> <LocationY>0</LocationY> <URL>http://192.168.10.11/image.png</URL> <SoftKeyItem> <Name>UPDATE</Name> <URL>http://192.168.10.11/cam1.php</URL> <Position>2</Position> </SoftKeyItem> <SoftKeyItem> <Name>BACK</Name> <URL>SoftKey:Exit</URL> <Position>5</Position> </SoftKeyItem> </CiscoIPPhoneImageFile> </SoftKeyItem> </CiscoIPPhoneImageFile> 6.1 The video surveillance prototype With that in mind PHP offers a big leverage (Deel, D. & Nelson, M., 2004). The actual operation of the application is depicted in Fig. 7. In the following we will describe a code example to give an insight how applications can be written. A script can be designed to automatically refresh the pictures on the phone. On top of the script we added a header for refresh. header("Refresh:3"); header("Refresh:3"); header("Refresh:3"); header("Refresh:3"); First we need to read the content from the folder where Fwink program saves the acquired pictures from the Web/IP camera. $image = file_get_contents($campath); Fig. 7. The test environment for video surveillance application Then we use the ImageMagick extension. First we create an object, and then we save the picture in that object. Then we resize and convert the picture. $image = file_get_contents($campath); Fig. 7. The test environment for video surveillance application Then we use the ImageMagick extension. First we create an object, and then we save the picture in that object. Then we resize and convert the picture. www.intechopen.com 184 New Technologies – Trends, Innovations and Research $im = new Imagick(); g $im->readimageblob($image); g $im->readimageblob($image); $im->resizeImage(296,166,Imagick::FILTER_LANCZOS,1); $im >setImageFormat("png"); $im->resizeImage(296,166,Imagick::FILTER_LANCZOS,1); $im->setImageFormat("png"); After that the picture is saved back to the disk into a server folder enabling the IP phone to have access to the acquired picture. $fp=fopen($imagepath,'w'); fwrite($fp,$im); fclose($fp); $fp=fopen($imagepath,'w'); fwrite($fp,$im); p fclose($fp); Now we have a converted and saved the picture on the Web server. Next we need to send it back to the phone. Proprietary XML language objects now come into play, as the phone has to display the image correctly. We used the CiscoIPPhoneImageFile XML object for that purpose. This object is used for displaying PNG pictures on IP phones with a color display. It is necessary that we include a header that instructs the phone of the content in XML format. Only if that header is included in the script the phone will correctly parse XML objects and display the picture. header("Content-type: text/xml"); header("Content-type: text/xml"); 6.2 Application testing Some tests were executed to determine the capabilities of the introduced improvised video surveillance application. Multiple parameters had to be considered – the speed of the Web camera snapshots and the speed of application execution. Additionally we had to consider www.intechopen.com 185 A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery the speed of the PHP script – it executes the conversion of the image and also reads/writes from/to the file. The last but most important was the IP phone processing time. We had to evaluate the time of picture processing and XML objects parsing. The IP phone is an embedded system and because of that limited with the memory and processor power (Deel, D. & Nelson, M., 2004; Cisco Systems, 2011b). That also means the IP phone cannot refresh a new picture as fast as a Web browser on a PC would. We had to consider all the limitations to determine the optimal time values when the script is refreshed and had to evaluate the minimum time interval between the sampled snapshots. the speed of the PHP script – it executes the conversion of the image and also reads/writes from/to the file. The last but most important was the IP phone processing time. We had to evaluate the time of picture processing and XML objects parsing. The IP phone is an embedded system and because of that limited with the memory and processor power (Deel, D. & Nelson, M., 2004; Cisco Systems, 2011b). That also means the IP phone cannot refresh a new picture as fast as a Web browser on a PC would. We had to consider all the limitations to determine the optimal time values when the script is refreshed and had to evaluate the minimum time interval between the sampled snapshots. For the test reference we used a 10 seconds picture refresh interval with a 5 seconds refresh of the script. This is how we were able to display every picture acquired. With this reference we also evaluated the time limit of the script execution and picture refresh. In the following table (Table 1) the results of the performed test acquired with the packet sniffer software are presented (Wireshark, 2007). Resolution Average time for loading the picture[s] Average time of script execution [s] 640x480 2,83 0,12 320x240 2,40 0,11 Table 1. www.intechopen.com 6.2 Application testing Average application loading time The average time of picture loading and all delays that contribute to the system are covered. With the lower resolution the execution time is app. half a second (0,43 s). From the column Average time of script execution, we see that the conversion itself on the server adds only a small portion of time to display the picture. It was evident that the speed of application execution on the IP phone is solely limited with its capability of image processing. There is also some unexpected delay in the system. In our examination we concluded that this is contributed by the TCP protocol accessed with the upper layer HTTP. TCP offers reliable transfer and consequently for every request from the IP phone a session is established with the generally tree way handshake. Between the sessions acknowledgments are sent for the segments of the picture between the Web server and the IP phone. Additionally the session has to be closed after the communication ends. Two separate sessions are established during the transfer of one image from the Web server – the first one for the request of script execution and the second one for actually transferring a single picture to the IP phone. That procedure contributes approximately a 0,7 s delay for every single refresh on the IP phone display. After all the tests in the introduced test environment we conclude that one can use a minimum time interval of 5 seconds between the snapshots if every picture has to be displayed on the phone. In a production environment careful testing is required. Because the pictures need approximately 3,5 s (average time for loading the picture in addition to TCP delay) to load a time higher than 3,5 s must be chosen for a refresh-rate. That corresponds to 320x240 resolution pictures. For the higher 640x480 resolution we have www.intechopen.com 186 New Technologies – Trends, Innovations and Research chosen 4 seconds because of the slightly increased load time (0,4 s). If we had chosen a time lower than 3,5 s not every snapshot is displayed, some are skipped. Because of that a new refresh happens before the previous one has finished and therefore the application may occasionally crash. This is because TCP sessions take too long to complete. Consequently the application times out (MaFiRa, 2011). 6.2 Application testing The best scenario conducted in our test is shown in the following table (Table 2). Resolution Optimized sampling time [s] PHP Script optimized refresh time [s] 640x480 5 3,5 320x240 5 4 Table 2. Optimal time interval settings If required we can still use lower than optimal time intervals between the snapshots. In that case not all the sampled pictures will be displayed. Nevertheless more precise movement detection is possible. Resolution Optimized sampling time [s] PHP Script optimized refresh time [s] 640x480 5 3,5 320x240 5 4 Table 2. Optimal time interval settings Table 2. Optimal time interval settings If required we can still use lower than optimal time intervals between the snapshots. In that case not all the sampled pictures will be displayed. Nevertheless more precise movement detection is possible. www.intechopen.com 7. Conclusion Our application works for all types of Web/IP cameras. With a help of an open source program and Apache Web server we changed a Web camera into an IP camera. Our application can be improved in many ways. Because there are many different types of IP phones available with different capabilities we have to adapt applications to the receiving phone model. For example some models do not have a color display. So we can write a decisive structure to choose a different conversion for a different phone type. We implement that with the help of the header the IP phone sends when initiating a session with the Web server. Pictures can also be saved by the user or automatically into a database. We can also implement a movement detection feature some IP cameras offer and for example an image on the phone only appears when motion in front of the camera is detected. Then only changes and appropriate time tags are saved with the image to the database. In the presented application we had to evaluate different values for the snapshots acquiring time interval and refresh rate to correctly display every picture acquired. With the PHP script we can synchronize those parameters which now operate individually. With that we could achieve shorter intervals for displaying the snapshot images on the IP phone. We presented an example of a multimedia application that can be written for the IP phone. We tried to demonstrate that an IP phone application involves many distributed processes. The application is intended as a demo and together with the Web server, IP-PBX and other equipment in our local IP & VoIP network forms a basis for educational application design approach. It enables numerous possibilities in application development and proof of context for prototype development in business environments. www.intechopen.com A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery 187 8. References Au, D.; Cho, B.; Haridas, R.; Hattingh, C.; Koulagi, R.; Tasker, M. & Xia, L., (2005). Cisco IP Communications Express: CallManager Express with Cisco Unity Express (1. edition), Cisco Press, ISBN 1-58705-180-X, Indianapolis, IN 46240 USA Cisco Systems, (2006). (n.d.). Cisco Unified IP Phone 7970 Series for Cisco Unified CallManager 4.2(3), Document ID: OL-10776-01, 2006. September 2011, Available from: http://www.cisco.com/en/US/products/hw/phones/ps379/products_user_guid e_list.html Cisco Systems, (2011c). (n.d.). Configure and Manage the CUE system auto Attendant, Document ID: 63986, September 2011, Available from: http://www.cisco.com/en/US/products/sw/voicesw/ps5520/products_configur ation_example09186a00803f82eb.shtml Document ID: 63986, September 2011, Available from: http://www.cisco.com/en/US/products/sw/voicesw/ps5520/products_configur ation_example09186a00803f82eb.shtml Cisco Systems, (2011). Cisco Unified Communications Manager Express System Administrator guide, Cisco Press, San Jose, CA 95134-1706 USA, 2011. Cisco Systems, (2011a). (n.d.). IP phone services –Resources, In: Cisco Developer Community – Resources, June 2011, Available from: http://developer.cisco.com/web/ipps/resources Cisco Systems, (2011b). (n.d.). IP phone services –Forum, In: Cisco Developer Community- Forums, June 2011, Available from:, Access: http://developer.cisco.com/web/ipps/forums Deel, D. & Nelson, M. and A. Smith, (2004). Developing Cisco IP Phone Services, ISBN: 978- 1587050602, Cisco Press, Indianapolis, IN 46240 Gilmore, W. J., (2010). Beginning PHP and MySQL: From Novice to Professional, 4.th Edition, ISBN: 978-1430231141 Springer, New York, NY 10013 Hatting, S., Sladden, D., & Swapan, Z.A., (2010). SIP Trunking, Migrating from TDM to IP for Business Communications, Cisco Press, ISBN 1-58705-944-4, Indianapolis, IN 46240 USA Holzner, S., (2007). PHP: The Complete Reference, McGraw Hill, ISBN: 978-0071508544, New York, NY 10121-2298 Kaza, R.; & Asadullah S., (2005). Cisco IP Telephony – Planning, Designing, Operation and Optimization, Cisco Press, ISBN 1-58705-157-5, Indianapolis, IN 46240 USA Lundie, C. (2011), Free webcam software, In: Fwink, July 2011, Available from: http://www.lundie.ca/fwink/ MaFiRaWiki TCP/IP, (n.d.). July 2011, Available from: http://wiki.fmf.uni-lj.si/ wiki/TCP/IP Powers, S., (2008). Painting the Web. O'Reilly Media, Inc., ISBN: 978-0596515096, Sebastopol, CA 95472 Still, M., (2005). The Definitive Guide to ImageMagick, 1. Edition, APress, ISBN: 978- 1590595909, Berkeley, CA 94710 The Apache Software Foundation. (n.d.). Apache Usage Statistics, July 2011, Available from: http://trends.builtwith.com/Web-Server/Apache The Apache Software Foundation. (n.d.). The Apache HTTP Server project. July 2011, Available from: http://httpd.apache.org/, july 2011 www.intechopen.com New Technologies – Trends, Innovations and Research 188 UC500.com. (n.d.). Custom XML IP phone services, March 2011, Available from: http://uc500.com/en/custom-xml-ip-phone-services-uc500-and-cisco-unified-call- manager-express Wireshark (2011). (n.d.). Go Deep, July 2011, Available from: http://www.wireshark.org/ 188 New Technologies – Trends, Innovations and Research UC500.com. (n.d.). Custom XML IP phone services, March 2011, Available from: http://uc500.com/en/custom-xml-ip-phone-services-uc500-and-cisco-unified-call- manager-express g p Wireshark (2011). (n.d.). Go Deep, July 2011, Available from: http://www.wireshark.org/ g Wireshark (2011). (n.d.). Go Deep, July 2011, Available from: http://www.wireshark.org/ www.intechopen.com ISBN 978-953-51-0480-3 Hard cover, 396 pages Publisher InTech Published online 30, March, 2012 Published in print edition March, 2012 The book "New Technologies - Trends, Innovations and Research" presents contributions made by researchers from the entire world and from some modern fields of technology, serving as a valuable tool for scientists, researchers, graduate students and professionals. Some practical applications in particular areas are presented, offering the capability to solve problems resulted from economic needs and to perform specific functions. The book will make possible for scientists and engineers to get familiar with the ideas from researchers from some modern fields of activity. It will provide interesting examples of practical applications of knowledge, assist in the designing process, as well as bring changes to their research areas. A collection of techniques, that combine scientific resources, is provided to make necessary products with the desired quality criteria. Strong mathematical and scientific concepts were used in the applications. They meet the requirements of utility, usability and safety. Technological applications presented in the book have appropriate functions and they may be exploited with competitive advantages. The book has 17 chapters, covering the following subjects: manufacturing technologies, nanotechnologies, robotics, telecommunications, physics, dental medical technologies, smart homes, speech technologies, agriculture technologies and management. www.intechopen.com www.intechopen.com New Technologies - Trends, Innovations and Research Edited by Prof. Constantin Volosencu New Technologies - Trends, Innovations and Research Edited by Prof. Constantin Volosencu ISBN 978-953-51-0480-3 Hard cover, 396 pages Publisher InTech Published online 30, March, 2012 Published in print edition March, 2012 ISBN 978-953-51-0480-3 Hard cover, 396 pages Publisher InTech Published online 30, March, 2012 Published in print edition March, 2012 How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Janez Stergar, Janez Klanjšek and Sibila Vadlja (2012). A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery, New Technologies - Trends, Innovations and Research, Prof. Constantin Volosencu (Ed.), ISBN: 978-953-51-0480-3, InTech, Available from: http://www.intechopen.com/books/new-technologies-trends-innovations-and-research/a-framework-for-voip- testability-and-functionality-extension-with-interactive-content-delivery Janez Stergar, Janez Klanjšek and Sibila Vadlja (2012). A Framework for VoIP Testability and Functionality Extension with Interactive Content Delivery, New Technologies - Trends, Innovations and Research, Prof. Constantin Volosencu (Ed.), ISBN: 978-953-51-0480-3, InTech, Available from: InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com © 2012 The Author(s). Licensee IntechOpen. This is an open access artic stributed under the terms of the Creative Commons Attribution 3.0 cense, which permits unrestricted use, distribution, and reproduction in ny medium, provided the original work is properly cited. © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
https://openalex.org/W1497830658	https://hal.inria.fr/hal-01055919/file/pro91.pdf	English	null	The Application of Incentive Mechanisms for the Participation of Enterprises in Collaborative Networks from an Economic Perspective	IFIP advances in information and communication technology	2,010	cc-by	3,525	To cite this version: Hendrik Jähn. The Application of Incentive Mechanisms for the Participation of Enterprises in Col- laborative Networks from an Economic Perspective. 11th IFIP WG 5.5 Working Conference on Vir- tual Enterprises (PRO-VE), Oct 2010, Saint-Etienne, France. pp.773-780, 10.1007/978-3-642-15961- 9_91. hal-01055919 The Application of Incentive Mechanisms for the Participation of Enterprises in Collaborative Networks from an Economic Perspective Hendrik Jähn Hendrik Jähn The Application of Incentive Mechanisms for the Participation of Enterprises in Collaborative Networks from an Economic Perspective Hendrik Jähn Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01055919 https://inria.hal.science/hal-01055919v1 Submitted on 25 Aug 2014 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License The Application of Incentive Mechanisms for the Participation of Enterprises in Collaborative Networks from an Economic Perspective Hendrik Jähn1, 1 Chemnitz University of Technology, Dept. of Economic Sciences, Thüringer Weg 7 09126 Chemnitz, Germany hendrik.jaehn@wirtschaft.tu-chemnitz.de 1 Chemnitz University of Technology, Dept. of Economic Sciences, Thüringer Weg 7 09126 Chemnitz, Germany hendrik.jaehn@wirtschaft.tu-chemnitz.de Abstract. This contribution is to be assigned to the operative production management of collaborative networks. By the application of incentive mechanisms, a competitive and effective management of production processes allows sustainable success. Incentives are relevant for all stages of a network lifecycle. In this paper, the problem is discussed that enterprises from a pool of cooperation-willing enterprises do not take part in a certain value-added process, even though it would be possible from the technical point of view and would also be economically reasonable. As a reason for this behavior, the utility calculus of an enterprise plays a decisive role. To convince these enterprises to collaborate in a value-added process, primarily incentive mechanisms are to be applied. In the following, this topic is taken on and is discussed extensively by means of two concrete examples. In this connection, a concrete calculation model both for the amount of incentives and the profit share is introduced. Keywords: Collaborative network, incentive mechanisms, operative network management, sustainable success. 2 Incentives in an Economic Environment The application of incentives in an economic environment is possible in various ways and for numerous situations. In relation to the analysis of principal-agent-relations [1] within the frame of the principal-agent-theory [2], [3], incentives can be applied as a possible solution approach for problems classes representing hidden action and hidden information. Here, the whole enterprise network represents the principal; the involved enterprises are the agents. In this context, incentives serve as alignment of interests between principal and agent after conclusion of a contract. At this point, a reduction or removal respectively of the moral hazard on the agent's side is achieved. Within the frame of the principal-agent-theory, these actions are tendentially agent- related. Here, incentive systems represent the combination of several single incentives. In this context, incentives also stand for a suitable measure for an alignment of interests of the agent to those of the principal [4]. In this context, incentives are understood as a situational condition, which is supposed to motivate the economic agents due to their individual needs, i.e. utility structure for a performance for the purpose of the principal. By means of incentives, agents are influenced to perform actions in the intensity desired by the principal (management) [5]. Incentive systems represent the sum of all working conditions designed purposely enforcing certain behavior patterns and reducing the probability of the occurrence of undesirable behavior patterns. Behavior patterns are made stronger by the application of positive incentives in form of rewards taken as an example. Undesirable behavior can be reduced by negative incentives in form of sanction mechanisms [4]. From that results the so-called “motivation problem“, which in its core statement says that an individual needs an incentive to produce a certain performance or to omit an action as long as this has no connection to the individual utility maximization [6]. By means of incentives, it is aspired to suppress self- interested behavior of agents and to adjust their behavior to the aims of the principal. However, this method does not lead to the fact that an agent does not maximize its individual utility, but it is rather attempted to bring the agent to actions by directed incentives for which, by maximizing of his individual utility, also the utility of the principal is maximized. In this connection, one also speaks of incentive compatibility [4]. This procedure can be summarized by the terms interest alignment or interest harmonization. 1 Motivation The implementation of value-added processes within networked production structures has strongly gained in importance. Integral advantages are a high degree of flexibility, sustainability and cost saving potential. However, not in every case value-added processes may be realized by collaboration although this would be reasonable from an economic perspective. So, the effort of an enterprise to cooperate primarily depends on the economic advantageousness of a project, i.e. when the utility can be maximized or a benefit can be achieved respectively. A project is, however, only reasonable if the overall benefit of the network is maximized or if a benefit results. In certain cases, enterprises do maximize their benefit without cooperation although participation is desirable if seen as a whole. Reasons for not cooperating may principally be found in a shortage of production capacity or missing financial attractiveness of an order. In these cases, the network management has the possibility to interfere in a regulating way. To achieve a sustainable outcome, incentive mechanisms can be applied to a certain degree for selected enterprises. 828 H. Jähn 828 H. Jähn 3 Incentive Design with insufficient Production Capacity To be able to guarantee and quantify incentives, reasons for this need to be identified. So, not taking part in a value-added process may for an individual enterprise be the best alternative, but not necessarily for the whole network. This would be the case if a core competency is missing in the value-added chain to be able to perform a service in a collaborative network. In such a case the “missing“ enterprise needs to be guaranteed suitable incentives for participating. In the following it is assumed that an enterprise does not cooperate due to insufficient production capacity (machine or personnel). Here incentives need to be paid for supplying additional capacities. There are several approaches to solve this problem. One was is to compensate the additional costs for the enterprise supplying additional capacities. The starting point is that an enterprise is not able to fulfill the respective requirements within the given time frame on the basis of a request of the network. As a reason for this it is assumed that insufficient production capacities lead to this decision. Although a possible completion date can be stated within the frame of the tender preparation, this should however be oriented at the desired date of the customer or, in case the customer does not give a desired date, the date should be within a reasonable time span for the fulfillment of an order. At this point, it must be decided whether the enterprise being the only one offering the demanded core competence can be made giving a (realistic) offer. A practicable solution would be, taken as an example, to create suitable capacities by replanning. This option marks the starting point for the following considerations. It is assumed that a certain enterprise aspires to be able to give an offer by suitable replanning. The costs accruing by replanning are difficult to quantify, which however is indispensable for the calculation of the incentives. Personnel costs resulting from the replanning taken as an example may be included in the costs of replanning. Additional costs resulting from overtime, nightshifts or weekend work can be included as well. Furthermore, costs accruing from delayed delivery caused by other replanned orders have to be calculated. These additional accruing costs need to be refunded to the affected enterprise, which can be interpreted as an incentive payment. Here, the affected enterprise has to raise a part of the incentive amount by itself. 2 Incentives in an Economic Environment In addition to these rather generally formulated functions of incentive systems, incentives can also be divided into single part functions [7]: Activation function: stimulation of motivation and • Controlling function: influence of the agents' behavior, • Controlling function: influence of the agents' behavior, Information function: agents get signals regarding desirable behavior, • Information function: agents get signals regarding desirable behavior, • Selection function: principals get information about agents’ performance and • Selection function: principals get information about agents’ performance and To ensure that these functions are fulfilled as good as possible, incentive systems shall meet different requirements. On the one hand, incentives need to be economically justifiable. Also the application of incentive mechanisms is only reasonable if the costs caused by the incentive system are lower than resulting (additional) earnings. These costs result from the measures conducted for the incentive system and also the administrative costs. 829 Application of Incentive Mechanisms for the Participation of Enterprises in CNs 4.1 Description of the Situation The financial aspect of an order is primarily represented by the profit to be gained for an enterprise. So the focus is laid on approaches for calculating enterprise-related profit shares gi. For this purpose various approaches have been developed [8] and are available. As a precondition, knowledge concerning the entire distributable profit G is essential. If no quote can be submitted for the concrete enquiry of a customer in the network from the network's side, this situation is unsatisfactory for all involved parties. Therefore, an alternative solution needs to be found. In the present case, it is assumed that the enterprise(s), which need(s) to be engaged for the considered value-added process possess(es) sufficient production capacity. So, the missing financial profitability of the order can be considered to be the only reason for the rejection. Therefore, it is the aim to convince the respective enterprises (here it is assumed that only one enterprise i* is concerned) regarding participation in the value-added process by means of suitable incentives. To be able to make a decision for or against an incentive payment, various results connected to the guaranteeing or omission of incentives, need to be analyzed and compared. Based on the situation that the single shares of profit gi were determined within the frame of an algorithm for determining the profit G, but no agreement could be found with enterprise i* regarding profit gi* and therefore it was not possible to submit a quote for a customer enquiry, suitable incentives need to be guaranteed. Quantification of these incentives is described in the following. For this purpose, an algorithm for the calculation of profit and incentive payments within networked production structures is introduced. Assuring a minimal profit for an enterprise represents a material incentive on the network's side. This incentive is guaranteed in cases if an enterprise rejects participation due to lacking profitability, although the respective core competency is urgently needed to be able to implement the value-added process in the network. This situation always occurs when the share of profit of an enterprise is lower than the expectation of profit (minimal profit) according to the model for distribution of profit. So, a guaranteed minimal profit is a security incentive. 3 Incentive Design with insufficient Production Capacity This is explained by the fact that the respective enterprise is entitled to get a share in profit when taking part in the value-added process, which would not be obtained when not participating Guaranteeing incentive payments is based on the assumption that all enterprises aspire individual utility maximization. So, it is always first goal to cooperate in a value-added process as long as benefit (profit) can be obtained. For the case that a customer order cannot be realized due to missing enterprises, no benefit can be achieved. As a general rule, abandoning the participation in a value-added process by an enterprise has high probability if the missed benefit is lower than additional costs. These include replanning costs or further costs for capacity expansion. The latter kind of costs includes costs for the purchasing or leasing of a machine, but also additional personnel costs like overtime payments or rewards for working on holidays or at the weekend. Such costs have to be taken into account for consideration of total costs for the calculation of possible incentives. 830 H. Jähn 830 H. Jähn 4.2 Calculation of the individual Share of Profit of an enterprise Assumptions. The calculation of the cash flows in the network may be conducted for a comprehensive model regarding the distribution of profit when applying a suitable calculation approach. For the modeling of this approach regarding determining participation incentives, the following five assumptions apply representing the general conditions for the application of incentive mechanisms: a) guaranteed incentive payments are paid by integration into the process of distribution of profits, a) guaranteed incentive payments are paid by integration into the process of distribution of profits, b) each enterprise receives an individual share of profit gi, which depends on the part in total costs and also consists of a fixed part, 831 Application of Incentive Mechanisms for the Participation of Enterprises in CNs c) all incentive and profit payments (ai or gi respectively) resulting from the concrete value-added process have to be financed from the realized network profit G of the considered value-added process, d) p p d) the model for distribution of profit can be modified by agreements about minimal profit shares between network and the single enterprises; the minimal profit share of an enterprise gi min has to be guaranteed by the regular share of profit and if necessary by an incentive payment. Modeling. Consecutively the modeling of the calculation approach is introduced. This calculation approach can then be applied if a finite number of enterprises take part in the value-added process, but only one enterprise receives incentive payments. Condition 1: The sum of the profit share of the enterprises gi results from the gained or distributable network profit G less all guaranteed incentive payments ai. This side condition implies that the guaranteed incentive payments ai in the present model are financed from profit G. Condition 2: The second condition contains the calculation regulation for the share of profit of enterprises gi. In the present case, a two-component-approach is applied with a distribution parameter according to [8]. The profit share consists of a fixed and a variable part. Both parts are calculated according to the respective formulas. Condition 3: The guaranteed minimal profit of an enterprise gi min consists of the individual realized share of profit of this enterprise gi and an incentive payment being possibly guaranteed to enterprise ai. In addition, a difference variable ui is introduced. Its function is to represent a potential difference between the individual share of profit gi and the guaranteed minimal profit gi min. 4.2 Calculation of the individual Share of Profit of an enterprise This can occur if the individual share of profit is higher than the guaranteed minimal profit because no incentive payment is necessary. Therefore, variable ui makes sure that the equation condition is valid also in such cases. Each enterprise represents a specific ui. Condition 4: This condition is based on the third condition. So, variable ui is only applied for cases in which no incentive payment ai is guaranteed to the respective enterprise. This results in the fact that in this case ui gets a value of not equal to zero if the incentive payment ai equals zero and vice versa. Therefore, the product from difference variable ui and incentive payment ai has to be zero in each case. Additional conditions: For completion of the modeling several more model conditions need to be determined. This also contains the respective definition and co domains of the variables. So, non-negativity conditions for the total profit G, the incentive payments ai, the individual share of profit gi, the guaranteed individual minimal profits gi min, the individual costs ci and the difference variable ui have to be introduced. Furthermore, it has to be stated that the weighting parameter α shall be between 0 and 1, as the incentive is supposed to be applied independently. Finally, the number of enterprises in the network n has to be defined. Calculation Approach. In the following, the previously described calculation approach is presented formally. For this modeling, it needs to be noted that only a selected model for distribution of profit according to condition 2 is applied. The calculation approach consists of four equations and three unknown variables ai , gi , ui. This constellation makes an unproblematic solving of the task possible. However, the complexity of the model increases with a rising number of enterprises taking part 832 H 832 H. Jähn 832 H. Jähn in the value-added process, as then n+3 equations need to be solved with n+2 unknown variables. Fig. 1. Calculation model for incentives Fig. 1. Calculation model for incentives Example. The calculation approach introduced in the section before can be applied for cases, in which a finite number of enterprises n takes part in a value-added process and only one enterprise receives a financial incentive. 5 Conclusions In the previous sections, it has been proven that the implementation of incentive mechanisms is an effective and promising instrument of the operative network controlling. For this purpose the maximization of the profit both for the network (principal) and for the single enterprises (agents) is realized by guaranteeing financial incentives. Possible criteria for the assessment of a contractual behavior of the enterprises are comparisons between target and actual performance when considering various performance parameters [9]. The provision of service according to the contractual duty has an essential importance for the network as these criteria have a considerable influence on customer satisfaction and decide on the continuance and sustainable success. Because of its structure the model is applicable for an unlimited number of network participants. In case a different approach for profit distribution is considered, the affected side conditions need to be modified. 4.2 Calculation of the individual Share of Profit of an enterprise Having a number of three enterprises in the network, when taken as an example and only one enterprise receiving an incentive, the following six equations result including five unknown variables ai, g1, g2, g3, ui. Fig. 2. Calculation model for incentives for three network participants Fig. 2. Calculation model for incentives for three network participants Application of Incentive Mechanisms for the Participation of Enterprises in CNs 833 In the following a quick solution finding is demonstrated. Here enterprise 1 was assumed to receive incentive payments. Consideration of this condition in the model leads to the result that the difference variable u1 is zero. It follows: After that, the formula for a calculation of the profit share has to be created. Then, equation (2´) can be included in equation (5). Then, equation (2´) can be included in equation (5). For calculation of the amount of incentive payment a1, equation (5´) has to be transposed to a1. To facilitate this process, the following two substitutions are conducted: So, transposition from equation (5´) to a1 can be realized as follows: So, transposition from equation (5´) to a1 can be realized as follows: Finally, the variables inserted by substitution can be replaced by the original terms again. The following formula results from that and by means of it, the amount of the incentive payment to enterprises 1 can be calculated: 834 H. Jähn 834 H. Jähn In addition to the realized individual share of profit g1, enterprise 1 receives an incentive payment a1. Consecutively the remaining profit shares can be calculated by means of equations (2), (3) and (4). Finally, all necessary values can be calculated. References 1. Ross, S.A.: The Economic Theory of Agency: The Principal’s Problem. American Economic Review 63, 134--139 (1973) 2. Furubotn, E.G., Richter, R.: Institutions and Economic Theory: The Contribution of the New Institutional Economics. 2nd ed., University of Michigan Press, Ann Arbor (2005) 3. Laffont, J.J., Martimort, D: The Theory of Incentives – The Principal-Agent-Model. Princeton University Press, Princeton, N.J. (2002) 4. Picot, A., Dietl, H., Franck, E.: Organisation - Eine ökonomische Perspektive. 4th ed., Schäffer-Poeschel, Stuttgart (2005) g 5. Macho-Stadler, I., Pérez-Castrillo, D.: An Introduction to the Economics of Information – Incentives and Contracts. 2nd ed., Oxford University Press, New York; Oxford (2001) 6. Milgrom, P., Roberts, J.: Economics, Organization and Management. Prentice Hall, Englewood Cliffs, New Jersey (1992) 7. Winter, S.: Prinzipien der Gestaltung von Managementanreizsystemen. Gabler, Wiesbaden (2002) 8. Jähn, H., Fischer, M., Teich, T.: Distribution of Network generated Profit by considering individual Profit Expectations. In: Camarinha-Matos, L.M. et al. (eds.) Establishing the Foundation of Collaborative Networks. pp. 337--344. Springer, New York (2007) 9. Jähn, H.: Value-added process-related performance analysis of enterprises acting in cooperative production structures. Production Planning & Control. 20, 178--190 (2009)
https://openalex.org/W4319294788	https://www.researchsquare.com/article/rs-2037224/latest.pdf	English	null	The important role of the interaction between manganese minerals and metals in environmental remediation: a review	Environmental science and pollution research international	2,023	cc-by	14,076	The important role of the interaction between manganese minerals and metals in environmental remediation: A review Meiqing Chen (  cmq2021@scut.edu.cn ) South China University of Technology Jiayan Wu South China University of Technology Xiaoshan Qiu South China University of Technology Lu Jiang South China University of Technology Pingxiao Wu South China University of Technology The important role of the interaction between manganese minerals and metals in environmental remediation: A review Meiqing Chen (  cmq2021@scut.edu.cn ) South China University of Technology Jiayan Wu South China University of Technology Xiaoshan Qiu South China University of Technology Lu Jiang South China University of Technology Pingxiao Wu South China University of Technology Research Article Keywords: Manganese minerals, Metal, Interaction, Synergistic effect, Environmental significance Posted Date: November 9th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2037224/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read F License Version of Record: A version of this preprint was published at Environmental Science and Pollution Research February 6th, 2023. See the published version at https://doi.org/10.1007/s11356-023-25575-8. The important role of the interaction between manganese minerals and metals in environmental remediation: A review Meiqing Chen (  cmq2021@scut.edu.cn ) South China University of Technology Jiayan Wu South China University of Technology Xiaoshan Qiu South China University of Technology Lu Jiang South China University of Technology Pingxiao Wu South China University of Technology Research Article Keywords: Manganese minerals, Metal, Interaction, Synergistic effect, Environmental significance Posted Date: November 9th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2037224/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read F License Version of Record: A version of this preprint was published at Environmental Science and Pollution Research February 6th, 2023. See the published version at https://doi.org/10.1007/s11356-023-25575-8. Research Article Keywords: Manganese minerals, Metal, Interaction, Synergistic effect, Environmental significance Posted Date: November 9th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2037224/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Environmental Science and Pollution Research on February 6th, 2023. See the published version at https://doi.org/10.1007/s11356-023-25575-8. Page 1/27 Abstract With illegal discharge of wastewater containing inorganic and organic pollutants, combined pollution is common and need urgent attention. Understanding the migration and transformation laws of pollutants in the environment has important guiding significance for environmental remediation. Due to the characteristics of adsorption, oxidation and catalysis, manganese minerals play important role in the environment fate of pollutants. This review summarizes the forms of interaction between manganese minerals and metals, the environmental importance of the interaction between manganese minerals and metals, and the contribution of this interaction in improving performance of Mn-based composite for environmental remediation. The literatures have indicated that the interactions between manganese minerals and metals involve in surface adsorption, lattice replacement and formation of association minerals. The synergistic or antagonistic effect resulted from the interaction influence the purification of heavy metal and organism pollutant. The synergistic effect benefited from the coordination of adsorption and oxidation, convenient electron transfer, abundant oxygen vacancies and fast migration of lattice oxygen. Based on the synergy, Mn-based composites have been widely used for environmental remediation. This review is helpful to fully understand the migration and transformation process of pollutants in the environment, expand the resource utilization of manganese minerals for environmental remediation. 1. Introduction Manganese minerals are rich on Earth with an average content of about 0.1% in the crust (Barceloux, 1999). They are particularly abundant in the crust, with an average abundance of 1000 mg⋅kg− 1 (Li et al., 2014). The world’s total average manganese in the soil is 850 mg⋅kg− 1 (Suttle, 2003). There are more than 30 different kinds of manganese minerals including birnessite, vemadite, todorokite, romanechite, cryptomelane and manganite, which are common in the soil (Robinson et al., 2013). Based on the structural type, manganese minerals can be divided into tunnel structure, layered structure and low-valence unordered manganese mineral. Mutual transformation occurs between different types of manganese minerals under the certain conditions (Wang et al., 2018; Wu et al., 2019a; Ruiz-Garcia et al., 2021). The combination of the [Mn(III)O6] octahedron and [MnO6] octahedron leads to the transformation of the layered structure into the tunnel structure (Liang et al., 2020). Because of high specific surface area, abundant hydroxyl groups and high chemical reaction activity, manganese minerals show strong interactions with metals (WegorzeWski et al., 2015; Wan et al., 2020; Yu et al., 2017a; Ibrahim et al., 2022; Jin et al., 2022). These interactions impact the environmental fate and biological toxicity of metals through adsorption and oxidation (Ma et al., 2015; Peacock and Moon, 2012; Xu et al., 2011). The interaction between manganese minerals and metals also affects the structure, morphology, the adsorption performance, photochemical properties and oxidation capacity of manganese minerals (Li et al., 2019a; Zhu et al., 2010a; Yin et al., 2020). Therefore, the interaction between manganese minerals and metals plays an important role in environmental behavior of element and environmental significance of manganese minerals (Atkins et al., 2014; Yin et al., 2020). The interaction between manganese minerals and metals can also produce associated minerals (Chigrin and Kirichenko, 2018; Najjar and Batis, 2016). Fe-Mn minerals and ferromanganese crusts are the most representative associated minerals (Reykhard and Shulga, 2019; Lu et al., 2019). Mn-based layered double hydroxides (Mn- LDHs) are important associated minerals in the environment (Chawla et al., 2017). These associated minerals further influence the migration and transformation of pollutants (Crowther, 1982; Spinks et al., 2017). The Page 2/27 Page 2/27 Page 2/27 oxidation degree of Tl(I) on ferromanganese crusts is determined by the proportion of birnessite, which controls the enrichment and isotopic fractionation of Tl(I) (Peacock and Moon, 2012). 2. The Interaction Mechanisms Between Manganese M Because of the large specific surface area, abundant surface hydroxyl groups, rich vacancies and strong oxidation, manganese minerals have strong affinity towards metals (Machado-Infante et al., 2016; Qin et al., 2019; Wu et al., 2019b). The interaction mechanisms between manganese minerals and metals include surface adsorption (Fig. 1a), lattice replacement (Fig. 1b) and formation of association minerals (Fig. 1c), which obviously affect the migration and transformation of metals (Kwon et al., 2010; Maeno et al., 2016; Nishi et al., 2017). 1. Introduction Fe-Mn minerals control the cycle of As in aquifer sediments by adsorption and fractions (Ma et al., 2015). The synergistic or antagonistic effect resulted from the interaction influence the purification of heavy metal and organism pollutant. Base on the synergistic effect of the interactions between manganese minerals with metals, Mn-based composites have been widely applied in the field of environmental protection in recent years (Tarjomannejad et al., 2017; Ma et al., 2021; Ruiz-Garcia et al., 2021; Shaheen et al., 2022). The contribution of the synergistic effect between Mn and metal to environment purification has been confirmed (Chen et al., 2017a; Guo et al., 2018; Kuang et al., 2019; Peng et al., 2020; Sotiles et al., 2019; Yang et al., 2018a). Mn0.52Co2.48O4 spinel has good oxidation activity because of its high specific surface area and the effective synergistic effect between Co and Mn during the cycles of different valence states of elements (Co2++Mn4+↔Mn3++Co3+; Co3++Mn2+↔Mn3++ Co2+)(Gao et al., 2019; Wang et al., 2019a). Because of the synergistic effect of Fe and Mn, the catalytic activity of Fe-Mn composite exhibits a five-fold increase in oxygen reduction activity (Wang et al., 2019b). Application of the density functional theory calculation showed that the synergistic effect of bimetallic sites reduces the energy reduction of ·OH barrier, which is beneficial for oxidation and generation of contaminants (Sarkar et al., 2020). The synergistic or antagonistic effect resulted from the interaction influence the purification of heavy metal and organism pollutant. Base on the synergistic effect of the interactions between manganese minerals with metals, Mn-based composites have been widely applied in the field of environmental protection in recent years This review firstly provides an overview the interaction mechanism between manganese minerals and metals, summarize the environmental importance of the interaction between manganese minerals and metals, and outline the role of synergistic effect between Mn and metal in improving performance of Mn-based composite for environmental remediation. This review not only helps to understand the migration and transformation processes of pollutants in the environment, but also expands the utilization of manganese minerals and Mn-based composite for environmental recovery. 2.1 Surface adsorption Previous studies have shown that a natural hexagonal birnessite with low crystallinity had a very high adsorption affinity to Pb(II) (Qin et al., 2019b; Wang et al., 2012). The adsorption capacity of birnessite for Pb(II) was 1.6–3.9 times higher than that of other metals. It was confirmed that Pb(II) formed two strong inter-sphere complex surfaces during adsorption: triple corner-sharing (TCS) on the Mn(IV) vacancy and double edge-sharing complexes (DES) on lateral edge surfaces (Kwon et al., 2010). The maximum adsorption capacity of Cd(II) was 104.17 mg·g− 1 (Huang et al., 2017), where the adsorption of Cd(II) on the manganese mineral was dominated by the formation of surface complexation in both outer and inner layers. According to the results of a surface complexation model, the adsorption of Cd(II) on manganese mineral can be well simulated by using the ion exchange at low pH and the inter-sphere surface complexation at high pH. Page 3/27 Page 3/27 In a biofilm of hydrated bacteria, biological birnessite adsorbed Zn(II) by tetrahedral coordination on the crystal position (Toner et al., 2006). Zn(II) was adsorbed on the biological manganese mineral by generating two types of tetrahedral coordination complexes: a monodentate-mononuclear complex with a regular/distorted MnO6 octahedron and a bidentate-binuclear complex with two MnO6 octahedrons (Zhang et al., 2014). Zn(II) was anchored as a TCS surface complex above the vernadite vacancy, the complex was tetrahedral at a low Zn/Mn mole ratio and octahedral at a high Zn/Mn mole ratio (Grangeon et al., 2012). As the result of the extended X-ray absorption fine structure spectrum (EXAFS) shown, there were two fixation modes of Ni on the biological manganese mineral: Ni was adsorbed by producing a triple-corner-sharing complex (Ni-TCS) at the vacancy when Ni concentration was low; Ni was embedded into the sheets of manganese mineral when Ni concentration was high (Peña et al., 2010). The maximum adsorption capacity of Ni(II) increases with the decrease of pH because the number of vacancies in biological manganese mineral increases with the increased acidity during the formation process (Zhu et al., 2010b). The adsorption process showed high selectivity for Cu(II) on the surface of the manganese mineral with a maximum adsorption capacity of 11.926 mg⋅g− 1 (Cano-Salazar et al., 2020). 2.1 Surface adsorption The result of the Cu-k-edge in EXAFS showed that most Cu(II) was adsorbed above the vacancy, Cu(II) was anchored at the edge of the birnessite in the form of polynuclear clusters when pH was 3.3–5.3 and the molar ratio of Cu/Mn increased from 0.08 to 0.23(Qin et al., 2017). Cu(II) was adsorbed on birnessite by forming an inner-sphere complexation on the surface of the {001} crystal plane, and the bond length of Cu and Mn was 3.4 Å(Sherman and Peacock, 2010). Todorokite has a high adsorption capacity for Cs(I), which influences the cycle of Cs(I) in the environment(Yu et al., 2017b). Although the proportion of the central metal vacancy in biological birnessite was higher than synthetic birnessite, the adsorption density of Cs(I) on biological birnessite was lower (Sasaki et al., 2014). Since Cs(I) adsorbed on the two minerals was mainly in the form of outer spherical complexes, it cannot easily occupy the central metal vacancy in biogenic minerals. In summary, Pb(II), Cd(II), Zn(II), Ni(II), Cu(II) and Cs(I) were adsorbed by preferentially combining with the surface hydroxyl group and form surface complexes on manganese minerals. 2 2 Lattice replacement 2.2 Lattice replacement The ability of metals to replace Mn(III)/Mn(IV) in layers has important influence on the removal efficiency of metals and the reactivity of manganese minerals (Holguera et al., 2018). Hexagonal birnessite can immobilize and oxidize Mn(II), and then transform it into Mn(III)-rich hexagonal birnessite, tripartite birnessite or tunnel manganese oxide, which significantly changes the environmental behavior of Mn oxide (Yang et al., 2019). Under the conditions of low Mn(II)/Mn (5–24%) and pH ≥ 8, δ-MnO2 and acid birnessite reacted with Mn(II) in solution and turned into orthogonal birnessite (Zhao et al., 2016). This transition was attributed to the co-distribution reaction between the Mn(II) fixed in the vacancy and the surrounding Mn(IV) in the layer. Therefore, the substitution Mn(IV) with generated Mn(III) impact importantly the structure of the birnessite. The effect of Cu(II) on the characteristics of manganese minerals varies among different fixation patterns (Li et al., 2019a). The replacement of Mn(II) with Cu(II) in the framework is considered to be a defect in the crystal structure but showed a stabilizing effect in the framework (Nicolas-Tolentino et al., 1999). Previous study found that part of Cu(II) was embedded in the layer structure of MnO2 by occupying vacancies at pH 8(Sherman and Peacock, 2010). Page 4/27 The substitution of Mn(III) with Co(III) in the MnO6 layer plays a key role in the conversion of birnessite to todorokite (Xu et al., 2011). Co(II) and Ni(II) enter the vacancy of hexagonal birnessite and become part of the hexagonal sheet structure by replacing the missing Mn(IV) (Kwon et al., 2013). The compatibility of metals in the birnessite layer decreases in the order Co2+ > Ni2+ > Fe2+(Yin et al., 2013; Yin et al., 2014). Co(II) and Fe(II) are immobilized on birnessite by replacing Mn(III) and Mn(IV); however, only the replacement of Mn(III) has been found in the Ni(II) immobilization process. The grain boundary coordination radius (CR) determines the substitution of metals with Mn(III)/Mn(IV) in the birnessite layer: the smaller the difference between CR of metals and Mn(IV) or Mn(III), the more compatible they are with the Mn layer (Wu et al., 2020). The particle size and vacancy in the structure of biological birnessite affect its fixing ability to Pb(II), Zn(II) and As(III, V), which determines the fate of metals in geochemical environment(Villalobos et al., 2014; Lee et al., 2013; Toner et al., 2006; Qin et al., 2018). 2.2 Lattice replacement The increase of internal vacancy causes negative electrostatic repulsion with the As(V) oxygen anion. XPS and XANES spectra indicated the enrichment mechanism of Pt(II) in marine ferromanganese crusts involved the oxidation reaction of Pt(II) and Mn(IV) on the Mn-O-Pt bond in [PtCl4n (OH)n]2− and Pt(IV) substitution on MnO2 structure (Maeno et al., 2016). There was a high correlation between thallium (Tl) and Mn, indicating a high geochemical affinity between Tl and manganese minerals(Cruz-Hernández et al., 2019). The adsorption of Tl(I) on the manganese minerals was through an irreversible oxidation mechanism. The produced Tl(III) was mainly retained in the vacancy. Manganese minerals show an extremely high capacity for Tl(I) oxidation, since Tl(I) can react with all internal Mn(IV) in the internal structure and form Tl2O3 under a high concentration of Tl(III) (Chen et al., 2019). 2.3 Formation of association minerals Associated minerals refer to a mixture of different kinds of minerals that appear in the same spatial range in nature (Marsh et al., 2019; Pirajno, 2015). Because of the interaction of manganese minerals and metals, associated minerals are generated during the formation of manganese minerals in the environment (Gao et al., 2015). Ferromanganese nodules on the seabed are the most representative associated minerals (Liu et al., 2017; Marcus et al., 2004; Jiang et al., 2009). There are three types of ferromanganese nodules: (i) 1–1.5 cm smooth irregular nodules, Mn/Fe = 2.50, trace metal abundance (Co + Ni + Cu = 2.10%); (ii) 2–4 cm smooth disc-shaped nodules, Mn/Fe = 3.65, trace metal abundance (Co + Ni + Cu = 2.35%); and (iii) 3–15 cm nodule, grape-like surface, Mn/Fe = 5.1, trace metal abundance (Co + Ni + Cu = 2.42%). The ferromanganese nodules fix metals through diagenesis during the growth of nodules under alternating oxygen and sulfite conditions (Reykhard and Shulga, 2019). Ferromanganese nodules present alternating layers of iron-rich and manganese-rich nodules, reflecting the changes of the redox property in the water system (Gao et al., 2015). Hetaerolite (ZnMn3O7.3H2O) is also a major associated mineral in nature (Chukanov et al., 2016). Zn is fixed by forming tetrahedral (ZnIV) and octahedral (ZnVI) triple-corner-sharing surface complexes (TCS) at Mn(IV) vacancy sites in hexagonal birnessite (Yin et al., 2018). The Zn-Mn octahedral complexes further convert to ZnMn3O7.3H2O (Kwon et al., 2009). 2.3 Formation of association minerals In summary, the properties of manganese minerals, the concentration of metals and the reaction condition (pH, ion strength and temperature) affect the way of interaction between manganese minerals and metals, including Page 5/27 Page 5/27 surface adsorption (Table 1), lattice replacement and formation of associated minerals Page 6/27 Table 1 The forms of interaction between manganese minerals and metals Manganese minerals Metals Condition Forms of interaction Reference Mn oxide Cd(II) pH = 6.0, C0 = 1.0–100 mg/L, I = 0.01 mol/L; m/v = 1 g/L, T = 298 K Surface adsorption: outer-sphere and inner- sphere surface complexation (Huang et al., 2017a) Biogenic bixbyite-like Mn2O3 Zn(II) pH = 4.0 or 6.0, C0 = 0.2– 4.0 mM, I = 0.1 mol/L, m/v = 1.67 g/L, T = 298 K Surface adsorption: tetrahedral coordination complexes (Zhang et al., 2014) Biological manganese mineral Ni(II) pH = 6.0, C0 = 0.13 mM Surface adsorption: triple-corner- sharing complex (Peña et al., 2010) Hight Ni concentrations, pH = 6 or pH = 8 Lattice replacement: incorporation of Ni into the birnessite sheet Birnessite Cu(II) pH = 4.0, C0 = 33–167mg/L, I = 0.1 mol/L, m/v = 3.33 g/L, Surface adsorption: inner-sphere complexation, (Sherman and Peacock, 2010; Nicolas- Tolentino et al., 1999) pH = 8.0, C0 = 33-167mg/L, I = 0.1 mol/L, m/v = 3.33 g/L, Lattice replacement: incorporated into the MnO2 layer by occupying the vacancy sites Hexagonal turbostratic birnessite Cu(II) pH = 3.3–5.3, Cu/Mn = 0.08–0.23 Surface adsorption: polynuclear clusters (Qin et al., 2017) pH = 4.6–6.4 Surface adsorption: outer spherical complexes (Qin et al., 2017) Hexagonal birnessite Mn(II) C0 = 0–7.5 mM, pH = 7–9 Lattice replacement: occupying i (Zhao et al., 2016) pH = 8.0, C0 = 33-167mg/L, I = 0.1 mol/L, m/v = 3.33 g/L, C0 = 0–7.5 mM, pH = 7–9 Page 7/27 Manganese minerals Metals Condition Forms of interaction Reference Birnessite Pb(II) pH = 5.0, C0 = 0–15 mM, I = 0.15 mol/L, m/v = 1.67g/L, Lattice replacement: anchored on the vacanties (Zhao et al., 2009) Birnessite Co(II), Ni(II) and Fe(II) M(Fe, Co, Ni)/Mn = 0.05 and 0.10 Lattice replacement: enter the vacancy by replacing Mn(IV) or Mn(III) (Yin et al., 2013) δ-MnO2 Pt(II) pH = 8.0, C0 = 5–500 ppm, I = 0.12 mol/L, m/v = 0.5g/L Lattice replacement: substitution on MnO2 structure (Maeno et al., 2016). 2.3 Formation of association minerals Birnessite Tl(I) pH = 8.0, C0 = 3 mg/L, m/v = 0.012 g/L, T = 298 K Lattice replacement: mainly retained in the vacancy (Chen et al., 2019a; Cruz- Hernández et al., 2019) Ferromanganese nodules Fe(II) Sampled from the sea and analyzed Forming associated minerals (Liu et al., 2017; Marcus et al., 2004; Jiang et al., 2009) Hetaerolite Zn(II) pH = 4.0, C = 0.5–5.0 ppm, m/v = 1.67g/L Forming associated minerals (Yin et al., 2018; Kwon et al., 2009) 3. Environmental Purification Of The Interaction Between Manganese Minerals And Metals Manganese minerals are critical components in the Earth system. Due to large specific surface area and high surface reactivities, they are recognized as ubiquitous geo-sorbents and geo-catalysts, regulating the immigration, transformation, crystallization, and bioavailability of metal ions in the environment through adsorption or redox reactions. In turn, metals can change the microstructural morphology, surface functional groups, electronic structure, adsorption and oxidation catalysis of manganese minerals (Wang et al., 2019; Li et al., 2019b). Thus, environmental purification of the interaction between manganese minerals and metals includes two aspects: (1) the immobilization of metals; (2) metals anchored on manganese minerals affect the environmental geochemical M(Fe, Co, Ni)/Mn = 0.05 and 0.10 3. Environmental Purification Of The Interaction Between Manganese Minerals And Metals 3. Environmental Purification Of The Interaction Between Manganese Minerals And Metals Manganese minerals are critical components in the Earth system. Due to large specific surface area and high surface reactivities, they are recognized as ubiquitous geo-sorbents and geo-catalysts, regulating the immigration, transformation, crystallization, and bioavailability of metal ions in the environment through adsorption or redox reactions. In turn, metals can change the microstructural morphology, surface functional groups, electronic structure, adsorption and oxidation catalysis of manganese minerals (Wang et al., 2019; Li et al., 2019b). Thus, environmental purification of the interaction between manganese minerals and metals includes two aspects: (1) the immobilization of metals; (2) metals anchored on manganese minerals affect the environmental geochemical processes of other toxic pollutants (Timár et al., 2017; Yu et al., 2017a). 3.1 Interaction between manganese mineral and Mn 3.1 Interaction between manganese mineral and Mn Page 8/27 A positive correlation has been observed between the adsorbed Mn(II) and the structural Mn(III) in the whole formation process of bio-manganese oxide (BMO)(Watanabe et al., 2012). The structural Mn(III) played a leading role in the reactivity of BMO, since Mn(III) is not only main adsorption site for Mn(II), Mn(III) is also a highly active intermediates in manganese minerals (Sun et al., 2015). The adsorption performance of coexisting As(V) for manganese minerals was closely related to the structural Mn(III) and adsorption of Mn(II) (Liu et al., 2019). This shows that the interaction of manganese minerals and Mn(II)/Mn(III) affect the circulation of As(V) in the environment. In the absence of Mn(II), Zn(II) was adsorbed as a tetrahedral and TCS complex at the layer vacancy of hexagonal birnessite (Lefkowitz and Elzinga, 2015). However, the Zn(II) fixed on hexagonal birnessite was transformed to spinel (Zn(II)1−xMn (II)x Mn(III) 2O4) after the addition of Mn(II). This conversion was driven by the electron transfer from the adsorbed Zn(II) to the structural Mn(IV), then generated Mn(III) surface species for co-precipitation with Zn(II) and Mn(II). For the absence of Mn(II) in aqueous solution, the adsorbed Ni(II) located at the vacancy of hexagonal birnessite in the form of a TCS complex (Lefkowitz and Elzinga, 2017). The induction of Mn(II) to solution led to desorption of Ni(II) and formation of the edge-sharing Ni(II) complex, which was attribute to the competitive displacement between Ni(II) and Mn(II). The interaction of birnessite and Mn(II) at pH 7.5 caused the transformation of birnessite into secondary feitknechtite, which enhanced Ni(II) removal from the solution by incorporating Ni(II). In the absence of Mn(II), Zn(II) was adsorbed as a tetrahedral and TCS complex at the layer vacancy of hexagonal birnessite (Lefkowitz and Elzinga, 2015). However, the Zn(II) fixed on hexagonal birnessite was transformed to spinel (Zn(II)1−xMn (II)x Mn(III) 2O4) after the addition of Mn(II). This conversion was driven by the electron transfer from the adsorbed Zn(II) to the structural Mn(IV), then generated Mn(III) surface species for co-precipitation with Zn(II) and Mn(II). For the absence of Mn(II) in aqueous solution, the adsorbed Ni(II) located at the vacancy of hexagonal birnessite in the form of a TCS complex (Lefkowitz and Elzinga, 2017). The induction of Mn(II) to solution led to desorption of Ni(II) and formation of the edge-sharing Ni(II) complex, which was attribute to the competitive displacement between Ni(II) and Mn(II). 3.1 Interaction between manganese mineral and Mn The interaction of birnessite and Mn(II) at pH 7.5 caused the transformation of birnessite into secondary feitknechtite, which enhanced Ni(II) removal from the solution by incorporating Ni(II). The stability of Cd(II) fixed on birnessite was slightly improved because of the addition of Mn(II) (Bochatay and Persson, 2000; Elzinga, 2011). Under alkaline conditions, the addition of low-concentration Mn(II) to the vacancy- rich birnessite caused the migration of Cd(II) from the vacancy to the edge site; In contrast, the high concentration of Mn(II) reacted with birnessite and generated abundant Mn(III), which co-precipitated with Cd(II) to form an amorphous Cd(II)-Mn(III) complex(Sun et al., 2019). This co-precipitation process reduces the mobility of Cd(II) in the environment. In summary, the interaction of manganese mineral and Mn(II) impacts the geochemical circulation of Mn(II) and other coexisted heavy metals in the environment. 3.2 Interaction between manganese minerals and Fe 3.2 Interaction between manganese minerals and Fe Interactions between Fe and Mn/manganese minerals commonly occur in nature and produce ferromanganese nodules, which impact the migration and transformation of pollutants (Wu et al., 2019c; Sun et al., 2018; Liu et al., 2017). Because of the similar octahedral Sb(V)(OH6) structure to MnO6 of Mn oxides, Sb(V) preferentially adsorbed on Mn oxides in ferromanganese nodules (Qin et al., 2019a); In contrast, tetrahedral AsVO4 3− was adsorbed on ferrite and manganese oxides, mainly through the formation of bidentate-binuclear complexes. The Mn/Fe ratio and mineral composition controls their distribution on the ferromanganese nodule. Platinum group elements (platinum, iridium and ruthenium) were significantly related to some redox sensitive trace metals (Co, Ce and Tl) in ferromanganese nodules(Berezhnaya et al., 2018; Marcus et al., 2018); Similar to the behavior of Co and Ce, platinum group elements were also removed from seawater by suspended ferromanganese nodules. The enrichment mechanism of platinum group elements was due to adsorption and oxidation on the surface of δ- MnO2. Fe-Mn binary oxides (BMFO) as one of production of interactions between Fe and Mn/manganese minerals, are also widely found in the natural environment(Foroutan et al., 2019; Pranudta et al., 2020). Because of their strong affinity to As(III), Fe-Mn binary oxides significantly impact the environmental behavior of As. The synergistic effect between hematite and manganese oxide in Fe-Mn binary oxide exhibited a synergistic effect on the removal of Page 9/27 Page 9/27 As(III)(Zheng et al., 2020). The coordination of adsorption-oxidation plays an important role in As(III) removal(You et al., 2020). The in-situ formation of BFMO can eliminate the species of Fe(II), Mn(II) and As(III & V) simultaneously in the microbial oxidation ecosystem (Bai et al., 2016). The BMO in the BMFO was mainly responsible for Sb(III) oxidation and FeOOH mainly adsorbed Sb(V)(Bai et al., 2017). In systems with coexisting As(III) and Sb(III), in-situ BMFO preferentially oxidized Sb(III) rather than As(III), but the adsorption efficiency of As was higher (Kashiwabara et al., 2008). As(III)(Zheng et al., 2020). The coordination of adsorption-oxidation plays an important role in As(III) removal(You et al., 2020). The in-situ formation of BFMO can eliminate the species of Fe(II), Mn(II) and As(III & V) simultaneously in the microbial oxidation ecosystem (Bai et al., 2016). The BMO in the BMFO was mainly responsible for Sb(III) oxidation and FeOOH mainly adsorbed Sb(V)(Bai et al., 2017). 3.2 Interaction between manganese minerals and Fe In systems with coexisting As(III) and Sb(III), in-situ BMFO preferentially oxidized Sb(III) rather than As(III), but the adsorption efficiency of As was higher (Kashiwabara et al., 2008). The interaction between Fe and Mn/manganese minerals also formed two-dimensional Fe-Mn layered double hydroxide (Fe-Mn-LDH)(Hou et al., 2019). Fe-Mn-LDH had a large internal surface area and abundant surface hydroxyls. The adsorption capacity of As(III) and As(V) was 1.5 and 0.7 mmol·g− 1, respectively, which was larger than that of other oxides(Otgonjargal et al., 2012). Fe-Mn-LDH has also been shown to be conducive to the adsorption of Sb(V) (Cao et al., 2017). FTIR and XPS results showed that Sb(V) was removed mainly through adsorption on the surface of Fe-Mn-LDH. It has been found that Fe-MnMg-LDH showed good performance for Pb(II) and Cd(II) removal (Cyprian Y. Abasi, 2019). When a variety of heavy metals exist at the same time, Fe- MnMg-LDH adsorption capacity for heavy metals decreased in the order Cd(II) > Cu(II) > Pb(II). Heavy metals were removed from aqueous solution through surface adsorption, surface precipitation and ion exchange. The process also changed the morphology and electronegativity of LDH (He et al., 2018; Zhou et al., 2018). M-Mn-LDHs (M = Mg2+, Zn2+, Cu2+, Ni2+, Co2+, Fe3+ and Cr3+) have shown a high catalytic activity for alcohol oxidation(Du et al., 2017). The oxidation ability of the catalysts was related to the manganese valence and lattice oxygen in the LDH. In summary, the interaction between manganese mineral and Fe shows outstanding environmental purification by immobilizing and oxidizing pollutants. 3.3 Interaction between manganese minerals and other metals 3.3 Interaction between manganese minerals and other metals 4. Environmental Functional Materials Based On Synergy Of Mn And Metals Based on the understanding of the synergistic effect among heavy metals, the synthesis of bimetallic composites has been recognized as an effective technology to improve the reactivity of materials for environmental remediation applications(Du et al., 2020; Feng et al., 2016; Li et al., 2020; Lin et al., 2018; Zhao et al., 2018). In turn, the development and use of bimetallic materials for environmental remediation gives a good insight into the synergistic effects derived from metal-mineral interactions (Huang et al., 2017; Chen et al., 2020a; Tang et al., 2014). Recently, massive Mn-based composites have been designed and applied, the number of articles increased year by year from 2010 to 2019, although there was a slight decline in 2020 and 2021, the number of published articles remained at more than 3500 (Fig. 3). 3.3 Interaction between manganese minerals an Co is mainly present in the birnessite structure in the form of Co(III)OOH, replacement of Mn4+ by Co3+ not only increased the negative charge of the film and the hydroxyl content, but also decreased the Mn average oxidation state (Mn-AOS) of birnessite(Yin et al., 2011a). The adsorption capacity of Pb2+ and the oxidation of As(III) was improved by the synergistic effect of Co and Mn(Yin et al., 2011b). Cryptomelane can rapidly oxidize Cr3+ to HCrO4 − and remove 45–66% of total Cr, its adsorption capacity for Pb2+ was also improved due to the increase of the specific surface area and Mn-AOS of cryptomelane after adsorption Cr3+ (Li et al., 2015a). Nickel is rich in manganese minerals, the incorporation of Ni into manganese mineral also affects the environmental behavior of (Javier S.E., and Iñaki Yusta.,2019). The adsorption capacity of Ni-rich birnessite for Pb(II) and Zn(II) decreased, mainly due to the vacancies and edge sites becoming occupied by Ni(II)(Yin et al., 2012). However, the Ni-rich birnessite has a high oxidation capacity and can completely oxidize As(III) in solution at a fast reaction rate. The valence state of V in birnessite was + 5 in the form of oxygen anion (V6O16 2−, VO4 3−), which had tetrahedral symmetry and formed a monodentate-corning shared complex(Yin et al., 2015). The adsorption capacity of V-rich cryptomelane for Pb(II) was significantly improved: At high V contents (V/Mn > 0.07), Pb(II) combined with the V- oxygen anion and formed Pb5(VO4)3Cl precipitate; With the increase of V content, the binding affinity of V-rich birnessite to Pb(II) significantly increased (Chen et al., 2018). These findings indicate that V-rich cryptomelane plays an important role in the in-situ remediation of environments pollution. Page 10/27 In summary, the interaction between manganese minerals and metals might exhibit a synergistic or inhibitory effect on the migration and transformation of pollutants (Fig. 2). The synergy of manganese minerals and metals results from coordination of adsorption and oxidation, convenient electron transfer, abundant oxygen vacancies and fast migration of lattice oxygen. These findings suggest that in the heavy metal–organic compound pollution system, the interface interaction processes of heavy metals and manganese minerals can change their catalytic properties, thus regulating the migration and transformation of toxic organic pollutants. 4.2 Elimination of organic pollutants in water Because of the synergy between metals, Mn-based bimetallic oxides exhibit superior performance for degradation of organic pollutants (Li et al., 2015; Afzal et al., 2017; Hou et al., 2019b; Chen et al., 2019b; Zhang and Wu, 2013; Pan et al., 2021; Peng et al., 2021). The catalytic performance of Mn1.8Fe1.2O4 nanospheres was obviously higher than that of Mn or Fe single-metal oxide (Huang et al., 2017b). Mn was inferred to be the primary active site, with Fe(III) acting as the main adsorption site for BPA, The synergy between Fe and Mn plays a key role in activating peroxymonosulfate (PMS) for Bisphenol A degradation. Compared with Fe3O4-a and MgMnLDO-a, 0.3Ma- MgMnLDO-a exhibited higher paracetamol removal with a high first-order rate constant of 0.116 min− 1 and TOC removal (67.7%), which was ascribed to the synergy of Fe and Mn for the activation of PMS(Chen et al., 2020b). CuO-MgMn-LDO-300 showed good catalytic performance; almost 97% of sulfamethoxazole was removed within 30 min (Chen et al., 2020a). This was attributed to the oxygen vacancies and synergy between Cu and Mn that favored electron transfer. FMO-73 could effectively degrade TC through the transfer of electrons not only from Mn(II) to Mn(III) or from Mn(III) to Mn(IV), but also from Fe(II) to the lattice of Mn(III) or Mn(IV) (Yang et al., 2018c). The synergistic effect of Fe(III)/Mn(IV) promoted the adsorption process and reactive oxygen species that generation for the complete degradation of organic pollutants. At pH 4.0, the maximum adsorption capacity of p- ASA on the Fe-Mn framework and cubic Fe(OH)3 was 1.3 and 0.72 mmol·g− 1, respectively (Joshi et al., 2017). In the FMBO and H2O2 system, the synergistic effect of Fe and Mn mediated the generation of the ·OH radical for sulfamethoxazole degradation (Wu et al., 2019b). Fe-Mn diatomite had a high catalytic oxidation for phenol and its main intermediates (catechol and hydroquinone), and the degradation efficiency was as high as 100% within 50 min (Son et al., 2017). 4.1 Removal of heavy metals from water Based on the synergistic effect of Mn and metals, various Mn-based composites have been designed for heavy metal removal (Xu et al., 2016; Li et al., 2017a; McCann et al., 2018; Wu et al., 2022; Yang et al., 2021). The synergistic effect of oxidation-adsorption between manganese dioxide and other metal oxides have shown good application prospects for As(III) removal (Zhang et al., 2019; Shan and Tong, 2013; Du et al., 2022; Imran et al., 2021). The manganese dioxide in the Fe-Mn binary composite oxidized As(III) to As(V), and the resultant As(V) was adsorbed by iron oxide (Zhang et al., 2007; Zhang et al., 2012). The oxidation of the manganese oxide layer in zero-valent manganese and the adsorption of the iron oxide layer in zero-valent iron showed an outstanding synergistic effect for As(III) oxidation-removal (Amulya et al., 2020). Ce-Mn binary oxide effectively oxidized As(III) to As(V) (Chen et al., 2018b). The synergistic effect between Mn and Ce played important role in As(III) oxidation. The resultant As(V) was adsorbed by cerium oxide. Fe-Mn bimetal oxide (FMBO) is a promising adsorbent for Sb removal in drinking water and wastewater (Liu et al., 2015; Li et al., 2022). The manganese oxide in FMBO dominated the oxidation process from Sb(III) to Sb(V), whereas iron oxide was responsible for the adsorption of Sb(III) and Sb(V) (Xu et al., 2011). These studies revealed the mechanism of oxidative-adsorption for Sb(III) by FMBO. Moreover, the addition of Mn(II) greatly improves the performance of FMBO for Sb(V) removal, because Mn(II) promotes the formation of amorphous iron oxide in FMBO, which play a crucial part in the immobilization process of Sb(V) (Yang et al., 2018b). A complex formed by the interaction of MnO2 and polyhydroxy iron (FeOOH) exhibited a stronger affinity for Tl(I) than pure MnO2 and FeOOH (Chen et al., 2017b). Because the oxidation of MnO2 played an important role for Tl(I) removal, and the adsorption of FeOOH loaded on MnO2 enhanced Tl(I) purification at the same time. Fe-Mn binary oxides showed high efficiency removal capacity for Tl(I) removal, which was ascribed to the strong oxidative power of manganese dioxide and the high adsorption capacity of iron oxides, Tl(I) was removed by the combined surface complexation, oxidation, precipitation and adsorption (Li et al., 2017b). Page 11/27 Page 11/27 Page 11/27 The Mn-based Layered double hydroxides (LDH) and its derivatives showed an excellent ability for heavy metal removal. 4.1 Removal of heavy metals from water The MgMn-LDH prepared by the homogeneous co-precipitation method had an excellent adsorption capacity for Cu(II), and the maximum immobilization capacity for Cu(II) reached 668 mg·g− 1(Chen et al., 2020a). MgMn-LDH and its calcination product (MgMn-LDO) also showed good performance for Cd(II) immobilization (Chen et al., 2019b). The maximum adsorption capacity of a magnetic Mn-based composite (0.3Ma-MgMnLDO-a) for Cd(II) was 3.76 mmol·g− 1 (422.62 mg·g− 1) (Chen et al., 2020b). The Cd(II) adsorption equilibrium was reached within 5 min. The Cd(II) removal activity of 0.3Ma-MgMnLDO-a was significantly higher than Fe3O4-a and MgMnLDO-a. These findings indicate that the synergistic effect of Mn/manganese minerals and Fe is essential for heavy metal removal. 4.3 Purification of atmosphere pollution The synergistic effect in Mn-Co composites greatly contributed to its low-temperature reducibility and fast conversion rate for volatile organic carbon oxidation (Li et al., 2021). Compared with pure MnOx and Co3O4, the oxidation activity of Mn-Co composite for ethyl acetate and n-hexane was highest (Todorova et al., 2011). Because of the synergy of metals, the catalytic performance of Mn-Co composite with a varied mole ratio was much higher than that of the single MnOx and Co3O4 (Tang et al., 2014). The Mn5Co5 composite showed the best activity with T90% for benzene conversions into CO2 were low to 237°C with a high space velocity of 120,000 mL·g− 1·h− 1. Page 12/27 Page 12/27 Page 12/27 The synergistic effect of Mn and Ce led to low temperature reducibility, high capture capability, fast electron transport and abundant oxygen vacancy (Guo et al., 2018; Li et al., 2018; Hyok Ri et al., 2021). The doping of Ce obviously improved the performance of birnessite for methanal removal at low-temperatures (Zhu et al., 2017). Ce- MnO2 with a Ce:Mn mole ratio of 1:10 showed the best activity and achieved complete conversion of methanal at 100°C. Because of the synergistic effect of MnOx and CeO2, the performance of CeO2-MnOx composite in catalytic oxidation of benzene was higher than that of pure CeO2 or MnOx (Yang et al., 2019b, Cuo et al., 2018). The excellent catalytic performance of MnO2@NiO nanocomposites for oxidation of benzene was also related to the synergistic effect between Mn and Ni (Deng et al., 2018). The conversion temperature of 100% benzene was 320°C under 1000 ppm benzene with a space velocity of 120 L·g− 1·h− 1, whereas 380°C was needed for pure MnO2. The doping of Ni significantly improved the catalytic performance of α-MnO2:Ni-MnO2 catalysts not only showed a low-temperature performance (T90 = 199°C), but also showed high catalytic stability and strong water resistance in the toluene oxidation (Dong et al., 2020). Moreover, loading Ni2+ in the octahedral MnO6 framework can not only improve redox performance, lattice oxygen mobility and formation of active lattice oxygen, but also promote toluene adsorption. The close contact of Cu with Mn and synergistic effect of Cu and Mn in Cu-Mn composites played a key role in promoting the catalytic activity (Luo et al., 2019). 4.3 Purification of atmosphere pollution Because Cu doping significantly improved the lattice oxygen activity of ramsdellite MnO2, Cu-doped ramsdellite MnO2 had higher catalytic activity than pure ramsdellite MnO2 for CO oxidation under full solar spectral radiation (Yang et al., 2018d). The presence of Cu in composite improved the reducibility and the adsorption of methanal, which accelerated the oxidation of methanol (Liu et al., 2018). The Cu/Mn composite showed a synergistic effect in terms of activity and selectivity to CO2, the water resistance of the composite also increased with the increase of Cu content (Ibrahim et al., 2019). The combination of Cu(II) and Mn(III) in CuMn-composites favored the interface effect, which enhanced benzene catalytic oxidation (Zhang et al., 2020). The major synergy between Mn and Fe is important for improving the catalytic oxidation of Mn-Fe composites. Compared with pure metal oxides, p-Mn/Fe3O4 exhibits high catalytic performance for benzene oxidation (Du et al., 2019). Fe doping in Mn3 − xFex4 spinel increased lattice defects, oxygen vacancy concentration, specific surface area and mesoporous rate, these features endow Mn3 − xFe4 spinel with high reactivity for benzene oxidation (Liu et al., 2019b). 5. Research Perspectives Therefore, revealing the effect of dissolved organic matter on the synergistic effect of manganese minerals and metals should be consider in in-situ environment remediation. 6. Conclusion Manganese minerals are active in chemical cycling within the environment and have an important role in migration and transformation of metals. The interaction with metals affects the microstructure and properties of manganese minerals, which further regulates the environmental geochemical processes of toxic pollutants. The following conclusions can be drawn from this review: (1) Manganese minerals show strong affinity to metals. The interaction mechanism between manganese minerals and metals includes surface adsorption, lattice replacement and formation of associated minerals. (2) The interaction of manganese minerals and metals might exhibit synergistic or inhibitory effects on the migration and transformation of pollutants in the environment. (3) Based on the synergistic effect between Mn and metals, a large number of Mn-based composites have been designed for environmental remediation. (4) The development and use of Mn-based composites for environmental remediation gives a good insight into the synergistic effect derived from metal-mineral interactions. (5) The synergistic effect between manganese and metals results from the coordination of adsorption and oxidation, convenient electron transfer, abundant oxygen vacancies and fast migration of lattice oxygen. In the further research, it should pay more attention to the role of manganese minerals in regulating the geochemical fate of combined pollutants in environment. The synergistic effect between manganese and metals should also be fully utilized in expanding the resource utilization of manganese minerals and Mn-based composites for environmental remediation. 5. Research Perspectives There are several different forms of interaction between manganese minerals and metals. The synergy or antagonism derived from the interaction shows important environmental significance. However, few studies have focused on the relationship between the forms of interaction and synergy/antagonism effect. The synergistic or antagonism processes of manganese minerals and metals also need to be further clarified, as they will be of considerable importance for understanding the environmental effect and resource utilization of manganese minerals. Page 13/27 In some situations, the interaction of manganese minerals with metals has an inhibitory effect on the reactivity of the manganese minerals because the active sites on the manganese minerals are masked by the metals. In order to achieve in-situ self-purification of polluted environment, measures need to be taken to weaken these inhibitory Page 13/27 effects. It is important to remediate heavy metal-organic polluted environment by in-situ utilization of manganese minerals. The synergistic effect of manganese minerals and metals is conducive to in-situ utilization of manganese minerals. Based on the synergistic effect, a large number of Mn-based composites have been synthesized for environmental remediation, and it has been verified that the synergy plays an important role in pollutant removal. More effective technologies are needed to further strengthen the synergistic effect and expand the utilization of metal-rich manganese minerals. There are many technologies that could be considered, including external energy reinforcement (ultraviolet light, microwave, PMS, H2O2), acid or alkali activation, and high temperature calcination. Metals that have a synergistic effect with manganese minerals, such as Co, Ni, Cu, and Ce, might also cause environmental pollution. Therefore, monitoring and preventive measures are needed in the in-situ utilization of manganese minerals. The risk of metal dissolution should first be evaluated when Mn-based composites is used in practical application. The practice application of environmentally friendly Mn-based composites is encouraged. The actual environmental factors have an important impact on the interaction of metals. The function of the synergistic effect might be affected by environmental factors as using Mn-based composites for environmental remediation. To our knowledge, there are few studies have focused on this. Dissolved organic matter, as active components in the environment, might affect the features of manganese minerals, metals and pollutants. Therefore, revealing the effect of dissolved organic matter on the synergistic effect of manganese minerals and metals should be consider in in-situ environment remediation. Author contributions All authors contributed to the study conception and design. References collection and analysis were performed by Meiqing Chen, Jiayan Wu, Xiaoshan Qiu and Jiang Lu. The first draft of the manuscript was written by Meiqing Chen and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Declarations Acknowledgments Page 14/27 Page 14/27 The authors appreciate financial support from the National Natural Science Foundation of China (Grant Nos. 41972037, 42172042), the Basic and Applied Basic Research Foundation of Guangdong Province (2021A1515110371, 2019B1515120015), Guangdong Special Support Program for Local Innovative and Research Teams Project (2019BT02L218), the Guangzhou Science and Technology Program (202206010055), the Guangdong Science and Technology Program (2020B121201003), and China postdoctoral science foundation (2022M711191). Declaration of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and materials The datasets used during the current study are available from the corresponding author on reasonable request. The datasets used during the current study are available from the corresponding author on reasonable request. he datasets used during the current study are available from the corresponding au References Metal Ion Coordination at the Water–Manganite (γ-MnOOH) Interface. J. Colloid Interf. Sci. 229, 593-599. 9. Cano-Salazar, L. F. , A Martínez-Luévanos, Claudio-Rizo, J. A. , Carrillo-Pedroza, F. R. , Montemayor, S. M. , Rangel-Mendez, J. R. 2020. Synthesis, structural characterization and Cu(II) adsorption behavior of manganite (g-MnOOH) nanorods. Rsc. Adv. 10, 179-186. 10. Cao, D., Zeng, H., Yang, B., Zhao, X., 2017. Mn assisted electrochemical generation of two-dimensional Fe-Mn layered double hydroxides for efficient Sb(V) removal. J. Hazard. Mater. 336, 33-40. 11. Chawla, M., Dubey, R., Singh, G., Sengupta, S.K., Siril, P.F., 2017. Controlling the morphology of layered double hydroxides of Mn and Co and their exceptional catalytic activities. Thermochim. Acta 654, 130-139. 12. Chen, M., Wu, P., Li, S., Yang, S., Lin, Z., Dang, Z., 2019a. The effects of interaction between vermiculite and manganese dioxide on the environmental geochemical process of thallium. Sci. Total. Environ. 669, 903-910. 13. Chen, S., Chen, H., Li, C., Fan, M., Lv, C., Tian, G., Shu, K., 2017a. Tuning the electrochemical behavior of Cox Mn3−x sulfides by varying different Co/Mn ratios in supercapacitor. J. Mater. Sci. 52, 6687-6696. 14. Chen, M., Wu, P., Yu, L., Liu, S., Ruan, B., Hu, H., Zhu, N., Lin, Z., 2017b. FeOOH-loaded MnO2 nano-composite: An efficient emergency material for thallium pollution incident. J. Environ. Manage. 192, 31-38. 15. Chen, M., Wu, P., Zhu, N., Dang, Z., Bi, Y., Pei, F., 2020a. Re-utilization of spent Cu2+-immobilized MgMn-layered double hydroxide for efficient sulfamethoxazole degradation: Performance and metals synergy. Chem. Eng. J. 392, 123709. 16. Chen, M., Liu, J., Bi, Y., Rehman, S., Dang, Z., Wu, P., 2020b. Multifunctional magnetic MgMn-oxide composite for efficient purification of Cd2+ and paracetamol pollution: Synergetic effect and stability. J. Hazard. Mater. 388, 122078. 17. Chen, M., Wu, P., Huang, Z., Liu, J., Li, Y., Zhu, N., Dang, Z., Bi, Y., 2019b. Environmental application of MgMn- layered double oxide for simultaneous efficient removal of tetracycline and Cd pollution: Performance and mechanism. J. Environ. Manage. 246, 164-173. 18. Chen, S., Zhang, S., Wang, T., Lei, Z., Zhu, M., Dai, X., Liu, F., Li, J., Yin, H., 2018a. Structure and properties of vanadium-doped α-MnO2 and enhanced Pb2+ adsorption phenol/photocatalytic degradation. Mater. Chem. Phys. 208, 258-267. 19. Chen, J., Wang, J., Zhang, G., Wu, Q., Wang, D., 2018b. Facile fabrication of nanostructured cerium- manganese binary oxide for enhanced arsenite removal from water. Chem. Eng. J. 334, 1518-1526. 20. References 1. Afzal, S., Quan, X., Zhang, J., 2017. High surface area mesoporous nanocast LaMO3 (M = Mn, Fe) perovskites for efficient catalytic ozonation and an insight into probable catalytic mechanism. Appl. Catal. B-Environ. 206, 692-703. 1. Afzal, S., Quan, X., Zhang, J., 2017. High surface area mesoporous nanocast LaMO3 (M = Mn, Fe) perovskites for efficient catalytic ozonation and an insight into probable catalytic mechanism. Appl. Catal. B-Environ. 206, 692-703. 2. Amulya, P.P., Priyanka, R., Sanjukta, A., Kumar, B.J., Swain, S.K., 2020. Enhanced performance of a core–shell structured Fe(0)@Fe oxide and Mn(0)@Mn oxide (ZVIM) nanocomposite towards remediation of arsenic contaminated drinking water. J. Mater. Chem. A 8, 4318-4333. 2. Amulya, P.P., Priyanka, R., Sanjukta, A., Kumar, B.J., Swain, S.K., 2020. Enhanced performance of a core–shell structured Fe(0)@Fe oxide and Mn(0)@Mn oxide (ZVIM) nanocomposite towards remediation of arsenic contaminated drinking water. J. Mater. Chem. A 8, 4318-4333. 3. Atkins, A.L., Shaw, S., Peacock, C.L., 2014. Nucleation and growth of todorokite from birnessite: Implications for trace-metal cycling in marine sediments. Geochim. Cosmochim. Acta 144, 109-125. 3. Atkins, A.L., Shaw, S., Peacock, C.L., 2014. Nucleation and growth of todorokite from birnessite: Implications for trace-metal cycling in marine sediments. Geochim. Cosmochim. Acta 144, 109-125. 4. Bai, Y., Jefferson, W.A., Liang, J., Yang, T., Qu, J., 2017. Antimony oxidation and adsorption by in-situ formed biogenic Mn oxide and Fe–Mn oxides. J. Environ. Sci-China 54, 126-134. 4. Bai, Y., Jefferson, W.A., Liang, J., Yang, T., Qu, J., 2017. Antimony oxidation and adsorption by in-situ formed biogenic Mn oxide and Fe–Mn oxides. J. Environ. Sci-China 54, 126-134. 5. Bai, Y., Yang, T., Liang, J., Qu, J., 2016. The role of biogenic Fe-Mn oxides formed in situ for arsenic oxidation and adsorption in aquatic ecosystems. Water Res. 98, 119-127. 5. Bai, Y., Yang, T., Liang, J., Qu, J., 2016. The role of biogenic Fe-Mn oxides formed in situ for arsenic oxidation and adsorption in aquatic ecosystems. Water Res. 98, 119-127. 6. Barceloux, D.G., 1999. manganese. Clinical Toxicology 37, 293-307. 7. Berezhnaya, E., Dubinin, A., Rimskaya-Korsakova, M., Safin, T., 2018. Accumulation of Platinum Group Elements in Hydrogenous Fe–Mn Crust and Nodules from the Southern Atlantic Ocean. Minerals-Basel 8, 275. 7. Berezhnaya, E., Dubinin, A., Rimskaya-Korsakova, M., Safin, T., 2018. Accumulation of Platinum Group Elements in Hydrogenous Fe–Mn Crust and Nodules from the Southern Atlantic Ocean. Minerals-Basel 8, 275. Page 15/27 Page 15/27 8. Bochatay, L., Persson, P., 2000. References Chigrin, P.G., Kirichenko, E.A., 2018. Catalytic Properties of Zinc Manganites for Carbon Oxidation. Inorg. Mater. 54, 1131-1135. 21. Chukanov, N.V., Aksenov, S.M., Jančev, S., Pekov, I.V., Göttlicher, J., Polekhovsky, Y.S., Rusakov, V.S., Nelyubina, Y.V., Van, K.V., 2016. A new mineral species ferricoronadite, Pb[Mn6 4+(Fe3+, Mn3+)2]O16: mineralogical characterization, crystal chemistry and physical properties. Phys. Chem. Miner. 43, 503-514. 22. Crowther, J.G.D.A., 1982. The oxidation states of cobalt and selected metals in Pacific ferromanganese nodules. Geochim. Cosmochim. Acta 46, 755-759. 23. Cruz-Hernández, Y., Villalobos, M., Marcus, M.A., Pi-Puig, T., Zanella, R., Martínez-Villegas, N., 2019. Tl(I) sorption behavior on birnessite and its implications for mineral structural changes. Geochim. Cosmochim. Acta 248, 356-369. Page 16/27 Page 16/27 24. Cuo, Z., Deng, Y., Li, W., Peng, S., Zhao, F., Liu, H., Chen, Y., 2018. Monolithic Mn/Ce-based catalyst of fibrous ceramic membrane for complete oxidation of benzene. Appl. Surf. Sci. 456, 594-601. 25. Cyprian Y. Abasi, I.P.E.E., 2019. Synthesis, characterisation of ternary layered double hydroxides (LDH) for sorption kinetics and thermodynamics of Cd2+. Int. J. Biometeorol. 76, 441-455. 26. Deng, Y., Tang, W., Li, W., Chen, Y., 2018. MnO2-nanowire@NiO-nanosheet core-shell hybrid nanostructure derived interfacial Effect for promoting catalytic oxidation activity. Catal. Today 308, 58-63. 27. Dong, Y., Zhao, J., Zhang, J., Chen, Y., Yang, X., Song, W., Wei, L., Li, W., 2020. Synergy of Mn and Ni enhanced catalytic performance for toluene combustion over Ni-doped α-MnO2 catalysts. Chem. Eng. J. 388, 124244. 28. Du, J., Bao, J., Liu, Y., Kim, S.H., Dionysiou, D.D., 2019. Facile preparation of porous Mn/Fe3O4 cubes as peroxymonosulfate activating catalyst for effective bisphenol A degradation. Chem. Eng. J. 376, 119193. 29. Du, Y., Wang, Q., Liang, X., Yang, P., He, Y., Feng, J., Li, D., 2017. The role of various oxygen species in Mn- based layered double hydroxide catalysts in selective alcohol oxidation. Catal. Sci. Technol. 7, 4361-4365. 30. Du, Y., Zhen, S., Wang, J., Ma, Y., Wu, J., Dai, H., 2022. FeOOH-MnO2/Sepiolite and Fe2O3-MnO2/Diatomite: Highly efficient adsorbents for the removal of As(V). Appl. Clay. Sci. 222, 106491. 30. Du, Y., Zhen, S., Wang, J., Ma, Y., Wu, J., Dai, H., 2022. FeOOH-MnO2/Sepiolite and Fe2O3-MnO2/Diatomite: Highly efficient adsorbents for the removal of As(V). Appl. Clay. Sci. 222, 106491. 31. Du, Z., Li, K., Zhou, S., Liu, X., Yu, Y., Zhang, Y., He, Y., Zhang, Y., 2020. Degradation of ofloxacin with heterogeneous photo-Fenton catalyzed by biogenic Fe-Mn oxides. Chem. Eng. J. 380, 122427. References 31. Du, Z., Li, K., Zhou, S., Liu, X., Yu, Y., Zhang, Y., He, Y., Zhang, Y., 2020. Degradation of ofloxacin with heterogeneous photo-Fenton catalyzed by biogenic Fe-Mn oxides. Chem. Eng. J. 380, 122427. 32. Elzinga, E.J., 2011. Reductive Transformation of Birnessite by Aqueous Mn(II). Environ. Sci. Technol. 45, 6366-6372. 33. Feng, Y., Wu, D., Deng, Y., Zhang, T., Shih, K., 2016. Sulfate Radical-Mediated Degradation of Sulfadiazine by CuFeO2 Rhombohedral Crystal-Catalyzed Peroxymonosulfate: Synergistic Effects and Mechanisms. Environ. Sci. Technol. 50, 3119-3127. 34. Foroutan, R., Mohammadi, R., Adeleye, A.S., Farjadfard, S., Esvandi, Z., Arfaeinia, H., Sorial, G.A., Ramavandi, B., Sahebi, S., 2019. Efficient arsenic(V) removal from contaminated water using natural clay and clay composite adsorbents. Environ. Sci. Pollut. R. 26, 29748-29762. 35. Gao, F., Chu, C., Zhu, W., Tang, X., Yi, H., Zhang, R., 2019. High-efficiency catalytic oxidation of nitric oxide over spherical Mn Co spinel catalyst at low temperature. Appl. Surf. Sci. 479, 548-556. 35. Gao, F., Chu, C., Zhu, W., Tang, X., Yi, H., Zhang, R., 2019. High-efficiency catalytic oxidation of nitric oxide over spherical Mn Co spinel catalyst at low temperature. Appl. Surf. Sci. 479, 548-556. 36. Gao, T., Shen, Y., Jia, Z., Qiu, G., Liu, F., Zhang, Y., Feng, X., Cai, C., 2015. Interaction mechanisms and kinetics of ferrous ion and hexagonal birnessite in aqueous systems. Geochem. T. 16, 1-14. 36. Gao, T., Shen, Y., Jia, Z., Qiu, G., Liu, F., Zhang, Y., Feng, X., Cai, C., 2015. Interaction mechanisms and kinetics of ferrous ion and hexagonal birnessite in aqueous systems. Geochem. T. 16, 1-14. 37. Grangeon, S., Manceau, A., Guilhermet, J., Gaillot, A., Lanson, M., Lanson, B., 2012. Zn sorption modifies dynamically the layer and interlayer structure of vernadite. Geochim. Cosmochim. Acta 85, 302-313. 38. Guo, H., Kou, X., Zhao, Y., Wang, S., Sun, Q., Ma, X., 2018. Effect of synergistic interaction between Ce and Mn on the CO2 capture of calcium-based sorbent: Textural properties, electron donation, and oxygen vacancy. Chem. Eng. J. 334, 237-246. 39. He, X., Qiu, X., Hu, C., Liu, Y., 2018. Treatment of heavy metal ions in wastewater using layered double hydroxides: A review. J. Disper. Sci. Technol. 39, 792-801. 40. Holguera, J.G., Etui, I.D., Jensen, L.H.S., Peña, J., 2018. Contaminant loading and competitive access of Pb, Zn and Mn(III) to vacancy sites in biogenic MnO2. Chem. Geol. 502, 76-87. 41. References Hou, L., Li, X., Yang, Q., Chen, F., Wang, S., Ma, Y., Wu, Y., Zhu, X., Huang, X., Wang, D., 2019a. Heterogeneous activation of peroxymonosulfate using Mn-Fe layered double hydroxide: Performance and mechanism for organic pollutant degradation. Sci. Total. Environ. 663, 453-464. Page 17/27 Page 17/27 42. Hou, Z., Ma, J., Fan, C., Peng, M., Komarneni, S., 2019b. Iron activated sodium bisulfite enhances generation of Mn(III) species through the MnO2/bisulfite catalytic process. Ceram. Int. 45, 892-898. 43. Huang, X., Chen, T., Zou, X., Zhu, M., Chen, D., Pan, M., 2017a. The Adsorption of Cd(II) on Manganese Oxide Investigated by Batch and Modeling Techniques. Int. J. Env. Res. Pub. He. 14, 1145. 44. Huang, G., Wang, C., Yang, C., Guo, P., Yu, H., 2017b. Degradation of Bisphenol A by Peroxymonosulfate Catalytically Activated with Mn1.8Fe1.2O4 Nanospheres: Synergism between Mn and Fe. Environ. Sci. Technol. 51, 12611-12618. 45. Hyok Ri, S., Bi, F., Guan, A., Zhang, X., 2021. Manganese-cerium composite oxide pyrolyzed from metal organic framework supporting palladium nanoparticles for efficient toluene oxidation. J. Colloid Interf. Sci. 586, 836- 846. 46. Ibrahim, M., Labaki, M., Nuns, N., Giraudon, J.M., Lamonier, J.F., 2019. Cu-Mn Hydroxyapatite Materials for Toluene Total Oxidation. Chemcatchem. 12, 550-560. 47. Ibrahim, Y., Wadi, V.S., Ouda, M., Naddeo, V., Banat, F., Hasan, S.W., 2022. Highly selective heavy metal ions membranes combining sulfonated polyethersulfone and self-assembled manganese oxide nanosheets on positively functionalized graphene oxide nanosheets. Chem. Eng. J. 428, 131267. 48. Imran, M., Iqbal, M.M., Iqbal, J., Shah, N.S., Khan, Z.U.H., Murtaza, B., Amjad, M., Ali, S., Rizwan, M., 2021. Synthesis, characterization and application of novel MnO and CuO impregnated biochar composites to sequester arsenic (As) from water: Modeling, thermodynamics and reusability. J. Hazard. Mater. 401, 123338 Synthesis, characterization and application of novel MnO and CuO impregnated biochar composites to sequester arsenic (As) from water: Modeling, thermodynamics and reusability. J. Hazard. Mater. 401, 12333 49. Javier S.E., Iñaki Yusta., 2019. Coprecipitation of Co2+, Ni2+ and Zn2+ with Mn(III/IV) Oxides Formed in Metal- Rich MineWaters. Minerals-Basel. 9, 926-947. 50. Jiang, X., Yao, D., Lin, X., Zhai, S., 2009. Factors controlling the distribution of transitional metal elements in marine hydrogenic ferromanganese crusts. J. Ocean U. China 8, 57-64. 51. Jin, C., Li, Z., Huang, M., Ding, X., Zhou, M., Cai, C., Chen, J., 2022. Cadmium immobilization in lake sediment using different crystallographic manganese oxides: Performance and mechanism. J. Environ. Manage. 313, 114995. 52. References Joshi, T.P., Zhang, G., Koju, R., Qi, Z., Liu, R., Liu, H., Qu, J., 2017. The removal efficiency and insight into the mechanism of para arsanilic acid adsorption on Fe-Mn framework. Sci. Total Environ. 601-602, 713-722. 53. Kashiwabara, T. , Mitsunobu, S. , Das, A. , Itai, T. , Tanimizu, M. , & Takahashi, Y. 2008. Oxidation States of Antimony and Arsenic in Marine Ferromanganese Oxides Related to Their Fractionation in Oxic Marine Environment. Chem. Lett. 37, 756-757. 54. Kuang, J., Ba, Z., Li, Z., Jia, Y., Wang, Z., 2019. Fabrication of a superhydrophobic Mg-Mn layered double hydroxides coating on pure magnesium and its corrosion resistance. Surf. Coat. Tech. 361, 75-82. 55. Kwon, K.D., Refson, K., Sposito, G., 2009. Zinc surface complexes on birnessite: A density functional theory study. Geochim. Cosmochim. Acta 73, 1273-1284. 56. Kwon, K.D., Refson, K., Sposito, G., 2010. Surface complexation of Pb(II) by hexagonal birnessite nanoparticles. Geochim. Cosmochim. Acta 74, 6731-6740. 57. Kwon, K.D., Refson, K., Sposito, G., 2013. Understanding the trends in transition metal sorption by vacancy sites in birnessite. Geochim. Cosmochim. Acta 101, 222-232. 58. Lee, J., Ju, J.B., Cho, W.I., Cho, B.W., Oh, S.H., 2013. Todorokite-type MnO2 as a zinc-ion intercalating material. Electrochim. Acta 112, 138-143. Page 18/27 Page 18/27 59. Lefkowitz, J.P., Elzinga, E.J., 2015. Impacts of Aqueous Mn(II) on the Sorption of Zn(II) by Hexagonal Birnessite. Environ. Sci. Technol. 49, 4886-4893. 60. Lefkowitz, J.P., Elzinga, E.J., 2017. Structural alteration of hexagonal birnessite by aqueous Mn(II): Impacts on Ni(II) sorption. Chem. Geol. 466, 524-532. 61. Li, H., Chen, Y., Long, J., Li, X., Jiang, D., Zhang, P., Qi, J., Huang, X., Liu, J., Xu, R., Gong, J., 2017a. Removal of thallium from aqueous solutions using Fe-Mn binary oxides. J. Hazard. Mater. 338, 296-305. 62. Li ,H., Chen, Y., Long, J., Li, X., Jiang D., Zhang, P., Qi, J., Huang, X., Liu, J., Xu, R. 2017b. Removal of thallium from aqueous solutions using Fe-Mn binary oxides. Journal of Hazardous Materials, 338, 296-305. 63. Li, H., Liu, F., Zhu, M., Feng, X., Zhang, J., Yin, H., 2015a. Structure and properties of Co-doped cryptomelane and its enhanced removal of Pb2+ and Cr3+ from wastewater. J. Environ. Sci-China 34, 77-85. 64. Li, G., Li, N., Sun, Y., Qu, Y., Jiang, Z., Zhao, Z., Zhang, Z., Cheng, J., Hao, Z., 2021. Efficient defect engineering in Co-Mn binary oxides for low-temperature propane oxidation. Appl. Catal. B. 282, 119512. References Liu, W., Sun, B., Qiao, J., Guan, X., 2019a. Influence of Pyrophosphate on the Generation of Soluble Mn(III) from Reactions Involving Mn Oxides and Mn(VII). Environ. Sci. Technol. 53, 10227-10235. 76. Liu, L., Sun, J., Ding, J., Zhang, Y., Sun, T., Jia, J., 2019b. Highly Active Mn3–xFexO4 Spinel with Defects for Toluene Mineralization: Insights into Regulation of the Oxygen Vacancy and Active Metals. Inorg. Chem. 58, 13241-13249. 77. Liu, P., Wei, G., Liang, X., Chen, D., He, H., Chen, T., Xi, Y., Chen, H., Han, D., Zhu, J., 2018. Synergetic effect of Cu and Mn oxides supported on palygorskite for the catalytic oxidation of formaldehyde: Dispersion, microstructure, and catalytic performance. Appl. Clay Sci. 161, 265-273. 78. Liu, R., Liu, F., Hu, C., He, Z., Liu, H., Qu, J., 2015. Simultaneous removal of Cd(II) and Sb(V) by Fe–Mn binary oxide: Positive effects of Cd(II) on Sb(V) adsorption. J. Hazard. Mater. 300, 847-854. 79. Lu, A., Li, Y., Ding, H., Xu, X., Li, Y., Ren, G., Liang, J., Liu, Y., Hong, H., Chen, N., Chu, S., Liu, F., Li, Y., Wang, H., Ding, C., Wang, C., Lai, Y., Liu, J., Dick, J., Liu, K., Hochella, M.F., 2019. Photoelectric conversion on Earth’s surface via widespread Fe- and Mn-mineral coatings. P. Natl. Acad. Sci. USA 116, 9741-9746. 80. Luo, M., Cheng, Y., Peng, X., Pan, W., 2019. Copper modified manganese oxide with tunnel structure as efficient catalyst for low-temperature catalytic combustion of toluene. Chem. Eng. J. 369, 758-765. 81. Ma, J., Guo, H., Lei, M., Zhou, X., Li, F., Yu, T., Wei, R., Zhang, H., Zhang, X., Wu, Y., 2015. Arsenic Adsorption and its Fractions on Aquifer Sediment: Effect of pH, Arsenic Species, and Iron/Manganese Minerals. Water, Air, & Soil Poll. 226, 260.1-260.15. 82. Ma, Y., Ma, Y., Wang, Q., Schweidler, S., Botros, M., Fu, T., Hahn, H., Brezesinski, T., Breitung, B., 2021. High- entropy energy materials: challenges and new opportunities. Energ. Environ. Sci. 14, 2295-2883. 83. Machado-Infante, J., Ramírez-Caballero, G., Barajas Meneses, M.J., 2016. Study of the adsorption capacity of Fe(II) dissolved in water by using a mineral rich in Manganese Dioxide (MnO2) from Colombia. Dyna-Bilbao. 83, 223-228. 84. Maeno, M.Y., Ohashi, H., Yonezu, K., Miyazaki, A., Okaue, Y., Watanabe, K., Ishida, T., Tokunaga, M., Yokoyama, T., 2016. References 65. Li, L., Zhang, C., Yan, J., Wang, D., Peng, Y., Li, J., Crittenden, J., 2020. Distinctive bimetallic oxides for enhanced catalytic toluene combustion: Insights into the tunable fabrication of Mn‐Ce hollow structure. Chemcatchem. 12, 2872-2879. 66. Li, M., Kuang, S., Kang, Y., Ma, H., Dong, J., Guo, Z., 2022. Recent advances in application of iron-manganese oxide nanomaterials for removal of heavy metals in the aquatic environment. Sci. Total. Environ. 819, 153157. 67. Li, P., Qian, H., Howard, K.W.F., Wu, J., Lyu, X., 2014. Anthropogenic pollution and variability of manganese in alluvial sediments of the Yellow River, Ningxia, northwest China. Environ. Monit. Assess. 186, 1385-1398. 68. Li, W., Liu, H., Ma, X., Mo, S., Li, S., Chen, Y., 2018. Fabrication of silica supported Mn–Ce benzene oxidation catalyst by a simple and environment-friendly oxalate approach. J. Porous. Mat. 25, 107-117. 69. Li, W., Wu, P., Zhu, Y., Huang, Z., Lu, Y., Li, Y., Dang, Z., Zhu, N., 2015. Catalytic degradation of bisphenol A by CoMnAl mixed metal oxides catalyzed peroxymonosulfate: Performance and mechanism. Chem. Eng. J. 279, 93-102. 70. Li, Y., Liu, F., Xu, X., Liu, Y., Li, Y., Ding, H., Chen, N., Yin, H., Lin, H., Wang, C., Lu, A., 2019a. Influence of heavy metal sorption pathway on the structure of biogenic birnessite: Insight from the band structure and photostability. Geochim. Cosmochim. Acta 256, 116-134. 71. Li, Y., Wang, X., Li, Y., Duan, J., Jia, H., Ding, H., Lu, A., Wang, C., Nie, Y., Wu, X., 2019b. Coupled anaerobic and aerobic microbial processes for Mn-carbonate precipitation: A realistic model of inorganic carbon pool formation. Geochim. Cosmochim. Acta 256, 49-65. 72. Liang, X., Post, J.E., Lanson, B., Wang, X., Zhu, M., Liu, F., Tan, W., Feng, X., Zhu, G., Zhang, X., De Yoreo, J.J., 2020. Coupled morphological and structural evolution of δ-MnO2 to α-MnO2 through multistage oriented assembly processes: the role of Mn(III). Environ. Sci.: Nano 7, 238-249. 73. Lin, J., Guo, Y., Chen, X., Li, C., Lu, S., Liew, K.M., 2018. CO Oxidation over Nanostructured Ceria Supported Bimetallic Cu–Mn Oxides Catalysts: Effect of Cu/Mn Ratio and Calcination Temperature. Catal. Lett. 148, 181-193. 74. Liu, H., Lu, X., Li, J., Chen, X., Zhu, X., Xiang, W., Zhang, R., Wang, X., Lu, J., Wang, R., 2017. Geochemical fates and unusual distribution of arsenic in natural ferromanganese duricrust. Appl. Geochem. 76, 74-87. Page 19/27 Page 19/27 75. References Sorption behavior of the Pt(II) complex anion on manganese dioxide (δ-MnO2): a model reaction to elucidate the mechanism by which Pt is concentrated into a marine ferromanganese crust. Miner. Deposita. 51, 211-218. 85. Marcus, M.A., Manceau, A., Kersten, M., 2004. Mn, Fe, Zn and As speciation in a fast-growing ferromanganese marine nodule. Geochim. Cosmochim. Acta 68, 3125-3136. 86. Marcus, M.A., Toner, B.M., Takahashi, Y., 2018. Forms and distribution of Ce in a ferromanganese nodule. Mar. Chem. 202, 58-66. 86. Marcus, M.A., Toner, B.M., Takahashi, Y., 2018. Forms and distribution of Ce in a ferromanganese nodule. Mar. Chem. 202, 58-66. 87. Marsh, A., Heath, A., Patureau, P., Evernden, P., Walker, P., 2019. Influence of clay minerals and associated minerals in alkali activation of soils. Constr. Build. Mater. 229, 116816. 87. Marsh, A., Heath, A., Patureau, P., Evernden, P., Walker, P., 2019. Influence of clay minerals and associated minerals in alkali activation of soils. Constr. Build. Mater. 229, 116816. 88. McCann, C.M., Peacock, C.L., Hudson-Edwards, K.A., Shrimpton, T., Gray, N.D., Johnson, K.L., 2018. In situ arsenic oxidation and sorption by a Fe-Mn binary oxide waste in soil. J. Hazard. Mater. 342, 724-731. 88. McCann, C.M., Peacock, C.L., Hudson-Edwards, K.A., Shrimpton, T., Gray, N.D., Johnson, K.L., 2018. In situ arsenic oxidation and sorption by a Fe-Mn binary oxide waste in soil. J. Hazard. Mater. 342, 724-731. 89. Najjar, H., Batis, H., 2016. Development of Mn-based perovskite materials: Chemical structure and applications. Catalysis Reviews 58, 371-438. 90. Nicolas-Tolentino, E., Tian, Z., Zhou, H., Xia, G., Suib, S.L., 1999. Effects of Cu2+ Ions on the Structure and Reactivity of Todorokite- and Cryptomelane-Type Manganese Oxide Octahedral Molecular Sieves. Chem. Mater .11, 1733-1741. Page 20/27 Page 20/27 91. Nishi, K., Usui, A., Nakasato, Y., Yasuda, H., 2017. Formation age of the dual structure and environmental change recorded in hydrogenetic ferromanganese crusts from Northwest and Central Pacific seamounts. Ore. Geol. Rev. 87, 62-70. 92. Otgonjargal, E., Kim, Y., Park, S., Baek, K., Yang, J., 2012. Mn-Fe Layered Double Hydroxides for Adsorption of As(III) and As(V). Sep. Sci. Technol. 47, 2192-2198. 92. Otgonjargal, E., Kim, Y., Park, S., Baek, K., Yang, J., 2012. Mn-Fe Layered Double Hydroxides for Adsorption of As(III) and As(V). Sep. Sci. Technol. 47, 2192-2198. 93. Pan, F., Ji, H., Du, P., Huang, T., Wang, C., Liu, W., 2021. References Insights into catalytic activation of peroxymonosulfate for carbamazepine degradation by MnO2 nanoparticles in-situ anchored titanate nanotubes: Mechanism, ecotoxicity and DFT study. J. Hazard. Mater. 402, 123779. 94. Peacock, C.L., Moon, E.M., 2012. Oxidative scavenging of thallium by birnessite: Explanation for thallium enrichment and stable isotope fractionation in marine ferromanganese precipitates. Geochim. Cosmochim. Acta 84, 297-313. 95. Peña, J., Kwon, K.D., Refson, K., Bargar, J.R., Sposito, G., 2010. Mechanisms of nickel sorption by a bacteriogenic birnessite. Geochim. Cosmochim. Acta 74, 3076-3089. 95. Peña, J., Kwon, K.D., Refson, K., Bargar, J.R., Sposito, G., 2010. Mechanisms of nickel sorption by a bacteriogenic birnessite. Geochim. Cosmochim. Acta 74, 3076-3089. 96. Peng, C., Xing, H., Xue, Y., Wang, J., Li, J., Wang, E., 2020. Ratiometric sensing of alkaline phosphatase based on the catalytical activity from Mn-Fe layered double hydroxide nanosheets. Nanoscale 12, 2022-2027. 97. Peng, Y., Tang, H., Yao, B., Gao, X., Yang, X., Zhou, Y., 2021. Activation of peroxymonosulfate (PMS) by spinel ferrite and their composites in degradation of organic pollutants: A Review. Chem. Eng. J. 414, 128800. 98. Pirajno, F., 2015. Intracontinental anorogenic alkaline magmatism and carbonatites, associated mineral systems and the mantle plume connection. Gondwana. Res. 27, 1181-1216. 99. Pranudta, A., Klysubun, W., El-Moselhy, M.M., Padungthon, S., 2020. Synthesis optimization and X-ray absorption spectroscopy investigation of polymeric anion exchanger supported binary Fe/Mn oxides nanoparticles for enhanced As(III) removal. React. Funct. Polym. 147, 104441. 100. Qin, H., Uesugi, S., Yang, S., Tanaka, M., Kashiwabara, T., Itai, T., Usui, A., Takahashi, Y., 2019a. Enrichment mechanisms of antimony and arsenic in marine ferromanganese oxides: Insights from the structural similarity. Geochim. Cosmochim. Acta 257, 110-130. 101. Qin, Z., Liu, F., Lan, S., Li, W., Yin, H., Zheng, L., Zhang, Q., 2019b. Effect of γ-manganite particle size on Zn2+ coordination environment during adsorption and desorption. Appl. Clay Sci. 168, 68-76. 102. Qin, Z., Xiang, Q., Liu, F., Xiong, J., Koopal, L.K., Zheng, L., Ginder-Vogel, M., Wang, M., Feng, X., Tan, W., Yin, H., 2017. Local structure of Cu2+ in Cu-doped hexagonal turbostratic birnessite and Cu2+ stability under acid treatment. Chem. Geol. 466, 512-523. 103. Qin, Z., Yin, H., Wang, X., Zhang, Q., Lan, S., Koopal, L.K., Zheng, L., Feng, X., Liu, F., 2018. The preferential retention of ZnVI over ZnIV on birnessite during dissolution/desorption. Appl. Clay Sci. 161, 169-175. 104. Reykhard, L.Y., Shulga, N.A., 2019. References Fe-Mn nodule morphotypes from the NE Clarion-Clipperton Fracture Zone, Pacific Ocean: Comparison of mineralogy, geochemistry and genesis. Ore. Geol. Rev. 110, 102933. 105. Robinson, D.M., Go, Y.B., Mui, M., Gardner, G., Zhang, Z., Mastrogiovanni, D., Garfunkel, E., Li, J., Greenblatt, M., Dismukes, G.C., 2013. Photochemical Water Oxidation by Crystalline Polymorphs of Manganese Oxides: Structural Requirements for Catalysis. J. Am. Chem. Soc. 135, 3494-3501. 106. Ruiz-Garcia, M., Villalobos, M., Voegelin, A., Pi-Puig, T., Martínez-Villegas, N., Göttlicher, J., 2021. Transformation of hexagonal birnessite upon reaction with thallium(I): Effects of birnessite crystallinit d h lli i E i S i T h l 55 4862 4870 106. Ruiz-Garcia, M., Villalobos, M., Voegelin, A., Pi-Puig, T., Martínez-Villegas, N., Göttlicher, J., 2021. 106. Ruiz-Garcia, M., Villalobos, M., Voegelin, A., Pi-Puig, T., Martínez-Villegas, N., Göttlicher, J., 2021. Transformation of hexagonal birnessite upon reaction with thallium(I): Effects of birnessite crystallinity, pH, and thallium concentration. Environ. Sci. Technol. 55, 4862-4870. Transformation of hexagonal birnessite upon reaction with thallium(I): Effects of birnessite crystallinity, pH, and thallium concentration. Environ. Sci. Technol. 55, 4862-4870. Page 21/27 Page 21/27 107. Sarkar, S., Biswas, A., Purkait, T., Das, M., Kamboj, N., Dey, R.S., 2020. Unravelling the Role of Fe–Mn Binary Active Sites Electrocatalyst for Efficient Oxygen Reduction Reaction and Rechargeable Zn-Air Batteries. Inorg. Chem. 59, 5194-5205. 108. Sasaki, K., Yu, Q., Momoki, T., Kaseyama, T., 2014. Adsorption characteristics of Cs+ on biogenic birnessite. Appl. Clay Sci. 101, 23-29. 109. Shan, C., Tong, M., 2013. Efficient removal of trace arsenite through oxidation and adsorption by magnetic nanoparticles modified with Fe–Mn binary oxide. Water Res. 47, 3411-3421. 110. Shaheen, S.M., Natasha, Mosa, A., El-Naggar, A., Faysal Hossain, M., Abdelrahman, H., Khan Niazi, N., Shahid, M., Zhang, T., Fai Tsang, Y., Trakal, L., Wang, S., Rinklebe, J., 2022. Manganese oxide-modified biochar: production, characterization and applications for the removal of pollutants from aqueous environments - a review. Bioresource. Technol. 346, 126581. 10. Shaheen, S.M., Natasha, Mosa, A., El-Naggar, A., Faysal Hossain, M., Abdelrahman, H., Khan Niazi, N., Shahid, M., Zhang, T., Fai Tsang, Y., Trakal, L., Wang, S., Rinklebe, J., 2022. Manganese oxide-modified biochar: 111. Sherman, D.M., Peacock, C.L., 2010. Surface complexation of Cu on birnessite (δ-MnO2): Controls on Cu in the deep ocean. Geochim. Cosmochim. Acta 74, 6721-6730. 112. Son, B.H.D., Mai, V.Q., Du, D.X., Phong, N.H., Cuong, N.D., Khieu, D.Q., 2017. Catalytic wet peroxide oxidation of phenol solution over Fe–Mn binary oxides diatomite composite. J. References Porous. Mat. 24, 601-611. 113. Sotiles, A., Gomez, N., Da Silva, S., Wypych, F., 2019. Layered Double Hydroxides with the Composition Mn/Al- SO4-A (A = Li, Na, K; Mn:Al ca. 1:1) as Cation Exchangers. J. Brazil. Chem. Soc. 30, 1807-1813. 114. Spinks, S.C., Uvarova, Y., Thorne, R., Anand, R., Reid, N., White, A., Ley-Cooper, Y., Bardwell, N., Gray, D., Meadows, H., LeGras, M., 2017. Detection of zinc deposits using terrestrial ferromanganese crusts. Ore. Geol. Rev. 80, 484-503. 115. Sun, B., Guan, X., Fang, J., Tratnyek, P.G., 2015. Activation of Manganese Oxidants with Bisulfite for Enhanced Oxidation of Organic Contaminants: The Involvement of Mn(III). Environ. Sci. Technol. 49, 12414-12421. 116. Sun, Q., Cui, P., Fan, T., Wu, S., Zhu, M., Alves, M.E., Zhou, D., Wang, Y., 2018. Effects of Fe(II) on Cd(II) immobilization by Mn(III)-rich δ-MnO2. Chem. Eng. J. 353, 167-175. 117. Sun, Q., Cui, P., Zhu, M., Fan, T., Ata-Ul-Karim, S.T., Gu, J., Wu, S., Zhou, D., Wang, Y., 2019. Cd(II) retention and remobilization on δ-MnO2 and Mn(III)-rich δ-MnO2 affected by Mn(II). Environ. Int. 130, 104932. 118. Suttle, N.F., Bell, J., Thornton, I., Agyriaki, A., 2003. Predicting the risk of cobalt deprivation in grazing livestock from soil composition data. Environ. Geochem. Hlth. 25, 33-39. 119. Tang, W., Li, W., Li, D., Liu, G., Wu, X., Chen, Y., 2014. Synergistic Effects in Porous Mn–Co Mixed Oxide Nanorods Enhance Catalytic Deep Oxidation of Benzene. Catal. Lett. 144, 1900-1910. 120. Tarjomannejad, A., Farzi, A., Niaei, A., Salari, D., 2017. NO reduction by CO over LaB0.5B′0.5O3 (B = Fe, Mn, B′=Fe, Mn, Co, Cu) perovskite catalysts, an experimental and kinetic study. J. Taiwan Inst. Chem. E. 78, 200- 211. 121. Timár, Z., Varga, G., Muráth, S., Kónya, Z., Kukovecz, Á., Havasi, V., Oszkó, A., Pálinkó, I., Sipos, P., 2017. Synthesis, characterization and photocatalytic activity of crystalline Mn(II)Cr(III)-layered double hydroxide. Catal. Today 284, 195-201. 122. Todorova, S., Naydenov, A., Kolev, H., Tenchev, K., Ivanov, G., Kadinov, G., 2011. Effect of Co and Ce on silica supported manganese catalysts in the reactions of complete oxidation of n-hexane and ethyl acetate. J. Mater. Sci. 46, 7152-7159. Page 22/27 123. Toner, B., Manceau, A., Webb, S.M., Sposito, G., 2006. Zinc sorption to biogenic hexagonal-birnessite particles within a hydrated bacterial biofilm. Geochim. Cosmochim. Acta 70, 27-43. 124. Villalobos, M., Escobar-Quiroz, I.N., Salazar-Camacho, C., 2014. References The influence of particle size and structure on the sorption and oxidation behavior of birnessite: I. Adsorption of As(V) and oxidation of As(III). Geochim. Cosmochim. Acta 125, 564-581. 125. Wan, W., Xing, Y., Qin, X., Li, X., Liu, S., Luo, X., Huang, Q., Chen, W., 2020. A manganese-oxidizing bacterial consortium and its biogenic Mn oxides for dye decolorization and heavy metal adsorption. Chemosphere. 253, 126627. 126. Wang, Q., Yang, P., Zhu, M., 2018. Structural transformation of birnessite by fulvic acid under anoxic conditions. Environ. Sci. Technol. 52, 1844-1853. 127. Wang, Y., Yang, Y., Jia, S., Wang, X., Lyu, K., Peng, Y., Zheng, H., Wei, X., Ren, H., Xiao, L., Wang, J., Muller, D.A., Abruña, H.D., Hwang, B.J., Lu, J., Zhuang, L., 2019a. Synergistic Mn-Co catalyst outperforms Pt on high-rate oxygen reduction for alkaline polymer electrolyte fuel cells. Nat. Commun. 10. 128. Wang, B., Zou, J., Shen, X., Yang, Y., Hu, G., Li, W., Peng, Z., Banham, D., Dong, A., Zhao, D., 2019b. Nanocrystal supracrystal-derived atomically dispersed Mn-Fe catalysts with enhanced oxygen reduction activity. Nano. Energy. 63, 103851. 129. Wang, Q., Yang, P., Zhu, M., 2019c. Effects of metal cations on coupled birnessite structural transformation and natural organic matter adsorption and oxidation. Geochim. Cosmochim. Acta 250, 292-310. 130. Wang, Y., Feng, X., Villalobos, M., Tan, W., Liu, F., 2012. Sorption behavior of metals on birnessite: Relationship with its Mn average oxidation state and implications for types of sorption sites. Chem. Geol. 292-293, 25-34. 131. Watanabe, J., Tani, Y., Miyata, N., Seyama, H., Mitsunobu, S., Naitou, H., 2012. Concurrent sorption of As(V) and Mn(II) during biogenic manganese oxide formation. Chem. Geol. 306-307, 123-128. 132. Wegorzewski, A. V., Kuhn, T., Dohrmann, R., Wirth R., Grangeon, S., 2015. Mineralogical characterization of individual growth structures of Mn-nodules with different Ni+Cu content from the central Pacific Ocean. Am. Mineral. 100, 2497-2508. , C.L., Lanson, B., Yin, H., Zheng, L., Chen, Z., Tan, W., Qiu, G., Liu, F., Feng, X., 2019a 133. Wu, Z., Peacock, C.L., Lanson, B., Yin, H., Zheng, L., Chen, Z., Tan, W., Qiu, G., Liu, F., Feng, X., 2019a. Transformation of Co-containing birnessite to todorokite: Effect of Co on the transformation and implications for Co mobility. Geochim. Cosmochim. Acta 246, 21-40. Transformation of Co-containing birnessite to todorokite: Effect of Co on the transformation and implications for Co mobility. Geochim. Cosmochim. Acta 246, 21-40. 134. References Hazard. Mater. 354, 107-115. 145. Yang, X., Liu, L., Tan, W., Qiu, G., Liu, F., 2018c. High-performance Cu2+ adsorption of birnessite using electrochemically controlled redox reactions. J. Hazard. Mater. 354, 107-115. 146. Yang, Y., Li, Y., Zeng, M., Mao, M., Lan, L., Liu, H., Chen, J., Zhao, X., 2018d. UV–vis-infrared light-driven photothermocatalytic abatement of CO on Cu doped ramsdellite MnO2 nanosheets enhanced by a photoactivation effect. Appl. Catal. B-Environ. 224, 751-760. 147. Yin, H., Feng, X., Qiu, G., Tan, W., Liu, F., 2011a. Characterization of Co-doped birnessites and application for removal of lead and arsenite. J. Hazard. Mater. 188, 341-349. 147. Yin, H., Feng, X., Qiu, G., Tan, W., Liu, F., 2011a. Characterization of Co-doped birnessites and application for removal of lead and arsenite. J. Hazard. Mater. 188, 341-349. 148. Yin, H., Feng, X., Tan, W., Koopal, L.K., Hu, T., Zhu, M., Liu, F., 2015. Structure and properties of vanadium(V)- doped hexagonal turbostratic birnessite and its enhanced scavenging of Pb2+ from solutions. J. Hazard. Mater. 288, 80-88. 149. Yin, H., Li, H., Wang, Y., Ginder-Vogel, M., Qiu, G., Feng, X., Zheng, L., Liu, F., 2014. Effects of Co and Ni co- doping on the structure and reactivity of hexagonal birnessite. Chem. Geol. 381, 10-20. 150. Yin, H., Liu, F., Feng, X., Hu, T., Zheng, L., Qiu, G., Koopal, L.K., Tan, W., 2013. Effects of Fe doping on the structures and properties of hexagonal birnessites – Comparison with Co and Ni doping. Geochim. Cosmochim. Acta. 117, 1-15. 151. Yin, H., Liu, F., Feng, X., Liu, M., Tan, W., Qiu, G., 2011b. Co2+-exchange mechanism of birnessite and its application for the removal of Pb2+ and As(III). J. Hazard. Mater. 196, 318-326. 152. Yin, H., Sun, J., Yan, X., Yang, X., Feng, X., Tan, W., Qiu, G., Zhang, J., Ginder-Vogel, M., Liu, F., 2020. Effects of Co(II) ion exchange, Ni(II)- and V(V)-doping on the transformation behaviors of Cr(III) on hexagonal turbostratic birnessite-water interfaces. Environ. Pollut. 256, 113462. 153. Yin, H., Tan, W., Zheng, L., Cui, H., Qiu, G., Liu, F., Feng, X., 2012. Characterization of Ni-rich hexagonal birnessite and its geochemical effects on aqueous Pb2+/Zn2+ and As(III). Geochim. Cosmochim. Acta. 93, 47 62. 154. Yin, H., Wang, X., Qin, Z., Ginder-Vogel, M., Zhang, S., Jiang, S., Liu, F., Li, S., Zhang, J., Wang, Y., 2018. References Wu, K., Si, X., Jiang, J., Si, Y., Sun, K., Yousaf, A., 2019b. Enhanced degradation of sulfamethoxazole by Fe– Mn binary oxide synergetic mediated radical reactions. Environ. Sci. Pollut. R. 26, 14350-14361. 135. Wu, F., Owens, J.D., Tang, L., Dong, Y., Huang, F., 2019c. Vanadium isotopic fractionation during the formation of marine ferromanganese crusts and nodules. Geochim. Cosmochim. Acta 265, 371-385. 136. Wu, P., Cui, P., Zhang, Y., Alves, M.E., Liu, C., Zhou, D., Wang, Y., 2022. Unraveling the molecular mechanisms of Cd sorption onto MnOx-loaded biochar produced from the Mn-hyperaccumulator Phytolacca americana. J. Hazard. Mater. 423, 127157. 137. Wu, Z., Lanson, B., Feng, X., Yin, H., Qin, Z., Wang, X., Tan, W., Chen, Z., Wen, W., Liu, F., 2020. Transformation of Ni-containing birnessite to tectomanganate: Influence and fate of weakly bound Ni(II) species. Geochim. Cosmochim. Acta 271, 96-115. 138. Xu, H., Qu, Z., Zong, C., Quan, F., Mei, J., Yan, N., 2016. Catalytic oxidation and adsorption of Hg0 over low- temperature NH3-SCR LaMnO3 perovskite oxide from flue gas. Appl. Catal. B-Environ. 186, 30-40. Page 23/27 Page 23/27 139. Xu, W., Wang, H., Liu, R., Zhao, X., Qu, J., 2011. The mechanism of antimony(III) removal and its reactions on the surfaces of Fe–Mn Binary Oxide. J. Colloid Interf. Sci. 363, 320-326. 140. Yang, J., Wang, J., Zhu, L., Gao, Q., Zeng, W., Wang, J., Li, Y., 2018a. Enhanced electrocatalytic activity of a hierarchical CeO2@MnO2 core-shell composite for oxygen reduction reaction. Ceram. Int. 44, 23073-23079. 141. Yang, K., Zhou, J., Lou, Z., Zhou, X., Liu, Y., Li, Y., Ali Baig, S., Xu, X., 2018b. Removal of Sb(V) from aqueous solutions using Fe-Mn binary oxides: The influence of iron oxides forms and the role of manganese oxides. Chem. Eng. J. 354, 577-588. 142. Yang, P., Post, J.E., Wang, Q., Xu, W., Geiss, R., McCurdy, P.R., Zhu, M., 2019a. Metal adsorption controls stability of layered manganese oxides. Environ. Sci. Technol. 53, 7453-7462. 143. Yang, M., Shen, G., Liu, M., Chen, Y., Wang, Z., Wang, Q., 2019b. Preparation of Ce–Mn Composite Oxides with Enhanced Catalytic Activity for Removal of Benzene through Oxalate Method. Nanomaterials-Basel 9, 197. 144. Yang, R., Fan, Y., Ye, R., Tang, Y., Cao, X., Yin, Z., Zeng, Z., 2021. MnO2-Based Materials for Environmental Applications. Adv. Mater. 33, 2004862. 145. Yang, X., Liu, L., Tan, W., Qiu, G., Liu, F., 2018c. High-performance Cu2+ adsorption of birnessite using electrochemically controlled redox reactions. J. References Coordination geometry of Zn2+ on hexagonal turbostratic birnessites with different Mn average oxidation states and its stability under acid dissolution. J. Environ. Sci-China 65, 282-292. Page 24/27 Page 24/27 155. You, Y., Liang, Y., Peng, S., Lan, S., Lu, G., Feng, X., Shi, Z., 2020. Modeling coupled kinetics of arsenic adsorption/desorption and oxidation in ferrihydrite-Mn(II)/manganese (oxyhydr)oxides systems. Chemosphere 244, 125517. 156. Yu, C., Drake, H., Mathurin, F.A., Åström, M.E., 2017a. Cerium sequestration and accumulation in fractured crystalline bedrock: The role of Mn-Fe (hydr-)oxides and clay minerals. Geochim. Cosmochim. Acta 199, 370- 389. 157. Yu, Q., Ohnuki, T., Kozai, N., Sakamoto, F., Tanaka, K., Sasaki, K., 2017b. Quantitative analysis of radiocesium retention onto birnessite and todorokite. Chem. Geol. 470, 141-151. 158. Zhang, G., Liu, H., Qu, J., Jefferson, W., 2012. Arsenate uptake and arsenite simultaneous sorption and oxidation by Fe–Mn binary oxides: Influence of Mn/Fe ratio, pH, Ca2+, and humic acid. J. Colloid Interf. Sci. 366, 141-146. 159. Zhang, G., Qu, J., Liu, H., Liu, R., Li, G., 2007. Removal mechanism of As(III) by a novel Fe−Mn binary oxide adsorbent: Oxidation and sorption. Environ. Sci. Technol. 41, 4613-4619. 160. Zhang, L., Wu, Y., 2013. Sol-Gel synthesized magnetic MnFe2o4 spinel ferrite nanoparticles as novel catalyst for oxidative degradation of methyl orange. J. Nanomater. 2013, 1-6. 161. Zhang, M., Li, W., Wu, X., Zhao, F., Wang, D., Zha, X., Li, S., Liu, H., Chen, Y., 2020. Low-temperature catalytic oxidation of benzene over nanocrystalline Cu–Mn composite oxides by facile sol–gel synthesis. New. J. Chem. 44, 2442-2451. 162. Zhang, T., Wang, J., Zhang, W., Yang, C., Zhang, L., Zhu, W., Sun, J., Li, G., Li, T., Wang, J., 2019. Amorphous Fe/Mn bimetal–organic frameworks: Outer and inner structural designs for efficient arsenic(III) removal. J. Mater. Chem. A. 7, 2845-2854. 163. Zhang, Z., Yin, H., Tan, W., Koopal, L.K., Zheng, L., Feng, X., Liu, F., 2014. Zn sorption to biogenic bixbyite-like Mn2O3 produced by Bacillus CUA isolated from soil: XAFS study with constraints on sorption mechanism. Chem. Geol. 389, 82-90. 164. Zhao, H., Zhu, M., Li, W., Elzinga, E.J., Villalobos, M., Liu, F., Zhang, J., Feng, X., Sparks, D.L., 2016. Redox Reactions between Mn(II) and Hexagonal Birnessite Change Its Layer Symmetry. Environ. Sci. Technol. 50, 1750-1758. 165. Zhao, W., Feng, X., Tan, W., Liu, F., Ding, S., 2009. Relation of lead adsorption on birnessites with different average oxidation states of manganese and release of Mn2+/H+/K+. J. Environ. References Sci. (China) 21, 520-526. 166. Zhao, X., Niu, C., Zhang, L., Guo, H., Wen, X., Liang, C., Zeng, G., 2018. Co-Mn layered double hydroxide as an effective heterogeneous catalyst for degradation of organic dyes by activation of peroxymonosulfate. Chemosphere 204, 11-21. 167. Zheng, Q., Hou, J., Hartley, W., Ren, L., Wang, M., Tu, S., Tan, W., 2020. As(III) adsorption on Fe-Mn binary oxides: Are Fe and Mn oxides synergistic or antagonistic for arsenic removal? Chem. Eng. J. 389, 124470. 168. Zhou, H., Jiang, Z., Wei, S., Liang, J., 2018. Adsorption of Cd(II) from Aqueous Solutions by a Novel Layered Double Hydroxide FeMnMg-LDH. Water, Air, & Soil Pollut. 229,78.1-78.16. 169. Zhu, L., Wang, J., Rong, S., Wang, H., Zhang, P., 2017. Cerium modified birnessite-type MnO2 for gaseous formaldehyde oxidation at low temperature. Appl. Catal. B-Environ. 211, 212-221. 170. Zhu, M., Ginder-Vogel, M., Parikh, S.J., Feng, X., Sparks, D.L., 2010a. Cation effects on the layer structure of biogenic Mn-Oxides. Environ. Sci. Technol. 44, 4465-4471. Page 25/27 Page 25/27 171. Zhu, M., Ginder-Vogel, M., Sparks, D.L., 2010b. Ni(II) Sorption on Biogenic Mn-Oxides with Varying Mn Octahedral Layer Structure. Environ. Sci. Technol. 44, 4472-4478. 171. Zhu, M., Ginder-Vogel, M., Sparks, D.L., 2010b. Ni(II) Sorption on Biogenic Mn-Oxides with Varying Mn Octahedral Layer Structure. Environ. Sci. Technol. 44, 4472-4478. Figures Figures Figure 1 The interaction mechanisms between manganese minerals and metals: (a) surface adsorption; (b) lattice replacement; (c) formation of association minerals (represented by the ferromanganese nodule). Figure 1 The interaction mechanisms between manganese minerals and metals: (a) surface adsorption; (b) lattice replacement; (c) formation of association minerals (represented by the ferromanganese nodule). Figure 2 Page 26/27 Environment purification effect of interaction between manganese minerals and metals Environment purification effect of interaction between manganese minerals and metals Figure 3 Figure 3 Illustration of studies on the synthesis and application of Mn-based composites Illustration of studies on the synthesis and application of Mn-based composites Page 27/27 Page 27/27
https://openalex.org/W2125449237	https://ro-journal.biomedcentral.com/counter/pdf/10.1186/1748-717X-8-103	English	null	Selective use of postoperative neck radiotherapy in oral cavity and oropharynx cancer: a prospective clinical study	Radiation oncology	2,013	cc-by	6,017	Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 RESEARCH Open Access Keywords: Oral cavity and oropharynx cancer, Postoperative radiotherapy, Selective neck irradiation, Lymph node Keywords: Oral cavity and oropharynx cancer, Postoperative radiotherapy, Selective neck irradiation, Lymph node The therapeutic decision depends on the lymph node metastatic status, because tumor control requires treat- ment of both the primary tumor and involved regional nodes [3]. The therapeutic decision depends on the lymph node metastatic status, because tumor control requires treat- ment of both the primary tumor and involved regional nodes [3]. © 2013 Martínez Carrillo 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. Selective use of postoperative neck radiotherapy in oral cavity and oropharynx cancer: a prospective clinical study Miguel Martínez Carrillo1, Isabel Tovar Martín1, Ildefonso Martínez Lara2, José Mariano Ruiz de Almodóvar Rivera3 and Rosario Del Moral Ávila1 Correspondence: martinezcarrillo.miguel@gmail.com 1Department of Radiation Oncology, Virgen de las Nieves University Hospital, Avda. Fuerzas Armadas 4, Granada 18014, Spain Full list of author information is available at the end of the article Abstract Background: In cervical postoperative radiotherapy, the target volume is usually the same as the extension of the previous dissection. We evaluated a protocol of selective irradiation according to the risk estimated for each dissected lymph node level. Methods: Eighty patients with oral/oropharyngeal cancer were included in this prospective clinical study between 2005 and 2008. Patients underwent surgery of the primary tumor and cervical dissection, with identification of positive nodal levels, followed by selective postoperative radiotherapy. Three types of selective nodal clinical target volume (CTV) were defined: CTV0, CTV1, and CTV2, with a subclinical disease risk of <10%, 10-25%, and 25% and a prescribed radiation dose of <35 Gy, 50 Gy, and 66–70 Gy, respectively. The localization of node failure was categorized as field, marginal, or outside the irradiated field. Results: A consistent pattern of cervical infiltration was observed in 97% of positive dissections. Lymph node failure occurred within a high-risk irradiated area (CTV1-CTV2) in 12 patients, marginal area (CTV1/CTVO) in 1 patient, and non-irradiated low-risk area (CTV0) in 2 patients. The volume of selective lymph node irradiation was below the standard radiation volume in 33 patients (mean of 118.6 cc per patient). This decrease in irradiated volume was associated with greater treatment compliance and reduced secondary toxicity. The three-year actuarial nodal control rate was 80%. Conclusion: This selective postoperative neck irradiation protocol was associated with a similar failure pattern to that observed after standard neck irradiation and achieved a significant reduction in target volume and secondary toxicity. Materials and methods For this study, we recruited all consecutive patients diag- nosed with squamous cell cancer of the oral cavity or oropharynx between 2005 and 2008 who underwent surgi- cal resection of the primary tumor, without gross residual disease, including unilateral or bilateral neck dissection with a report of histological results by neck level, and who received postoperative radiation therapy prescribed by a multidisciplinary team of head and neck specialists. Writ- ten informed consent was obtained from all recruited patients, and the trial was approved by the hospital ethics committee (Clinical Research Ethical Committe Virgen de las Nieves Hospital). Out of the 80 patients recruited, 5 were subsequently excluded after application of the following exclusion criteria: receipt of <90% of the planned radiotherapy dose (1 patient), interruption of > 2 weeks (1 patient), and the detection of cervical (2 patient), or local (1 patient) relapse during the course of the Surgeons were the first to use the concept of selective treatment, performing selective dissections of the neck to remove only high-risk lymph node levels. This prac- tice led surgeons to divide the neck into different levels according to anatomical references that can be easily identified during neck dissection procedures (i.e. major vessels, muscles, cartilages), known as the Robbins clas- sification [7,8]. Selective neck dissection is currently the procedure of choice for elective neck surgery and for a specific group of neck-positive patients (selected N1 and N2 patients) [9] and is associated with a comparable nodal relapse rate to that observed in patients undergo- ing radical dissection [10-12]. Table 1 Clinical and pathological features Clinical n % Pathological n % Gender Infiltration Male 56 75 Vascular 7 9 Female 19 25 Perineural 13 17 Tumor location Histological grade Oral cavity 58 77 G3 11 15 Oropharynx 17 23 G4 2 3 Performance status ECOG 0-1 70 93 Resection margin Close 13 17 Infiltrated 9 12 Clinical stage Pathological stage I 4 5 I - - II 13 17 II - - III 21 28 III 24 32 IVa 30 40 IVa 48 64 IVb 7 9 IVb 3 4 Clinical N+ 47 63 Pathological N+ 75 100 Extracapsular spread 31 41 Median age of 62 years; range, 39–78 years. ECOG = Eastern Cooperative Oncology Group; N + = Positive lymph node. Table 1 Clinical and pathological features The applicability of the Robbins classification to the definition of radiation target volumes remains controversial. Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Page 2 of 8 neck dissection was carried out in 39 of the 736 patients with clinically positive nodes. Metastatic disease was confirmed in 33% of elective neck dissections and 82% of therapeutic neck dissections. The distribution of patho- logically confirmed metastatic lymph nodes varied according to the primary tumor site. In clinically N0 patients, metastatic lymph nodes were generally observed in levels I-III for oral cavity tumors (20%, 17% and 9% respectively) and in levels II-IV for oropharyngeal tumors (25%, 19% and 8% respectively). A similar pattern was observed in the patients who underwent therapeutic neck dissection except for a higher density of adenopathies and significant pathologic infiltration of an additional level; among tumors of the oral cavity, 44% were in level I, 32% in level II, 16% in level III, and 3% in levels IV; in oropha- ryngeal tumors, 15% were in level I, 71% in level II, 42% in level III, 27% in level IV, and 9% in level V. Pathological infiltration of level V was low, observing a single infiltration of level V in only one patient; it was below 1% when a single pathologically confirmed positive node was detected in levels I-III but increased to 16% when a single positive node was located in level IV. When more than one level was infiltrated, the likelihood of level V involvement progressively increased, reaching 40% when levels I-IV were all involved. Pathological involvement of level I was observed in only 2% of clinical N0 patients with oropharyn- geal tumors, increasing to 15% after therapeutic dissection. These observations illustrate the gradual and orderly infil- tration of node levels in the neck. Target volume dose-dependent toxicity (e.g., oromu- cositis and dysphagia) can limit the correct uninter- rupted administration of adjuvant radiation therapy [21]. Therapeutic compliance would be enhanced if target volumes and, therefore, secondary toxicity could be reduced while maintaining tumor control. Standardized neck dissection by levels can yield data that allow clini- cians to adapt target volumes according to the estimated risk of involvement for each node level. Introduction Squamous cell cancer of the head and neck is diagnosed in 650,000 individuals annually worldwide and accounts for 5-10% of all malignant tumors [1]. The region with the highest incidence is Southern Europe, where there has been a gradual reduction in oral cancer and an increase in oropharyngeal cancer [2]. Standard guidelines for selecting target volumes are based on knowledge of neck tumor distribution and dissemination patterns. The Memorial Sloan-Kettering Cancer Center described the pattern of cervical lymph node metastasis in 1077 naïve patients with head and neck cancer after performing 1118 neck dissections [4-6]. Neck dissection was performed in 343 of the 341 patients with a clinically N0 neck cancer, i.e., bilateral dissection was only performed in two patients. Bilateral Squamous cell cancer of the head and neck follows a consistent and well-defined locoregional growth pattern. Median age of 62 years; range, 39–78 years. ECOG = Eastern Cooperative Oncology Group; N + = Positive lymph node. Materials and methods The standardization of target volumes is challenging, because the relevant anatomical-surgical references are not always identifiable on CT or MR scans. References pro- posed in the 1990s for the radiological definition of neck lymph node levels were inconsistent [13-18], leading to the publication of consensus guidelines for N0 patients in 2003 [19]. In 2006, Gregoire et al. [20] proposed a modification of the definition of neck regions for postoperative radiation therapy, which otherwise appears to have received little research attention. Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Page 3 of 8 Table 2 Nodal surgical procedure Cervical dissection (levels) Ipsilateral Contralateral Total RND (I-V) 7 - 7 MRND (I-V) 33 5 38 SOHND (I-III) 30 9 39 LND (II-IV) 5 1 6 Total 75 15 90 Abbreviations: RND = radical neck dissection; MRND = modified radical neck dissection; SOHND = supraomohyoid neck dissection; LND = lateral neck dissection. Table 2 Nodal surgical procedure by Gregoire et al. for postoperative neck radiotherapy [20]. This radiotherapy was administered to patients with at least two involved lymph nodes or with one involved lymph node generally associated with an unfavorable prognosis (at this level we only identified the infiltrated node, involved lymph nodes with a dia- meter >3 cm, or any extracapsular spread at any other dissected nodal level). N-pathological dissected levels adjacent to nodal levels with extracapsular spread and/ or pN3 were considered high-risk levels. When the involved lymph node was located at the boundary of two levels, both levels were considered high-risk. Levels situ- ated between two or more high-risk levels were included in the target volume, regardless of their pathological status. Abbreviations: RND = radical neck dissection; MRND = modified radical neck dissection; SOHND = supraomohyoid neck dissection; LND = lateral neck dissection. Abbreviations: RND = radical neck dissection; MRND = modified radical neck dissection; SOHND = supraomohyoid neck dissection; LND = lateral neck dissection. radiotherapy. The clinical-pathological characteristics of the patients in the final sample are described in Table 1. Out of these 75 patients, 15 underwent both ipsilateral and contralateral node dissection. The type of dissection depended on the nodal stage, performing selective dissection in N0-N1 patients and dissection of all levels (I-V) in N2-N3 patients (Table 2). Materials and methods According to these criteria and the estimated risk for subclinical disease, we used three types of selective nodal CTV (Table 3) and two types of standard nodal CTV (all dissected neck levels) routinely used at our center for comparison of the results. Nodal levels were intraoperatively identified following standard guidelines and then stained and delineated using surgical clips. After en bloc resection, nodal levels were coded and sent for individual histopathological study with hematoxylin-eosin staining. Adenopathies >3 mm in diameter were identified, and a topographical diagram was created of the involved levels, the number and size of positive nodes, and the presence of extra- capsular spread. The Pinnacle 8.0 version 3, 3D planning system was used, and the treatment was administered by means of a Varian Clinac-D 2100C linear accelerator equipped with multileaf collimator. An isotropic margin of 5 mm was applied to the respective CTVs to generate the appropriate planning target volume (PTV). The dose variation in a PTV was not allowed to exceed +7% or −5% of the prescribed dose. Brain, brainstem, spinal cord, lens, retina, optic nerves, optic chiasm, parotid gland, and mandible, among other structures, were delineated on the CT image. The prescribed dose to the PTV was 50 Gy to the isocenter for PTV1 and a cumulative dose of 66–70 Gy for PTV2. Concomitant cisplatin-based chemotherapy was received by 36 patients with extracapsular spread and/or positive surgical margin (100 mg/m2 cisplatin every 3 weeks by 28 patients and 40 mg/ m2 weekly by 8). Following the standard protocol, postoperative radio- therapy was administered to the site of the primary tumor (close or infiltrated surgical margin, pT4, vascu- lar/perineural invasion, histologically undifferentiated), the dissected side of the neck (pN1 with perinodal inva- sion, skip metastases or inadequate dissection, i.e., total nodal yield ≤5, pN2-N3), and the clinical N0 contrala- teral non-dissected side of the neck with significant risk of subclinical involvement (considered for ipsilateral neck irradiation and/or primary in midline). Post-treatment neck CT examinations with intraven- ous contrast were performed at 2, 6, 12, 18, 24 months and then annually, or when a relapse was suspected. Post-treatment nodal relapse was defined by the appear- ance of an adenopathy in the neck. Materials and methods A nodal relapse The clinical tumor volume (CTV) for elective neck radiotherapy without dissection (clinical contralateral N0) was delineated using an adaptation of consensus guidelines [19] with the above-mentioned modification Table 3 Selection of selective nodal clinical target volume (CTV) Nodal CTV0 Nodal CTV1 Nodal CTV2 Subclinical disease risk <10% 10%-25% >25% Prescribed radiation dose <35 Gy (a) 50 Gy 66-70 Gy Levels dissected Negative level with sufficient dissection Negative and/or positive level without risk factors with insufficient dissection Positive level with risk factors Positive level without risk factors Adjacent negative level to nodal CTV2 (ECS and/or pN3) Levels not dissected - Ipsilateral: Non-dissected level adjacent to infiltrated dissected level - - Contralateral: Levels with risk factors - (a) Referred to mean dose < 35 Gy. Abbreviations: ECS = Extracapsular spread; pN = pathological nodal stage. Table 3 Selection of selective nodal clinical target volume (CTV) Table 3 Selection of selective nodal clinical target volume (CTV) Nodal CTV0 Page 4 of 8 Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Nodal Recurrence CTV1 CTV2 Nodal Recurrence CTV2 CTV1 A B CTV0 CTV1 Nodal Recurrence CTV1 CTV0 Nodal Recurrence C Nodal Recurrence CTV2 CTV2 CTV1 CTV2 CTV1 Nodal Recurrence Figure 1 The radiologic imaging of nodal recurrence volume was co-registered with the pre-treatment CT data set used for treatment planning. (A) DVH analysis of nodal failure “in field”. (B) DVH analysis of nodal failure “marginal field”. (C) DVH analysis of nodal failure “out-field”. Nodal Recurrence CTV1 CTV2 A CTV2 CTV0 CTV1 Nodal Recurrence B CTV1 CTV0 Nodal Recurrence CTV2 CTV1 Nodal Recurrence C Figure 1 The radiologic imaging of nodal recurrence volume was co-registered with the pre-treatment CT data set used for treatment planning. (A) DVH analysis of nodal failure “in field”. (B) DVH analysis of nodal failure “marginal field”. (C) DVH analysis of nodal failure “out-field”. Actuarial locoregional control and survival rates were calculated by using the Kaplan-Meier method. SPSS version 19 (IBM, Chicago IL) was used for all data analyses. detected on the CT scan was confirmed by histological study, and a new CT-simulation scan was performed using the same thermoplastic mask and parameters (isocenter, thickness and number of radiological slices) to calculate the nodal relapse volume (NRV). Materials and methods After obtaining the dose-volume histograms, nodal relapse was classified as: within the irradiated field (>95% of NRV within CTV1 or CTV2); within the margins of the irradiated field (20%-95% of NRV within CTV1 or CTV2); or outside the irradiated field (<20% of NRV within CTV1 or CTV2) (Figure 1). Results As depicted in Figure 2, 1,545 lymph nodes were dissected: 1,294 nodes in ipsilateral dissections and 251 in contralateral dissections, distributed over the five neck levels. Metastasis to at least one of the examined lymph nodes was observed in all 75 ipsilateral necks Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Page 5 of 8 Ipsilateral Contralateral Figure 2 Quantification of nodal levels dissected. Figure 2 Quantification of nodal levels dissected. dissected, which evidenced a total of 121 involved (posi- tive) levels, i.e., a mean of 1.6 positive ipsilateral node levels per patient (1–4). These 121 levels contained a total of 183 metastatic adenopathies, i.e., a mean of 1.5 positive nodes per involved level (1–7). Level II was the most frequently involved in the oral cavity (16/31) and oropharynx (37/90) tumors; among the oral cavity cancers, level Ib was most frequent in tumors of the floor of the mouth (14/27). A regular neck dissemination pattern to levels I to III was observed in 73 (97%) of the 75 ipsilateral dissections, starting in levels Ia/Ib in 39 dissections, level II in 30, and level III in 4. In the other two ipsilateral dissections, one showed involvement of levels Ib, II and IV but not of level III and the other involvement of levels II and V but not of levels III or IV. area (nodal CTV0) in the remaining 2 patients, who therefore did not receive post-treatment radiation thera- py in this zone (Table 5). The dosimetric study of nodal failures revealed a mean NRV of 4.4 cc (1.9-10.2 cc). Mean, minimal, and max- imal doses were, respectively, 6518 cGy, 6420 cGy, and 6894 cGy for patients with nodal failure in the irradiated field; 3091 cGy, 590 cGy, and 5101 cGy for those with failure within the margins of the field; and 2357 cGy, 320 cGy, and 4421 cGy for patients with nodal failure outside the radiation field (Table 5). Out of the 75 patients in the study, 33 (44%) received postoperative selective nodal irradiation, with a mean reduction in target volume of 118.6 cc per patient (21.9 cc-234.7 cc), i.e., nodal CTV0, with a mean reduction of 1.8 levels per patient. Dose stratification by levels allowed us to administer dose-escalated radiotherapy from a dose of 50 Gy, only applying 66–70 Gy in high- risk levels. Results Out of the total of 186 irradiated levels, 40% received a high dose (66–70 Gy: nodal CTV2) and 60% a low dose (50 Gy: nodal CTV1). Only one case of metastasis to a contralateral node was detected (to levels II and Ib). Post-treatment nodal failure was observed in 15 of the 75 patients, local failure in 9, distal failure in 7, and a second primary tumor in 3 (Table 4). All nodal relapse cases were located in the ipsilateral dissected neck. Nodal failure was within high-risk irradiated areas (nodal CTV1, CTV2) in 12 patients, in a marginal area (nodal CTV1/CTV0) in 1 patient, and outside the irradiation After a mean follow-up of 32 months, 45 patients (60%) were alive and disease-free, 2 were alive with disease (3%), 22 patients died from the tumor (29%), and Table 4 Pattern of treatment failure Failure n (%) Post-treatment months (range) Prognostic factors Localization (n) Nodal failure 15 (20) 10.2 (6–19) Age > 62 yrs (p 0.002) - Local failure 9 (12) 11.4 (7–23) Oral c. (tongue) (p = 0.042) - Distal failure 7 (9) 11.7 (6–20) - Lung (5) Bone (1) Skin (1) Second primary tumor 3 (4) 25 (11–46) - Lung (2) Bladder (1) Table 4 Pattern of treatment failure Martínez Carrillo et al. Results Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Page 6 of 8 Table 5 Failure pattern and dosimetric characteristics in patients with nodal recurrence Patient nº Localization nodal failure XRT volume Failure (month) Volume (cc) Minimum dose (cGy) Maximum dose (cGy) Mean dose (cGy) 1 II-III ipsilateral Nodal CTV2 6 9.1 6498.2 6980.0 6602.9 2 III-IV ipsilateral Nodal CTV2 12 3.45 6489.9 6995.9 6629.5 3 II ipsilateral Nodal CTV1 14 2.8 4399.0 6551.2 5554.1 (Nodal CTV2 marginal) 4 III ipsilateral Nodal CTV0 9 9.35 175.1 3113.2 1191.4 5 III ipsilateral Nodal CTV1 8 1.83 5685.5 7005.2 6535.1 II ipsilateral Nodal CTV2 8 2.51 6652.8 6741.6 6701.8 6 II ipsilateral Nodal CTV2 7 3.5 7009.1 7255.1 7149.8 7 V ipsilateral Nodal CTV0 19 3.1 195.0 5050.1 2789.8 8 Ib-II ipsilateral Nodal CTV2 8 3.35 6561.1 7171.2 6805.5 9 II-III ipsilateral Nodal CTV0 17 2.9 589.9 5101.0 3091.0 (nodal CTV1 marginal) 10 Ib ipsilateral Nodal CTV1 11 2.8 6491.0 7001.0 6682.2 II ipsilateral Nodal CTV2 11 1.9 4901.0 6281.2 5402.3 11 II ipsilateral Nodal CTV2 7 9.8 6595.3 6798.4 6639.0 12 Ib ipsilateral Nodal CTV2 7 2.9 6598.2 6792.0 6689.3 13 II ipsilateral Nodal CTV2 8 2.1 6601.4 6850.1 6602.2 14 Ib ipsilateral Nodal CTV2 11 2.8 6599.0 6899.4 6598.0 15 Ib ipsilateral Nodal CTV2 10 10.2 6278.7 7195.5 6658.9 Abbreviations: CTV0 = low-risk clinical target volume; CTV1 = moderate-risk clinical target volume; CTV2 = high-risk clinical target volume. Table 5 Failure pattern and dosimetric characteristics in patients with nodal recurrence Abbreviations: CTV0 = low-risk clinical target volume; CTV1 = moderate-risk clinical target volume; CTV2 = high-risk clinical target volume. 6 died from other causes (8%). Nodal relapse-free survival rates were 84% at 12 months and 79% at 36 months. high-dose volume (CTV2), suggesting the need to dis- cern a radiation-resistant subpopulation within CTV2. Given the likely relationship of radiotherapy-induced toxicity with the target volume and administered dose, this toxicity might be significantly reduced by a selective irradiation approach. In the TROG 91:01 trial, which included 350 head and neck cancer patients, Poulsen et al. Results [23] reported that the target volume in the second phase of treatment had the strongest influence on the development of odynophagia and need for aggressive nutritional intervention (gastrotomy or insertion of naso- gastric tube), finding that the likelihood of developing grade-4 (G-4) odynophagia was 36% when the volume was > 82 mm and 16% when it was <18 mm (p = 0.0001). Author details 1 1Department of Radiation Oncology, Virgen de las Nieves University Hospital, Avda. Fuerzas Armadas 4, Granada 18014, Spain. 2Department of Maxillo- Facial Surgery, Virgen de las Nieves University Hospital, Granada, Spain. 3Department of Radiology and Physical Medicine, University of Granada, Granada, Spain. Received: 21 October 2012 Accepted: 16 February 2013 Published: 28 April 2013 Radiotherapy oncologists have devoted considerable research efforts to measures for the protection of normal Authors’ contributions MMC d d d MMC conceived and developed the project and supervised RT applications. IML participated in this design and coordination. MMC, ITM, IML and RDA collected the data. JMRAR critically reviewed the manuscript. All authors read and approved the final manuscript. The histopathological report plays a key role in the assessment of the dissected specimen. The standard procedure for studying a dissected node after hematoxylin- eosin staining is to remove a longitudinal section for exa- mination under light microscopy to detect metastatic deposits [29]. These partial findings are then extrapolated to the remaining nodal volume. Although an incontrovert- ible diagnosis would be provided by examination of 5-μm sections of the whole node, as in sentinel-node techniques, this is a laborious and costly procedure, and the clinical relevance of the detection of occult subpathological meta- static deposits remains unclear [29]. Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 tissues from radiation, with a focus on minimizing their exposure. However, there have been few attempts to determine the target volume for irradiation in each specific situation, and no standardized patterns of lymph node relapse after postoperative neck radiation therapy have been established, which may be because target volumes generally include all lymph nodes on both sides of the neck. To our best knowledge, the present inves- tigation offers one of the few descriptions of a selective nodal irradiation protocol for patients undergoing sur- gery for oral cavity and pharyngeal cancer according to the estimated risk for each nodal level. the plan with less than six days of interruption; only 3.5% of interruptions were secondary to toxicity, a lower percentage than in previous reports [24,25]. In the study based on SEER’s (Surveillance, Epidemiology and End Results) [26] database, which included 5,086 patients with head-and-neck cancer, 29.6% of the patients treated with surgery and radiotherapy showed treatment discon- tinuity and/or non-adherence (33.3% of those with oral cancer and 33.5% of those with oropharyngeal cancer). The development and incorporation of postoperative selective nodal irradiation techniques into clinical practice requires the routine performance of neck dissection by level. The possibility that the results may be inferior to those obtained with en bloc dissection was refuted by Upile et al., who found no difference in total yield between specimens sent to the histopathologist en bloc or divided into levels [27]. Acknowledgements This work was supported, in part, by Grants-in-Aid for Scientific Research from the Health Andalusian Authority PI-SAS-209/04. Competing interests Th h d l h Competing interests The authors declare that they have no competing interest. Discussion ll Following positive nodal dissection, standard target volume radiotherapy includes all node levels considered to be high-risk by the surgeon, coinciding with the extent of the dissection and including retropharyngeal lymph nodes, which are not usually dissected [13,22]. Selective nodal irradiation hypothetically offers a decrease in target volume with no increase in marginal failures, considering the neck as a set of volumes for mapping high-risk neck regions. Evidently, selective nodal irradi- ation would not significantly reduce the target volume in patients with bulky metastasis and/or multiple neck level involvement. In our series, acute toxicity most frequently presented as mucositis and dysphagia in the patients undergoing radiotherapy (G-3 mucositis in 16%; G-3 dysphagia in 14%) and in those treated with concomitant chemora- diotherapy (G-3 mucositis in 27%; G-4 mucositis in 6%; G-3 dysphagia in 21%). Out of the 75 patients, only 1.3% received <95% of the prescribed radiation dose; the dose administered to neck volumes represented 98.4% of the prescribed dose (nodal CTV1: 99.6%, nodal CTV2: 98%), 90.6% completed their radiation therapy plan with no more than one day of interruption, and all completed It proved possible to administer postoperative selective nodal irradiation to nearly half of the present series of 75 head-and-neck cancer patients, obtaining a significant reduction in target volume. Dose-volume histogram analysis showed that the dose for recurrent nodal disease was comparable to or higher than the target prescrip- tion. Nodal failure was predominantly observed in the Page 7 of 8 Conclusion In conclusion, nodal involvement in oral cavity and pharyngeal cancer follows a predictable and gradual pattern across the different neck levels, supporting the selective neck irradiation approach. The availability of a reliable postoperative nodal-involvement pattern by level allows clinicians to determine target volumes according to the estimated risk for each nodal level. The neck failure pattern obtained after a postoperative selective nodal irradiation protocol does not differ from that obtained after a standard irradiation protocol. Nodal recurrence is most frequently detected in high-risk nodal levels receiving high irradiation doses. The target volume with selective nodal radiotherapy is significantly lower than the target volume with a standard nodal radiotherapy protocol. The variability in the number of nodes obtained from neck dissection can only be partially attributed to ana- tomical differences among patients, given that previous studies indicated a minimum of 28 nodes on each side of the dissected neck [28]. The primary cause of nodal variability is generally related to the surgical technique. Although the boundaries of radical dissection are defined in consensus guidelines [7,8], they may be modi- fied by surgeons based on their own professional expe- rience or because the terminology used to define the different types and dimensions of neck dissection is incon- sistent. A substantial reduction in nodal variability can be expected if surgeons strictly adhere to the anatomical boundaries established in consensus guidelines for dissec- tion procedures. The number of nodes is usually lower in selective procedures, such as supraomohyoid or lateral dissection, due to the smaller number of dissected levels [9-12]. The amount of lymphoid tissue dissected for histological study by level should always be the same, regardless of the type of dissection (radical, modified, or selective). In the present series, there was a minimal variability in the mean number of nodes identified in each level as a function of the type of dissection. Selective nodal irradiation of the neck is not yet a standard treatment. However, greater understanding of the natural history of the tumors and the availability of increasingly sophisticated radiotherapy techniques will facilitate further trials to support oncologists in the opti- mal selection of high-risk target volumes. Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 2. de Souza DL, de Camargo CM, Pérez MM, Curado MP: Trends in the incidence of oral cavity and oropharyngeal cancers in Spain. Head Neck 2012, 34:649–654. 24. Maciá I, Garau M, Solé Monné J, Cambra Serés MJ, Monfà Binefa C, Peraire LM: Compliance to the prescribed overall treatment time (OTT) of curative radiotherapy in normal clinical practice and impact on treatment duration of counteracting short interruptions by treating patients on Saturdays. Clin Transl Oncol 2009, 11:302–311. 2. de Souza DL, de Camargo CM, Pérez MM, Curado MP: Trends in the f 3. Ferlito A, Rinaldo A, Robbins KT, Silver CE: Neck dissection: Past, present and future? J Laryngol Otol 2006, 120:87–92. 25. James ND, Williams MV, Summers ET, Jones K, Cottier B: The management of interruptions to radiotherapy in head and neck cancer: an audit of the effectiveness of National Guidelines. Clin Oncol 2008, 20:599–605. 4. Candela FC, Kothari K, Shah JP: Patterns of cervical node metastases from squamous carcinoma of the oropharynx and hypopharynx. Head Neck 1990, 12:197–203. 5. Shah JP: Patterns of cervical lymph node metastasis from squamous carcinomas of the upper aerodigestive tract. Am J Surg 1990, 160:405–409. 26. Fesinmeyer MD, Mehta V, Tock L, Blough D, McDermott C, Ramsey SD: Completion of radiotherapy for local and regional head and neck cancer in Medicare. Arch Otolaryngol Head Neck Surg 2009, 135:860–867. 6. Shah JP, Candela FC, Poddar AK: The patterns of cervical lymph node metastases from squamous carcinoma of the oral cavity. Cancer 1990, 66:109–113. 27. Upile T, Jerjes W, Nouraei SA, Singh S, Clarke P, Rhys-Evans P, et al: How we do it: a method of neck dissection for histopathological analysis. BMC Surg 2007, 7:21. 7. Robbins KT, Medina JE, Wolfe GT, Levine PA, Sessions RB, Pruet CW: Standardizing neck dissection terminology. Official report of the Academy’s committee for head and neck surgery and oncology. Arch Otolaryngol Head Neck Surg 1991, 117:601–605. g 28. Friedman M, Lim JW, Dickey W, Tanyeri H, Kirshenbaum GL, Phadke DM, et al: Quantification of lymph nodes in selective neck dissection. Laryngoscope 1999, 109:268–370. 29. Debaney K, Rinaldo A, Ferlito A: Micrometastases in cervical lymph nodes from patients with squamous carcinoma of the head and neck: should they be actively sought? Maybe. Am J Otolaryngol 2007, 28:271–274. 8. Robbins KT, Clayman G, Levine PA, Medina J, Sessions R, Shaha A, et al: Neck dissection classification update. References GLOBOCAN 2008. 2008. http://www-dep.iarc.fr/. Page 8 of 8 Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Revisions proposed by American Head and Neck Society and the American Academy of Otolaryngology Head and Neck Surgery. Arch Otolaryngol Head Neck Surg 2002, 128:751–758. doi:10.1186/1748-717X-8-103 Cite this article as: Martínez Carrillo et al.: Selective use of postoperative neck radiotherapy in oral cavity and oropharynx cancer: a prospective clinical study. Radiation Oncology 2013 8:103. 9. Chepeha DB, Hoff PT, Taylor RJ, Bradford CR, Teknos TN, Esclamado RM: Selective neck dissection for the treatment of neck metastasis from squamous cell carcinoma of the head and neck. Laryngoscope 2002, 112:434–438. 10. Ferlito A, Rinaldo A, Silver CE, Gourin CG, Shah JP, Clayman GL, et al: Elective and therapeutic selective neck dissection. Oral Oncol 2006, 42:14–25. 11. Ferlito A, Buckley JG, Shaha AR, Rinaldo A: Rationale for selective neck dissection in tumors of the upper aerodigestive tract. Acta Otolaryngol 2001, 121:548–555. 12. Buckley JG, Feber T: Surgical treatment of cervical node metastases from squamous carcinoma of the upper aerodigestive tract: Evaluation of the evidence for modifications of neck dissections. Head Neck 2001, 23:907–915 13. Grégoire V, Coche E, Cosnard G, Hamoir M, Reychler H: Selection and delineation of lymph node target volume in head and neck conformal radiotherapy. Proposal for standardizing terminology and procedure based on the surgical experience. Radiother Oncol 2000, 56:135–150. 14. Som PM, Curtin HD, Mancuso AA: An imaging based classification for the cervical nodes designed as an adjunct to recent clinically based nodal classifications. Arch Otolaryngol Head Neck Surg 1999, 125:388–396. 15. Nowak PJ, Wijers OB, Lagerwaard FJ, Levendag PC: A three-dimensional CT-based target definition for elective irradiation of the neck. Int J Radiat Oncol Biol Phys 1999, 45:33–39. 16. Gregoire V, Braaksma M, Muller K, Gregoire V, Braaksma M, Muller K: CT-based delineation of the neck node CTV in head and neck conformal radiotherapy (3D-CRT/IMRT): The Bussels-Rotterdam consensus guidelines. Radiother Oncol 2002, 64(Suppl.1):S251–S252. 17. Palazzi M, Soatti C, Bianchi E, Alterio D, Baio A, Bocci C, et al: Guidelines for the delineation of nodal regions of the head and neck on axial tomography images. Tumori 2002, 88:355–360. 17. Palazzi M, Soatti C, Bianchi E, Alterio D, Baio A, Bocci C, et al: Guidelines for the delineation of nodal regions of the head and neck on axial tomography images. Tumori 2002, 88:355–360. 18. Van Triest B, Kaanders J, Jooster F: Optimization of the definition of cervical lymph node regions for 3D radiotherapy in head and neck cancer. Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Radiother Oncol 2002, 64(Suppl 1):S90. 18. Van Triest B, Kaanders J, Jooster F: Optimization of the definition of cervical lymph node regions for 3D radiotherapy in head and neck cancer. Radiother Oncol 2002, 64(Suppl 1):S90. 19. Grégoire V, Levendag P, Ang KK, Bernier J, Braaksma M, Budach V, et al: CT-based delineation of lymph node levels and related CTVs in the node-negative neck: DAHANCA, EORTC, GORTEC, NCIC, RTOG consensus guidelines. Radiother Oncol 2003, 69:227–236. 19. Grégoire V, Levendag P, Ang KK, Bernier J, Braaksma M, Budach V, et al: CT-based delineation of lymph node levels and related CTVs in the node-negative neck: DAHANCA, EORTC, GORTEC, NCIC, RTOG consensus guidelines. Radiother Oncol 2003, 69:227–236. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 20. Grégoire V, Eisbruch A, Hamoir M, Levendag P: Proposal for the delineation of the nodal CTV in the node positive and the postoperative neck. Radiother Oncol 2006, 79:15–20. • Convenient online submission 21. Russo G, Haddad R, Posner M, Machtay M: Radiation treatment breaks and ulcerative mucositis in head neck cancer. Oncologist 2008, 13:886–898. 22. Chao KS, Ozyigit G, Tran BN, Cengiz M, Dempsey JF, Low DA: Patterns of failure in patients receiving definitive and postoperative IMRT for head and neck cancer. Int J Radiat Oncol Biol Phys 2003, 55:312–321. 22. Martínez Carrillo et al. Radiation Oncology 2013, 8:103 http://www.ro-journal.com/content/8/1/103 Chao KS, Ozyigit G, Tran BN, Cengiz M, Dempsey JF, Low DA: Patterns of failure in patients receiving definitive and postoperative IMRT for head and neck cancer. Int J Radiat Oncol Biol Phys 2003, 55:312–321. 23. Poulsen MG, Riddle B, Keller J, Porceddu SV, Tripcony L: Predictors of acute grade 4 swallowing toxicity in patients with stages III and IV squamous carcinoma of the head and neck treated with radiotherapy alone. Radiother Oncol 2008, 87:253–259. 23. Poulsen MG, Riddle B, Keller J, Porceddu SV, Tripcony L: Predictors of acute grade 4 swallowing toxicity in patients with stages III and IV squamous carcinoma of the head and neck treated with radiotherapy alone. Radiother Oncol 2008, 87:253–259.
https://openalex.org/W2111203587	https://cancerci.biomedcentral.com/counter/pdf/10.1186/1475-2867-2-8	English	null	Anti-viral activity of red microalgal polysaccharides against retroviruses.	Cancer cell international	2,002	cc-by	6,066	Cancer Cell International 2002, 2 x Primary research Anti-viral activity of red microalgal polysaccharides against retroviruses Marina M Talyshinsky, Yelena Y Souprun and Mahmoud M Huleihel* Received: 24 January 2002 Accepted: 5 July 2002 Published: 5 July 2002 Cancer Cell International 2002, 2:8 This article is available from: http://www.cancerci.com/content/2/1/8 © 2002 Talyshinsky et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any purpose, provided this notice is preserved along with the article's original URL. Keywords: Red microalgae, polysaccharides, malignant cell transformation, retroviruses, an- tiviral activity Abstract Red microalgal polysaccharides significantly inhibited the production of retroviruses (murine leukemia virus- MuLV) and cell transformation by murine sarcoma virus(MuSV-124) in cell culture. The most effective inhibitory effect of these polysaccharides against both cell transformation and virus production was obtained when the polysaccharide was added 2 h before or at the time of infection. Although, addition of the polysaccharide post-infection significantly reduced the number of transformed cells, but its effect was less marked than that obtained when the polysaccharide was added before or at the time of infection.The finding that the inhibition of cell transformation by MuSV-124 was reversible after removal of the polysaccharide suggested that microalgal polysaccharides inhibited a late step after provirus integration into the host genome. In conclusion, our findings could support the possibility that the polysaccharide may affect early steps in the virus replication cycle, such as virus absorption into the host cells, in addition to its effect on a late step after provirus integration. A class of natural products with low mammalian toxicity that are currently regarded as having antitumor activity are polysaccharides of biological origin, e.g., polysaccharides from yeasts, algae, bacteria, higher plants and fungi [7–9]. Of interest in this context are polysaccharides produced by some species of red algae; these compounds have shown promising activity against a variety of animal virus- es [10–13]. In general, polysaccharides exhibiting antivi- ral potential are highly sulfated [10,14–17]. Dextran sulfate and polysaccharides from marine algae, for exam- ple, have been found to be potent in vitro inhibitors of HIV types 1 and 2 [15,18–20]. They inhibit HIV-1-in- duced cytopathogenicity and HIV-1 antigen expression Page 1 of 7 (page number not for citation purposes) BioMed Central C TIONAL INTERNA CANCER CELL Cancer Cell International BioMed Central C TIONAL INTERNA CANCER CELL Cancer Cell International Characterization of cell transformation by MuSV-124 and MuSV/MuLV NIH/3T3 cells grown in plastic dishes in RPMI medium with 2% NBCS appear as flat cells (Fig 1a); these cells are completely unable to grow in agar. When these cells were infected with an appropriate dilution of MuSV-124, tiny foci of transformed cells, with a highly refractile spindle shape, growing randomly in a criss-cross fashion, could be detected by microscopic observation within five days of infection (Fig. 1b). Later, these foci gradually increased in size and compactness until they became visible to the na- ked eye on day 12 to 14 after infection with MuSV-124. The number of foci remained unchanged in these cultures during the entire culture period. When the cells were in- fected with high titer of MuSV-124 (1 ffu/cell), most of the cells were transformed two-three days after infection. In MuSV/MuLV-infected cultures, the number of foci in- creased continuously, and at any time foci of various sizes (tiny to large) could be detected. Moreover, if these cul- tures were maintained for a sufficiently long time, all the cells eventually became transformed. Examination of the culture medium for the presence of viral reverse tran- scriptase revealed that MuSV/MuLV infection yielded vi- rus-producing cells, whereas MuSV-124 infection resulted in the formation of transformed cells not producing virus. It is therefore likely that the increasing number of foci in the productively infected cells resulted from multiple sec- ondary infections by the virus progenies, the smaller foci being formed by these infections. It was found that the lat- er the infection, the smaller the foci. Both MuSV-124- and MuSV/MuLV-transformed cells could grow efficiently in agar. Table 1: Inhibiting effect of red microalgal polysaccharides on for- mation of foci by MuSV-124 and on virus production, as measured by reverse transcriptase (RT) activity Algal polysaccharide Virus ffu50 protection (µg/ml) RT50 reduction (µg/ml) Porphyridium sp. MuSV-124 10 - MuSV/MuLV - 5 P. aerugineum MuSV-124 500 - MuSV/MuLV - 200 Rhodella reticulata MuSV-124 150 - MuSV/MuLV - 50 NIH/3T3 cells were infected with MuSV-124 or MuSV/MuLV in the presence of various concentrations of algal polysaccharides. ffu50 pro- tection and RT50 reduction represent the concentration of polysac- charide that offers 50% protection against the formation of foci of malignant cells and 50% reduction of RT activity, respectively. Data represent mean values of five separate experiments. Antiretroviral effect of red microalgal polysaccharides Antiretroviral effect of red microalgal polysaccharides Antiretroviral effect of red microalgal polysaccharides The polysaccharide extracted from Porphyridium sp. was more effective in inhibiting retrovirus replication and cell transformation by MuSV than the polysaccharides ob- tained from P. aerugineum or Rhodella reticulata (Table 1). The concentration of Porphyridium sp. polysaccharide re- quired for 50% protection against the formation of foci of transformed cells by MuSV or for a 50% reduction in MuLV production (as evaluated in terms of reverse tran- scriptase activity) was one or two orders of magnitude lower than that needed when P. aerugineum or R. reticulata polysaccharide was applied. We therefore focused our re- search on the anti-retroviral effects of Porphyridium sp. polysaccharide. Background g Retroviruses [1–3] – viruses that contain reverse tran- scriptase, an RNA-directed DNA polymerase [4,5] – have been implicated in various types of human and animal leukemia's and other tumors. Although there are many compounds that exhibit potent anti-viral, and possibly anti-tumor, activity in cell culture and in experimental an- imals, only very few synthetic compounds and one natu- ral product – alpha interferon – have so far been approved for treatment of viral infections in man. Alpha interferon has been approved for treatment of hairy cell leukemia, of Karposi's sarcoma and of genital warts caused by papilo- ma virus [6]. Page 1 of 7 (page number not for citation purposes) Page 1 of 7 (page number not for citation purposes) Cancer Cell International 2002, 2 http://www.cancerci.com/content/2/1/8 [13,18,21,22]. These sulfated polysaccharides also inhibit the activity of purified reverse transcriptase and RNase H, which are essential for retrovirus replication [18,20]. Some previous studies showed that algal polysaccharides exert their inhibitory action at a very early stage (adsorp- tion, fusion or penetration), in the viral infection cycle [9,18,20,23–25], whereas others showed that these polysaccharides did not interfere with virus attachment or penetration, but they did prevent viral protein synthesis [11,26,27]. [13,18,21,22]. These sulfated polysaccharides also inhibit the activity of purified reverse transcriptase and RNase H, which are essential for retrovirus replication [18,20]. Some previous studies showed that algal polysaccharides exert their inhibitory action at a very early stage (adsorp- tion, fusion or penetration), in the viral infection cycle [9,18,20,23–25], whereas others showed that these polysaccharides did not interfere with virus attachment or penetration, but they did prevent viral protein synthesis [11,26,27]. Figure 1 (a) Control uninfected NIH/3T3 cells. (b) NIH/3T3 cells infected and transformed by MuSV (5 days p.i.). The magnifi- cation of these photos is ×40. a b b a b b a Figure 1 Figure 1 (a) Control uninfected NIH/3T3 cells. (b) NIH/3T3 cells infected and transformed by MuSV (5 days p.i.). The magnifi- cation of these photos is ×40. Our previous research has shown potent antiviral activity of a highly sulfated polysaccharide extracted from red microalga. The polysaccharide, which consists mainly of xylose, glucose and galactose [28], exhibits antiviral activ- ity against various members of the herpes family of viruses [29]. In the current study, the activity of this red micro- algal polysaccharide against the replication and the trans- forming ability of the retroviruses, Moloney murine sarcoma virus (MuSV) and Moloney murine leukemia vi- rus (MuLV), was studied. Page 2 of 7 (page number not for citation purposes) Characterization of cell transformation by MuSV-124 and MuSV/MuLV (days) 0 2 4 6 8 1 0 1 2 1 4 %Transformed cels (%) 0 20 40 60 80 100 Ti (d ) 0 1 2 3 4 5 Cell/ Plate (x 106) 0 1 2 3 3 Time p.i. (days) Time (days) Figure 2 Kinetics of the development of MuSV-malignant cell transformation in the presence of Porphyridium sp. polysaccharide, dextran sulfate or carragenaan NIH/ 3T3 cells were infected with 105 ffu of MuSV-124 in the absence or presence of 10 µg/ml of Porhyridium sp. polysac- charide (), dextran sulfate (♦) and carrageenan k (). The treatment with the polysaccharide was continued up to the end of the experiment. Data are mean ± SD, (n = 4). Figure 2 Kinetics of the development of MuSV-malignant cell transformation in the presence of Porphyridium sp. polysaccharide, dextran sulfate or carragenaan NIH/ 3T3 cells were infected with 105 ffu of MuSV-124 in the absence or presence of 10 µg/ml of Porhyridium sp. polysac- charide (), dextran sulfate (♦) and carrageenan k (). The treatment with the polysaccharide was continued up to the end of the experiment. Data are mean ± SD, (n = 4). Figure 2 Kinetics of the development of MuSV-malignant cell transformation in the presence of Porphyridium sp. polysaccharide, dextran sulfate or carragenaan NIH/ 3T3 cells were infected with 105 ffu of MuSV-124 in the absence or presence of 10 µg/ml of Porhyridium sp. polysac- charide (), dextran sulfate (♦) and carrageenan k (). The treatment with the polysaccharide was continued up to the end of the experiment. Data are mean ± SD, (n = 4). Figure 3 Effect of polysaccharides on cell proliferation of NIH/ 3T3 cells NIH/3T3 at 37°C in the absence () or presence of 10 (), 100 () or 1000 µg/ml (♦) of Porphyridium sp. polysaccharide or 100µg/ml of either dextran cells were seeded at a concentration of 0.5 × 106 cells per 9.6-cm2 plate and incubated sulfate () or carrageenan (). Data are mean ± SD, (n = 4). Porphyridium sp. polysaccharide was also superior to other polysaccharides, such as carrageenan and dextran sulfate 500,000, in preventing the transformation of NIH/3T3 cells. Although the anti-transforming activity of Porphyrid- ium sp. polysaccharide did not seem to be much higher than that of carrageenan or dextran sulfate at a concentra- tion of 10 µg/ml (Fig. 2), at the higher concentrations of Porphyridium sp. Characterization of cell transformation by MuSV-124 and MuSV/MuLV polysaccharide was not toxic to the cells whereas the other two biopolymers were extremely toxic to the cells (Fig. 3). Porphyridium sp. polysaccharide had no effect on the proliferation of NIH/3T3 cells, even up to a concentration of 500 µg/ml (results not shown). How- ever, at a concentration of 1,000 µg/ml, Porphyridium sp. polysaccharide caused the cells to stop growing three days after the beginning of the treatment (Fig. 3). with = 100 µg/ml of Porphyridium sp. polysaccharide, from the time of infection until 14 days after infection (scoring time), fully inhibited formation of foci by both virus stocks. Effect of time of addition of Porphyridium sp. polysaccha- ride on cell transformation f The algal polysaccharide (100 µg/ml) was added to NIH/ 3T3 cells at various times before and after infection with either MuSV-124 or MuSV/MuLV. As can be seen from Ta- ble 3, the polysaccharide fully inhibited formation of foci in MuSV-124- and MuSV/MuLV-infected cultures if it was added before or at the time of infection. If the polysaccha- ride was added post-infection, it was less effective. In MuSV/MuLV-infected cultures the polysaccharide was still significantly inhibitory to the formation of foci when add- ed 48 h after infection. The protective effect of the polysac- charide in these cultures was lost only if it was added 72 h after infection, whereas in MuSV-124-infected cultures the effectiveness of the polysaccharide was lost when addition was as early as 48 h after infection. Microscopy showed that there were there were no changes in cell morphology in the presence of Porphyridium sp. polysaccharide, even at a concentration of 1000 µg/ml. Characterization of cell transformation by MuSV-124 and MuSV/MuLV Table 1: Inhibiting effect of red microalgal polysaccharides on for- mation of foci by MuSV-124 and on virus production, as measured by reverse transcriptase (RT) activity NIH/3T3 cells were infected with MuSV-124 or MuSV/MuLV in the presence of various concentrations of algal polysaccharides. ffu50 pro- tection and RT50 reduction represent the concentration of polysac- charide that offers 50% protection against the formation of foci of malignant cells and 50% reduction of RT activity, respectively. Data represent mean values of five separate experiments. Page 2 of 7 (page number not for citation purposes) Cancer Cell International 2002, 2 http://www.cancerci.com/content/2/1/8 Porphyridium sp polysaccharide was also superior to other Figure 2 Kinetics of the development of MuSV-malignant cell transformation in the presence of Porphyridium sp. polysaccharide, dextran sulfate or carragenaan NIH/ 3T3 cells were infected with 105 ffu of MuSV-124 in the absence or presence of 10 µg/ml of Porhyridium sp. polysac- charide (), dextran sulfate (♦) and carrageenan k (). The treatment with the polysaccharide was continued up to the end of the experiment. Data are mean ± SD, (n = 4). Time p.i. (days) 0 2 4 6 8 1 0 1 2 1 4 %Transformed cels (%) 0 20 40 60 80 100 Figure 3 Effect of polysaccharides on cell proliferation of NIH/ 3T3 cells NIH/3T3 at 37°C in the absence () or presence of 10 (), 100 () or 1000 µg/ml (♦) of Porphyridium sp. polysaccharide or 100µg/ml of either dextran cells were seeded at a concentration of 0.5 × 106 cells per 9.6-cm2 plate and incubated sulfate () or carrageenan (). Data are mean ± SD, (n = 4). Time (days) 0 1 2 3 4 5 Cell/ Plate (x 106) 0 1 2 3 Figure 3 Effect of polysaccharides on cell proliferation of NIH/ 3T3 cells NIH/3T3 at 37°C in the absence () or presence of 10 (), 100 () or 1000 µg/ml (♦) of Porphyridium sp. polysaccharide or 100µg/ml of either dextran cells were seeded at a concentration of 0.5 × 106 cells per 9.6-cm2 plate and incubated sulfate () or carrageenan (). Data are mean ± SD, (n = 4). Time (days) 0 1 2 3 4 5 Cell/ Plate (x 106) 0 1 2 3 Time p.i. Page 3 of 7 (page number not for citation purposes) Effect of time of addition of Porphyridium sp. polysaccha- ride on release of progeny virus from MuSV/MuLV-infected cells The algal polysaccharide probably exerted its effect by pre- venting secondary infections, which were the major source of foci scored in these cultures under our experi- mental conditions. To determine whether the effect of the polysaccharide was due to the arrest of virus release from the primary infected cells or merely from blocking the es- tablishment of secondary infections, the polysaccharide was added at various times before and after infection, and the release of progeny virus was followed by assaying viral reverse transcriptase activity in aliquots taken from the culture medium at different times post infection. The re- sults presented in Fig. 4 showed that the infection cycle is completed within 20–24 h after inoculation, this being the time at which the appearance of the first progeny could be detected. The polysaccharide had a significant in- hibitory effect on the release of virus progeny even when it was added 48 h after infection. Therefore, the significant prevention of formation of malignant foci by the polysac- charide at this late time was most likely due to its action against the subsequent secondary infection cycle. These results are in agreement with our suggestion that in MuSV/ MuLV-infected cultures, part of the inhibitory effect of the polysaccharide against cell transformation could be a re- sult of inhibiting secondary viral infections. Table 3: Effect of time of addition of Porphyridium sp. polysach- aride on NIH/3T3 cell transformation Timing of Porphyridiumsp. addition of polysaccharide Number of foci1 MuSV-124 MuSV/MuLV None 80 70 24 h before infection 0 0 2 h before infection 0 0 0 h at infection 0 0 2 h after infection 15 ± 4 6 ± 2 24 h after infection 50 ± 7 14 ± 4 48 h after infection 73 ± 6 30 ± 5 72 h after infection 75 ± 7 64 ± 6 1Means of five cultures ± SD Foci were scored 12 days after infection. h means hours. Table 3: Effect of time of addition of Porphyridium sp. polysach- aride on NIH/3T3 cell transformation Effect of dosage of Porphyridium sp. polysaccharide on cell transformation The algal polysaccharide significantly inhibited malignant transformation of NIH/3T3 cells by MuSV or MuSV/ MuLV. As can be seen from Table 2, continuous treatment Page 3 of 7 (page number not for citation purposes) Page 3 of 7 (page number not for citation purposes) Cancer Cell International 2002, 2 http://www.cancerci.com/content/2/1/8 Table 2: Effect of dosage of Porphyridium sp. polysaccharide on NIH/3T3 cell transformation by MuSV-124 and MuSV/MuLV Porphyridium sp. polysaccharide (µg/ml) Number of foci1 MuSV-124 MuSV/MuLV 0 200 ± 10 120 ± 8 5 150 ± 10 90 ± 7 10 110 ± 8 65 ± 5 50 15 ± 6 10 ± 3 100 0 0 250 0 0 1Mean of 5 cultures ± SD. Foci scored 14 days after infection. Table 2: Effect of dosage of Porphyridium sp. polysaccharide on NIH/3T3 cell transformation by MuSV-124 and MuSV/MuLV Page 4 of 7 (page number not for citation purposes) Effect of time of removal of Porphyridium sp. polysaccha- ride on cell transformation To determine whether it is necessary for Porphyridium sp. polysaccharide to remain in the culture during the whole time until scoring of foci, cells were treated with the polysaccharide 2 h before infection and polysaccharide was removed at various times after infection. Foci were scored 12 days after infection. As can be seen from Table 4, about 75 and 65% of the transforming capacity of MuSV-124 and MuSV/MuLV, respectively, were recovered when the polysaccharide was removed at the time of infec- tion. When Porphyridium sp. polysaccharide was removed 72 h post-infection, only 55% and 20% of transforming capacity of MuSV-124 and MuSV/MuLV, respectively, were recovered. 1Means of five cultures ± SD Foci were scored 12 days after infection. h means hours. The obtained differences between cell cultures infected with either of these virus stocks (MuSV/MuLV or MuSV- 124) are due to the different characteristics of these infec- tions. In the case of MuSV-124-infected cell cultures, the inhibitory effect of Porphyridium sp. polysaccharide could not be explained in terms of the inhibition of secondary viral infections, since this virus yielded a virus nonproduc- ing infection [30,31]. However, in MuSV/MuLV-infected cultures, part of the inhibitory effect of the polysaccharide against cell transformation could be a result of inhibiting secondary viral infections, since the majority of the foci that appeared in the cultures seemed to result from multi- ple secondary infections by the progeny of the primary in- fection. As a consequence of the different character of the infec- tion by these two virus stocks, their focus-forming capaci- ty responded quite differently to the timing of polysaccharide addition and removal. In the case of MuSV/MuLV infected cultures, focus formation was sig- nificantly inhibited even when the polysaccharide was added or removed at longer times after infection com- pared to MuSV-124 infected cultures. These findings indi- cate that the continuous presence of thepolysaccharide in the culture medium after infection with the virus was es- sential for full prevention of malignant transformation Page 4 of 7 (page number not for citation purposes) Cancer Cell International 2002, 2 http://www.cancerci.com/content/2/1/8 6 0 Table 4: Focus formation after removal of Porphyridium sp. polysaccharide Porphyridium sp. Effect of time of removal of Porphyridium sp. polysaccha- ride on cell transformation polysaccharide on cell malignant transformation by MuSV was not a result of a direct interaction between the polysaccharide and the vi- rus particles. In contrast, our previous data (29) showed a strong interaction between Herpes simplex virus (HSV 1 and HSV-2) particles and Porphyridium sp. polysaccharide. This contradiction could be due to differences in viral en- velope composition. Herpes viruses envelope is positively charged, whereas retroviruses are negatively charged. Therefore, the sulfate groups of the polysaccharide could easily interact with positively charged viruses. Figure 4 Effect of time of Porphyridium sp. polysaccharide addition on MuLV progeny release Polysaccharide (100 µg/ml) untreated cells (♦) or treated at the time of infection (), 24 h () or 48 h (♦) postinfection (p.i.), were inocu- lated with MuSV/MuLV. Two hours after inoculation, unad- sorbed virus was removed by three washes, and the cells were covered with fresh medium containing the polysaccha- ride in all treated cultures. Virus release was followed by assaying viral reverse transcriptase activity in aliquots taken from the culture medium at various times after inoculation. h means hours. Data are mean ± SD, (n = 4). Figure 4 Effect of time of Porphyridium sp. polysaccharide addition on MuLV progeny release Polysaccharide (100 µg/ml) untreated cells (♦) or treated at the time of infection (), 24 h () or 48 h (♦) postinfection (p.i.), were inocu- lated with MuSV/MuLV. Two hours after inoculation, unad- sorbed virus was removed by three washes, and the cells were covered with fresh medium containing the polysaccha- ride in all treated cultures. Virus release was followed by assaying viral reverse transcriptase activity in aliquots taken from the culture medium at various times after inoculation. h means hours. Data are mean ± SD, (n = 4). over the tested period (about two weeks). When the treat- ment with the polysaccharide was terminated immediate- ly post-infection (Table 4), there was a significant recovery in the appearance of malignant transformed cells for all tested concentrations of the polysaccharide. This reversi- bility strongly suggests that the polysaccharide, partially at least, exerted its inhibitory effect on a certain event occur- ring after proviral integration. In addition, this reversibil- ity could not be explained only by the possibility of preventing viral reinfections by the polysaccharide be- cause in the case of MuSV-124 infections there are no rei- fections [30,31]. Effect of time of removal of Porphyridium sp. polysaccha- ride on cell transformation polysaccharide Number of foci1 MuSV-124 MuSV/MuLV Not added 100 ± 6 70 ± 5 Not removed 0 0 Removed at: 0 h after infection 75 ± 7 65 ± 4 24 h after infection 64 ± 6 50 ± 5 72 h after infection 55 ± 20 ± 4 1Means of five cultures ± SD 100 µg/ml of Porphyridiumsp. polysach- aride were added 2 h before infection and removed at the indicated times. Foci were scored 12 days postinfection. h means hours. Time p.i. (h.) 0 1 0 2 0 3 0 4 0 5 0 6 0 R.T. (CPMX10 3) 0 2 4 6 8 10 12 Table 4: Focus formation after removal of Porphyridium sp. polysaccharide Porphyridium sp. polysaccharide Number of foci1 MuSV-124 MuSV/MuLV Not added 100 ± 6 70 ± 5 Not removed 0 0 Removed at: 0 h after infection 75 ± 7 65 ± 4 24 h after infection 64 ± 6 50 ± 5 72 h after infection 55 ± 20 ± 4 1Means of five cultures ± SD 100 µg/ml of Porphyridiumsp. polysach- aride were added 2 h before infection and removed at the indicated times. Foci were scored 12 days postinfection. h means hours. Time p.i. (h.) 0 1 0 2 0 3 0 4 0 5 R.T. (CPMX10 3) 0 2 4 6 8 10 12 Table 4: Focus formation after removal of Porphyridium sp. polysaccharide 6 0 1Means of five cultures ± SD 100 µg/ml of Porphyridiumsp. polysach- aride were added 2 h before infection and removed at the indicated times. Foci were scored 12 days postinfection. h means hours. 6 0 1Means of five cultures ± SD 100 µg/ml of Porphyridiumsp. polysach- aride were added 2 h before infection and removed at the indicated times. Foci were scored 12 days postinfection. h means hours. 0 Time p.i. (h.) was supported by our results showing that treatment of the cells with the polysaccharide post-infection caused a significant inhibition of cell transformation, but that this inhibition was less impressive than that obtained when treatment with the polysaccharide was started before or at the time of infection (Table 3). This possibility is also in agreement with various previous studies [9,17,18,22,24,32] that suggested that sulfated polysac- charides prevent early steps in the viral life cycle. In addi- tion, some of our data not presented here showed that the inhibitory effect of Porphyridium sp. Effect of time of removal of Porphyridium sp. polysaccha- ride on cell transformation The inhibitory effect does not seem to be mediated by interferon or by an interferon-like antiviral state, since interferon has been found to inhibit certain events occurring before proviral integration [31]. Page 5 of 7 (page number not for citation purposes) Materials and Methods Cells and viruses 2. Vogt PK: Genetics of RNA tumor viruses. In: Comprehensive Virol- ogy (Edited by: Frankel-Conrat H. Wagner R) New York, Plenum Press 1977, 341-455 NIH/3T3 cells (mouse fibroblast cells) were grown at 37°C in RPMI medium supplemented with 10% new born calf serum (NBCS) and the antibiotics penicillin, strepto- mycin and neomycin. Clone 124 of TB cells chronically releasing Moloney murine sarcoma virus (MuSV-124) (31) was used to prepare a virus stock that contained an approximately 30-fold excess of MuSV particles over Moloney murine leukemia virus (MuLV) particles. MuLV and MuSV used in this research were grown on NIH/3T3 cells. The virus concentration was determined by counting the number of foci (ffu-focus-forming units) in the case of MuSV and by the reverse transcriptase assay in the case of MuLV. 3. Li M, Huang X, Zhu Z, Gorelik E: Sequence and insertion site was of murine melanoma-associated retrovirus. J Virol 1999, 73:9178-9186 4. Baltimore D: RNA-dependent DNA polymerase in virions of RNA tumor viruses. Nature. 1970, 226:1209-1211 5. Temin HM, Mizutani S: RNA dependent DNA polymerase in vir- ions of Rous sarcoma virus. Nature 1970, 226:1211-1213 6. Prusoff W, Lin T, August E, Wood T, Marongiu M: Approaches to antiviral drug development. Yale Biol Med 1989, 62:215-225 4. Baltimore D: RNA-dependent DNA polymerase in virions of RNA tumor viruses. Nature. 1970, 226:1209-1211 6. Prusoff W, Lin T, August E, Wood T, Marongiu M: Approaches to antiviral drug development. Yale Biol Med 1989, 62:215-225 g p 7. Chen Y, Shiao M, Lee S, Wang S: Effect of Cordyceps sinensis on the proliferation and differentiation of human leukemic U937 cells. Life Sciences 1997, 60:2349-2353 8. 8. Kim H, Segoh G, Kim Y, Hong D, Hong N, Yoo I: Stimulation of humoral and cell mediates immunity by polysaccharide from mushroom Phellinus linteus. Int J Immunopharmacol 1996, 18:295- 303 9. Mao XW, Green LM, Gridley DS: Evaluation of polysaccharopep- tide effects against C6 glioma in combination with radiation. Oncology-Basel 2000, 61:243-253 Preparation and purification of microalgal polysaccharide Polysaccharides produced from three species of red micro- alga; Porphyridium sp., P. aerugineum and Rhodella reticula- ta, were used in this study. The polysaccharides were collected and purified as previously described [33]. Brief- ly, these polysaccharides are produced and secreted into the growth medium by the appropriate red microalgae. References 1. Keith H, Wells BA, Bernard J: Biology of retroviruses: Detection, molecular biology and treatment of retroviral infection. J Am Acad Dermatol 1990, 22:1175-1195 Conclusions The present data show that the red microalgal polysaccha- rides profoundly inhibited retroviral malignant cell trans- formation and retrovirus replication. Most effective inhibitory activity of these polysaccarides on cell transfor- mation was obtained when the cells were treated with polysaccharide before or at the time of infection. These re- sults support the possibility that at least part of the inhib- itory effect of the polysaccharide was due to blocking some of the viral receptors, thus interfering with the pen- etration of the virus into the cells. On the other hand, the reversibility of this inhibitory activity strongly suggests that the polysaccharide exerted its inhibitory effect also on Our results do not rule out the possibility that at least part of the inhibitory effect of the polysaccharide was due to blocking some of the viral receptors, thus interfering with the penetration of the virus into the cells. This possibility Page 5 of 7 (page number not for citation purposes) Cancer Cell International 2002, 2 http://www.cancerci.com/content/2/1/8 Cell infection and determination of viral infection 14. Takemoto K, Fahisch P: Inhibition of Herpes simplex virus by nat- ural and synthetic acid polysaccharide. Proc Soc Exp Biol Med 1964, 116:140-144 f f f A monolayer of NIH/3T3 cells was grown in 9-cm2 tissue culture plates and treated with 0.8 µg/ml of polybrene (a cationic polymer required for neutralizing the negative charge of the cell membrane) for 24 h before infection with the virus. Free polybrene was then removed, and the cells were incubated at 37°C for 2 h with the infecting vi- rus (MuSV-124) at various concentrations in RPMI medi- um containing 2% of NBCS. The unabsorbed virus particles were removed, fresh medium containing 2% NBCS was added, and the monolayers were incubated at 37°C. After 2–3 days, the cell cultures were examined for the appearance of malignant transformed cells. The amount of malignant transformed cells was expressed as the percentage of transformed cells in the inspection field or as the number of foci in the infected culture 10 days af- ter infection. 15. Schaeffer DJ, Krylov VS: Anti-HIV activity of extracts and com- pounds from algae and cyanobacteria. Ecotox Environ Safe 2000, 45:208-227 16. Piret J, Lamontagne J, Bestman Smith J, Roy S, Gourde P, Desormeaux A, Omar RF, Juhasz J, Bergeron MG: In vitro and in vivo evalua- tions of sodium lauryl sulfate and dextran sulfate as microbi- cides against herpes simplex and human immunodeficiency viruses. J Clin Microbiol. 2000, 38:110-9 J 17. Haslin C, Lahaye M, Pellegrini M, Chermann JC: In vitro anti-HIV activity of sulfated cell-wall polysaccharides from gametic, carposporic and tetrasporic stages of the Mediterranean red alga Asparagopsis armata. Planta Med. 2001, 67:301-305 18. Baba M, Schols D, DeClercq E: Novel sulfated polymers as highly potent and selective inhibitors of human immunodeficiency virus replication and giant cell formation. Antimicrob Agents Chemotherapy 1990, 34:134-138 19. Hansen J, Shulze T, Mellert W, Moelling K: Identification and char- acterization of HIV-specific RNase H by monoclonal anti- body. EMBO J. 1988, 7:239-243 20. 20. McClure M, Moore J, Blanco D, Scotting P, Cook G, Keyner R, J We- ber, Davies D, Weiss R: Investigations into the mechanism by which sulfated polysaccharides inhibit HIV infection in vitro. AIDS Res Human Retroviruses 1992, 8:19-26 We thank Ms Marion Milner for typing. and editorial review of the manu- script. We thank Ms Marion Milner for typing. and editorial review of the manu- script. Materials and Methods Cells and viruses The medium was collected, cells were removed by centrif- ugation and the supernatant containing the polysaccha- rides was dialyzed and lyophilized. gy 10. Ehresman DW, Deig EF, Hatch MT: Antiviral properties of algal polysaccharides and related compounds. In: Marine algae in pharmaceutical science. Berlin, W. de Gruyter 1979, 293-302 p y 11. Gonzalez M, Alarcon B, Carrasco L: Polysaccharides as antiviral agents: antiviral activity of carrageenan. Antimicrob Agents Chemotherapy 1987, 31:1388-1393 py 12. Marchetti M, Pisani S, Pietropaolo V, Seganti L, Nicoletti R, Orsi N: Inhibition of Herpes simplex virus infection by negatively charged and neutral carbohydrate polymers. J Chemother. 1995, 7:90-96 13. Witvrouw M, DeClerq E: Sulfated polysaccharides extracted from sea algae as potential antiviral drugs. Gen Pharmacol 1997, 29:497-511 Page 6 of 7 (page number not for citation purposes) Acknowledgements a certain event occurring after proviral integration. Thus, it appears that Porphyridium sp. polysaccharide has a plei- otropic mode of action during the infection cycle of MuSV. The exact steps (or step) during the viral replication cycle that are affected by Porphyridium sp. polysaccharide remain to be elucidated. a certain event occurring after proviral integration. Thus, it appears that Porphyridium sp. polysaccharide has a plei- otropic mode of action during the infection cycle of MuSV. The exact steps (or step) during the viral replication cycle that are affected by Porphyridium sp. polysaccharide remain to be elucidated. This research was supported by Ma'OF (established by the Kahanoff Foun- dation). Reverse transcriptase assay 21. Moelling K, Schulze T, Divinger H: Inhibition of human immuno- deficiency virus type 1 RNase H by sulfated polyanions. J Virol 1989, 63:5489-5491 21. Moelling K, Schulze T, Divinger H: Inhibition of human immuno- deficiency virus type 1 RNase H by sulfated polyanions. J Virol 1989, 63:5489-5491 Viral reverse transcriptase activity was assayed as previous- ly described [34]. Page 6 of 7 (page number not for citation purposes) Page 6 of 7 or citation purposes) Page 6 of 7 Cancer Cell International 2002, 2 Cancer Cell International 2002, 2 Carcinogenesis 1990, 11:2097-2102 Page 7 of 7 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMedcentral will be the most significant development for disseminating the results of biomedical research in our lifetime." Paul Nurse, Director-General, Imperial Cancer Research Fund Publish with BMC and your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours - you keep the copyright editorial@biomedcentral.com Submit your manuscript here: http://www.biomedcentral.com/manuscript/ BioMedcentral.com Publish with BioMed Central and every scientist can read your work free of charge "BioMedcentral will be the most significant development for disseminating the results of biomedical research in our lifetime." Paul Nurse, Director-General, Imperial Cancer Research Fund Publish with BMC and your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours - you keep the copyright editorial@biomedcentral.com Submit your manuscript here: http://www.biomedcentral.com/manuscript/ BioMedcentral.com Publish with BioMed Central and every scientist can read your work free of charge Cancer Cell International 2002, 2 http://www.cancerci.com/content/2/1/8 22. De Clercq E: Current lead natural products for the chemo- therapy of human immunodefiency virus (HIV) infection. Med Res Rev 2000, 20:323-349 , 23. Shanon WM: In: Antiviral agents and viral diseases of man (Edited by: Galasso GJ, Merigan TC, Buchanan RA) New York, Raven Press 1984 24. Neyts J, Reymen D, Letourneur D, Jozefonvicz J, Schols D, Este J, An- drei G, McKenna P, Witvrouw M, Ikeda S: Differential antiviral ac- tivity of derivatized dextrans. Biochem Pharmacol. 1995, 7:743-51 y 25. Ohta S, Ono F, Shiomi Y, Nakao T, Aozasa O, Nagate T, Kitamura K, Yamaguchi S, Nishi M, Miyata H: Anti-herpes virus substances produced by the marine green alga, Dunaliella primolecta. J. Appl. Phycol. 1998, 10:349-355 26. Biesert L, Adamski M, Zimmer G, Suhartono H, Fuchs J, Unkelbach U, Mehlhorn R, Hideg K, Milbradt R, Rubsamen-Waigmann H: Anti-hu- man immunodeficiency virus (HIV) drug HOE/BAY 946 in- creases membrane hydrophobicity of human lymphocytes and specifically supress HIV-protein synthesis. Med Microbiol Immunol Berl, 1990, 179:307-321 27. Nakashima H, Kido Y, Kobayashi N, Motoki Y, Neushul M, Yamamoto N: Purification and characterization of avian myeloblastosis and human immunodeficiency virus reverse transcriptase in- hibitor, sulphated polysaccharides extracted from sea algae. Antimicrob. Agents & Chemotherapy 1987, 31:1524-1528 28. Geresh S, Arad S Malis: The extracellular polysaccharides of the red microalgae: chemistry and rheology. Bioresource Technol. 1991, 38:195-201 29. Huleihel M, Ishanu V, Tal J, Arad S: Antiviral effect of microalgal polysaccharides on Herpes simplex and Varicella zoster virus- es. J. Appl. Phycol. 2001, 13:127-134 30. Ball JK, McCarter JA, Sunderland SM: Evidence for helper inde- pendent murine sarcoma virus. I. Segregation of replication defective and transformation defective viruses. Virology 1973, 56:268-284 31. Huleihel M, Aboud M: Effect of mouse interferon on cell trans- formation and virus production in rat cells exogenously in- fected with Moloney murine sarcoma and leukemia viruses. Int J Cancer 1982, 29:471-472 J 32. Yoshida T: Synthesis of polysaccharides having specific biolog- ical activities. Prog Polym SCI 2001, 26:379-441 g y 33. Dubinsky O, Simon O, Karamanos Y, Geresh S, Barak Z, Arad S Malis: Composition of the cell wall polysaccharide produced by the unicellular red algae Rhodella reticulata. Plant Physiology and Bio- chemistry 1992, 30:409-414 y 34. Hassan Y, Preil E, Huleihel M, Segal S, Aboud M: Chemical-retrovi- ral cooperative carcinogenesis and its molecular basis in NIH/3T3 cells. Page 7 of 7 (page number not for citation purposes) (page number not for citation purposes)
https://openalex.org/W3113642213	https://www.researchsquare.com/article/rs-123146/latest.pdf	English	null	The experience of Australian general practice patients at high risk of poor health outcomes with telehealth during the COVID-19 pandemic: a qualitative study	BMC family practice	2,021	cc-by	4,462	The Experience of Australian General Practice Patients at High Risk of Poor Health Outcomes with Telehealth during the COVID-19 Pandemic: A Qualitative Study Sara Javanparast Flinders University Leigh Roeger Flinders University Heilie Kwok Flinders University Richard Reed (  richard.reed@flinders.edu.au ) Flinders University Research Article Posted Date: December 29th, 2020 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Family Practice on April 8th, 2021. See the published version at https://doi.org/10.1186/s12875-021-01408-w. Page 1/11 Background The emergence of the COVID-19 pandemic has raised concerns about the potential decrease in access and utilisation of general practice services and its impact on patient care. In March 2020, the Australian Government introduced telehealth services to ensure that people more vulnerable to COVID-19 do not delay routine care from their general practitioners. Evidence about patients’ experience of telehealth and its impact on patient care is scarce. This study aimed to investigate the experience with telehealth by Australian general practice patients at high risk of poor health outcomes during the COVID-19 pandemic. Methods: Semi-structured telephone interviews were conducted with 30 patients from nine general practices in metropolitan Adelaide (May-June 2020). Participants were identified by their regular doctor as being at high risk of poor health outcomes. Interviews sought participants’ perspectives and experiences about telehealth services in the general practice setting during COVID-19, and the value of offering continued telehealth services post pandemic. Interviews were recorded and transcribed verbatim. Data were analysed using a coding structure developed based on deductive codes derived from the research questions and any additional concepts that emerged inductively from interviews. Results: Participants expressed satisfaction with telehealth including convenient and timely access to general practice services. Yet, participants identified challenges including difficulties in expressing themselves and accessing physical exams. Prescription renewal, discussing test results and simple follow-ups were the most common reasons that telehealth was used. Telehealth was mainly via phone that better suited those with low digital literacy. Participants indicated that an existing doctor-patient relationship was important for telehealth services to be effective. Subjects believed that telehealth services should be continued but needed to be combined with opportunities for face-to-face consultations after the COVID-19 pandemic was over. Conclusions: The expansion of telehealth supported access to general practice including chronic disease management during the COVID-19 pandemic. In the future, telehealth in Australia is likely to have a stronger place in primary healthcare policy and practice and an increased acceptance amongst patients. Background The emergence of the COVID-19 pandemic has raised concerns about the potential decrease in access and utilisation of general practice services and its impact on patient care. Older people and those with chronic diseases are at higher risk of complications and death from COVID-19 [1, 2]. Patients in higher risk groups are encouraged to socially isolate and avoid public places but this may result in avoidance of routine general practice care for health problems. Literature on access to health services during pandemics for vulnerable populations is sparse. Evidence from the 2002–2004 SARS outbreak suggests chronic-care hospitalisations for diabetes dropped during Page 2/11 the crisis but significantly increased afterwards, a concern that is highly relevant to the COVID-19 pandemic [3]. Reasons for sub-optimal chronic disease management during emergencies include diversion of healthcare resources, interruption to routine care and medication supply, increased stress, and changes in activity levels [2]. During Covid-19, many countries implemented or expanded telehealth services to facilitate continued access to primary care and general practice services. A study by Hollander and Carr (2020) suggested urgent action to promote and expand telehealth to protect patients, clinicians and community from exposure [4]. The UK implemented programs of home and mobile health monitoring, videoconferencing, and telecare during Covid-19 [5]. Likewise, the USA responded to the Covid-19 by the promotion and use of telehealth in primary health care setting with reimbursement provided by Medicaid for some types of live videos [6]. In Australia, to ensure that people did not delay routine care from their general practitioners (GPs) during COVID-19, in March 2020 the Australian Government introduced temporary telehealth services targeted to people more vulnerable to COVID-19 [7–9]. These services were offered at no charge to the patient (bulk billed). The key aim was to facilitate access to essential health services while reducing risk of exposure to COVID-19 [8]. A survey of over 1000 GPs conducted by the Royal Australian College of General Practitioners found that nearly all (99%) GPs were offering telehealth services during Covid-19, while also continuing to offer face- to-face consultations [10]. Medicare data shows that out of 35.2 million GP consultations (levels A to D) in Australia between April and June 2020, 34% were via telehealth [11]. The majority (97%) of telehealth consultations were by telephone with videoconferencing representing only a very small (3%) proportion of telehealth consultations [11]. Background Consumer survey also found positive views to telehealth with 43% of respondents preferring to have their usual appointments by telehealth during the COVID-19 outbreak [12]. The provision of telehealth on an ongoing basis is supported by professional bodies [13] and consumer organisations [12, 14]. In July 2020 the Australian government restricted access to Medicare-subsidised telehealth services to only patients who had a face-to-face consultation with their regular GP in the past 12 months, those living in COVID-19 hot spots, infants under 12 months and homeless people [15]. While some professional bodies welcomed this change as a way to prevent “low-value pop-up telehealth services” [16] there are concerns that tighter restrictions may exclude some high-risk patients. In November 2020, the Australian Health Minister confirmed that telehealth would remain in place after the Covid-19 pandemic [17]. There has been minimal research on the impact of these changes on clinical care or patient satisfaction with the services provided. This study investigated the experience with telehealth of Australian general practice patients at high risk of poor health outcomes during the COVID- 19 pandemic. Methods Page 3/11 We conducted semi-structured telephone interviews with 30 patients from nine general practices in metropolitan Adelaide (May-June 2020). The patients were former participants of a clinical trial of We conducted semi-structured telephone interviews with 30 patients from nine general practices in metropolitan Adelaide (May-June 2020). The patients were former participants of a clinical trial of Page 3/11 enhanced general practice services (Flinders QUEST) conducted in 2019. The eligibility criteria for Flinders QUEST were that patients had at least three on-site GP visits at a GP practice within the previous two years and be identified by their GP to be at high risk of poor health outcomes. Participants were drawn from two cohorts: adults 18–64 years of age with two or more chronic diseases and people 65 years of age and above from the Intervention practices of the QUEST trial. The eligibility criteria for Flinders QUEST were that patients had at least three on-site GP visits at a GP practice within the previous two years and be identified by their GP to be at high risk of poor health outcomes. Participants were drawn from two cohorts: adults 18–64 years of age with two or more chronic diseases and people 65 years of age and above from the Intervention practices of the QUEST trial. An invitation letter was mailed to 33 potential participants followed by phone calls one week after the mail-out. Of the 33 people approached, 30 agreed to participate and a telephone interview was arranged. Participants provided verbal consent for participation and for recording of interviews. The previous relationships with participants through the Flinders QUEST facilitated recruitment and rapport building. An interview guide was developed, discussed and refined by the research team. The guide included questions on participants’ experiences with telehealth, the benefits and challenges related to telehealth, and participant’s views about the continuation of telehealth services post pandemic. Interviews took approximately 20–30 minutes. Interviews were transcribed verbatim and imported into NVivo-12 software for data management and coding. A coding structure was developed based on deductive codes from the research questions. We also generated codes inductively to capture additional concepts emerged from interviews. Results Participants were between 54 and 88 years of age (17 females and 13 males). Key study findings are presented below: Access to general practice services and management of health conditions People are more thinking about how we react with each other, the distance, it’s made us think about a lot more things and what it impacts on. (female, 68 yrs. old) Access to general practice services and management of health conditions Participants on average reported 2-3 GP consultations (ranged from 0 to 6) either face-to-face or by telephone since COVID-19 commenced (an approximate three-month period). A small number of participants reported that they had postponed or stopped making new GP appointments for non-urgent medical issues. Consultations by telephone were viewed as an enabling factor in accessing general practice care: I’ve found the medical fraternity has really stepped up to the plate, as far as making available these [phone] appointments, and to keep going the regular health checks that they have with people. I’m pretty impressed… (female, 68 yrs. old) Page 4/11 Page 4/11 Well, it hasn’t been the same obviously, but I haven’t felt as though I’ve been deprived… certainly on the doctor’s part they were available, I’ve always been accommodated. (male, 73 yrs. old) Out of 30 participants, only two reported longer waiting time and less frequent GP consultations: I’ve got to physically go there, they would only allow so many people into surgery, having to have the constant temperature [checks]. It’s all valid but all these extra things…that didn’t enable us to see him [GP] as frequently as we normally would have been before COVID. (male, 59 yrs. old) I’ve got to physically go there, they would only allow so many people into surgery, having to have the constant temperature [checks]. It’s all valid but all these extra things…that didn’t enable us to see him [GP] as frequently as we normally would have been before COVID. (male, 59 yrs. old) Twenty-nine out of 30 participants felt that, although their health had been managed differently during COVID-19 it had been managed quite well: Twenty-nine out of 30 participants felt that, although their health had been managed differently during COVID-19 it had been managed quite well: I think my health has been managed very well. If I needed, I know that the doctors would be available at the end of the phone, just the way things have gone along, I haven’t had any big problems. (female, 80 yrs. old) Three participants even noted an improvement in their health management: Three participants even noted an improvement in their health management: hree participants even noted an improvement in their health management: I actually think it’s better… people are much more aware of health, cleanliness. We’ve had less cases of flu. Experience of telehealth services old) It was just a three-monthly diabetes check-up so it was nothing that couldn’t have been done over the phone anyway. (male, 64 yrs. old) The availability of telehealth was highly appreciated by one participant who was diagnosed with cancer and required cancer investigations and treatments: The availability of telehealth was highly appreciated by one participant who was diagnosed with cancer and required cancer investigations and treatments: I was extremely grateful for those phone calls. It is, of course, not as good as a face-to-face because they can't see how you're looking and how you're actually doing. Nevertheless, I would have been lost without the phone calls. I appreciated that hugely. (female, 77 yrs. old) There were differing views however on the usefulness of telehealth for mental health issues. While one participant felt: ‘if you had mental health problems that would probably be a difficult one to talk about over the phone, you might need to speak to somebody.’ (female, 68 yrs. old), another participant stated: ‘I see the benefit in the mental health space for argument’s sake where people are able to talk…to just be there privately in your own room with your computer talking to the person, it does assist a lot’ (male, 59 yrs. old). A few participants who normally paid an additional charge (gap fee) for face-to-face GP visits appreciated the no-additional fee services (bulkbilling) mandated by Medicare for telehealth services: A few participants who normally paid an additional charge (gap fee) for face-to-face GP visits appreciated the no-additional fee services (bulkbilling) mandated by Medicare for telehealth services: The GP appointments have been really good because they’re bulkbilling, also I’ve been able to get in easily for the phone appointments. Our doctor’s surgery, they’re charging us, and now we’re not being charged, that’s a relief. (female, 56 yrs. old) Many participants believed that ‘familiarity of the doctor with patient’ was crucial in telehealth communication: Because my doctor knows me, I could say, “I need antibiotics or prescription”, and he says, “Yeah, okay”… but if you are talking by phone with a doctor that hasn’t seen you or doesn’t know you. That’s a big difference, isn’t it? (female, 68 yrs. old) Finally, despite an emphasis in policy documents on videoconferencing as the preferred mode of communication, participants in this study were only offered telephone consultations with their GPs. Experience of telehealth services Twenty-five participants utilised telehealth (at least once) for GP consultations. Of the five who did not use telehealth, three did not need a GP appointment and two did need but preferred face-to-face GP consultations due to their health conditions. Those who had a telehealth consultation were very positive about it: If we needed something we rang there and he [GP] rang us on the phone. If it was something that he wasn’t sure about or needed to check out – an appointment given to us and we had to go in and come straight out. It was quite good, a new standard. (female, 77 yrs. old) The availability of telehealth was viewed as potentially increasing the frequency of GP visits ‘In fact, I’m probably seeing him [GP] more now via the phone’ (female, 56 yrs. old), or saving travel time ‘It would save the person having to go into the surgery’ (female, 80 yrs. old). Most felt that phone consultations were not rushed ‘business was done, what’s the point in hanging around. I didn’t need any more time than what I had from the doctor’ (female, 68 yrs. old). Most felt that phone consultations were not rushed ‘business was done, what’s the point in hanging around. I didn’t need any more time than what I had from the doctor’ (female, 68 yrs. old). Prescription renewal, discussing test results and simple follow-ups were the most common reasons for which telehealth was used: Prescription renewal, discussing test results and simple follow-ups were the most common reasons for which telehealth was used: Page 5/11 It helps if we don’t necessarily have to go face-to-face all the time, but a phone call. A renewal of prescriptions you’ve been on for a long time, and you’re finding them working well. (female, 68 yrs. old) It was just a three-monthly diabetes check-up so it was nothing that couldn’t have been done over the phone anyway. (male, 64 yrs. old) It helps if we don’t necessarily have to go face-to-face all the time, but a phone call. A renewal of prescriptions you’ve been on for a long time, and you’re finding them working well. (female, 68 yrs. old) It was just a three-monthly diabetes check-up so it was nothing that couldn’t have been done over the phone anyway. (male, 64 yrs. Continuation of telehealth services articipants felt that telehealth should be continued for issues that do not ne Absolutely, if I’m just doing that 6 or 12-monthly blood test thing, I’d be more than happy for him to mail me the paperwork and ring up for a report - no dramas. It’s only when I have something that I feel I need to see him about. (male, 73 yrs. old) Experience of telehealth services For older participants with lower digital literacy telephone conversation was more convenient’ I don’t get on with the computer, I’m a bit old-fashioned’ (female, 69 yrs. old), however a few mentioned that they would have preferred videoconferencing but this wasn’t offered: If it was perhaps online, such as Microsoft Meeting or Zoom, I probably would appreciate that a bit better, some GPs are offering just a phone consultation, I don't think that's satisfactory. (female, 76 yrs. old) Page 6/11 Page 6/11 Opportunity for face-to-face consultations Participants appreciated that they were still able to have face-to-face GP appointments if needed. Of 30 participants, 26 had had a face-to-face consultation. The COVID-19 safety measures adopted by general practices, mitigated patient’s fear of contracting the virus and this was not viewed as a major barrier to attending in person to the practice. I felt quite comfortable, they were not taking a lot of people in, it was very staggered…you weren’t sitting with a lot of people in a waiting room with everyone coughing and sneezing over you. The practice did it very well, I had no concern at all. (female, 80 yrs. old) I preferred to go there, I found that when you see the doctor you can sit there, you can read his face and I guess he can read yours. I found by phone it was less personal, less invasive, less whatever. (male, 73 yrs. old) Discussion Telehealth has been advocated in Australia for many years to support access to health services especially in rural and remote areas [18]. The COVID-19 pandemic required a rapid adoption of telehealth to reduce the likelihood of virus transmission in patients and health providers, and to ensure that access to general practice services was maintained. Our study found that the telehealth model appears to have mitigated many of the adverse consequences of Covid-19, providing access to routine services. These findings are supported by other studies showing patient and caregivers satisfaction and the role of telehealth in facilitating continued access to care [19, 20]. The convenience and timely access to general practice care was particularly appreciated for services that do not usually require physical contact including repeat prescriptions, reporting of test results and monitoring of less complex health conditions. Continued access to face-to-face consultations (along with telehealth) was felt by patients to be the optimal approach. However, a few patients chose not to use telehealth instead preferring face-to-face consultations. Consistent with other studies [5, 21], our participants identified some challenges in using telehealth including difficulties in expressing themselves and undertaking physical examination by GPs when needed. Page 7/11 Page 7/11 Our findings support the importance of provider-patient relationships for telehealth to be effective. New changes imposed by the government in July 2020 restricting access to telehealth to those who have an established relationship with a GP has already raised debates about its impact on equity of access to general practice services. Whether having a ‘face-to-face consultation with the regular GP in the past 12 months’ as identified in recently policy change [16] is a true measure of ‘doctor-patient relationship’ requires further investigation but appeared to work well with this group of older patients with chronic health conditions who already had established relationships with GPs. The use of telephone rather than video consultation (which is the preferred mode of consultation of the Australian Government) seems to be more acceptable for patients in older age with poor digital literacy. If telehealth technologies are to be included in the future health communication strategies, further investment and training to facilitate both health providers and patients in using such technologies will be important [5]. A combination of telehealth and face-to-face services that allows choice of mode to obtain general practice services would facilitate continuity, accessibility and equity of care. Discussion As noted by Fisk et al (2020), ‘telehealth must not be seen as an alternative form of healthcare.’ Lessons learnt during the current pandemic should be incorporated into future routine practices as well as professional training curricula [5, 22]. The study participant sample represented a cohort of patients likely to be at greatest risk of poor health outcomes from COVID-19 and from disruptions to their regular healthcare caused by COVID-19. The benefits of telehealth reported by our participants however may be not be generalisable to people without an established relationship to a general practitioner. In addition, the selection criteria excluded people who were not fluent in English. The communication barriers experienced by this group may make it more difficult for them to utilise telehealth services and needs further study. Overall, this blended model appeared to be satisfactory to this population group avoiding some face-to- face consultations and enhancing access for this vulnerable population. This study supports the use of this model beyond the Covid-19 pandemic. Conclusions The expansion of telehealth supported access to general practice including chronic disease management during the COVID-19 pandemic. In the future, telehealth is likely to have a stronger place in primary healthcare policy and practice and an increased acceptance amongst patients. Declarations Page 8/11 The study was approved by the Southern Adelaide Clinical Human Research Ethics Committee (114.20). All recruitment, data collection methods and reporting strategies used in this study were in accordance with the ethical guidelines and regulations. Participants were provided detailed information about the study (through participant information sheet and via phone) and informed consent was obtained from all participants (all participants were above 18 years of age). They were informed that their participation was voluntary and that they can withdraw from participation at any time. Participants provided informed consent verbally prior to interviews. Authors’ contributions All authors contributed to the design of the study and to the manuscript. SJ collected and analysed interview data. SJ drafted the manuscript, but it was reviewed and revised for intellectual content by all authors. All authors read and provided approval of the final version submitted to the journal. Availability of data and materials The data used and analysed during the current study are available from the corresponding author on reasonable request. Acknowledgments We would like to thank all participants for participation in this study and sharing their views and experiences with us. Funding The study was funded internally by the Department of General Practice at Flinders University. The study was funded internally by the Department of General Practice at Consent for publication Not applicable Page 9/11 Page 9/11 1. Baker Heart and Diabetes Institute, Coronavirus (COVID-19) and chronic disease. 2020, Baker Heart and Diabetes Institute. 2. Hartmann-Boyce, J. and K.R. Mahtani, Supporting people with long-term conditions (LTCs) during national emergencies. 2020, Centre for Evidence-Based Medicine. 2. Hartmann-Boyce, J. and K.R. Mahtani, Supporting people with long-term conditions (LTCs) during national emergencies. 2020, Centre for Evidence-Based Medicine. 3. Huang, Y.T., Y.C. Lee, and C.J. Hsiao, Hospitalization for ambulatory-care-sensitive conditions in Taiwan following the SARS outbreak: a population-based interrupted time series study. J Formos Med Assoc, 2009. 108(5): p. 386-94. 4. Hollander, J.E. and B.G. Carr, Virtually Perfect? Telemedicine for Covid-19. New England Journal of Medicine, 2020. 30(18): p. 1679-1681. 5. Fisk, M., A. Livingstone, and S.W. Pit, Telehealth in the Context of COVID-19: Changing Perspectives in Australia, the United Kingdom, and the United States. J Med Internet Res, 2020. 22(6): p. e19264. 6. American Hospital Association, Fact sheet: telehealth. 2020, American Hospital Association. 7. Australian Government, Primary Care Package - MBS Telehealth Services and increased Practice Incentive Payments, D.o. Health, Editor. 2020: Canberra. 8. Desborough, J., et al., Australia’s National COVID-19 Primary Care Response. The Medical Journal of Australia, 2020. 9. Australian Government, COVID-19 Temporary MBS Telehealth Services - MBS changes factsheet, D.o. Health, Editor. 2020: Canberra. 10. RACGP, RACGP survey reveals strong take up of telehealth but face to face consultations still available. 2020. 11. Australian Government. Medicare Item Reports. 2020 [cited 2020 28 August]. 11. Australian Government. Medicare Item Reports. 2020 [cited 2020 28 August]. ustralian Government. Medicare Item Reports. 2020 [cited 2020 28 August]. 12. Consumers Health Forum of Australia, Pandemic offers guide to future continuity of care. 2020. 12. Consumers Health Forum of Australia, Pandemic offers guide to future continuity of care. 2020. 13. Woodley, M., Expanded access to telehealth could continue after pandemic, in NewsGP. 2020, Royal Australian College of General Practice. 13. Woodley, M., Expanded access to telehealth could continue after pandemic, in NewsGP. 2020, Royal Australian College of General Practice. 14. Duckett, S., et al., Coming out of COVID-19 lockdown: The next steps for Australian health care. 2020, Grattan Institute. 14. Duckett, S., et al., Coming out of COVID-19 lockdown: The next steps for Australian health care. 2020, Grattan Institute. 15. Minister of Health, Continuous care with telehealth stage seven 2020: Ca 16. Tsirtsakis, A., Government restricts telehealth MBS access to a patient’s regular GP in NewsGP. 2020, RACGP. 16. Page 9/11 Tsirtsakis, A., Government restricts telehealth MBS access to a patient’s regular GP in NewsGP. 2020, RACGP. 17. McHugh, F., Medicare: Health Minister Greg Hunt confirms telehealth here to stay. 2020: news.com.au. 17. McHugh, F., Medicare: Health Minister Greg Hunt confirms telehealth here to stay. 2020: news.com.au. 18. Gray, L.C., et al., Telehealth Assessment Final Report. 2011, UniQuest Pty Limited. 19. Atreya, S., et al., Patients'/Caregivers' Perspectives on Telemedicine Service for Advanced Cancer Patients during the COVID-19 Pandemic: An Exploratory Survey. Indian J Palliat Care, 2020. 26(Suppl 1): p. S40-S44. 20. Hasson, S.P., et al., Perception of patients regarding telemedicine at times of COVID-19: Did they miss the personal touch? Journal of Clinical Oncology, 2020. 38(29). Page 10/11 Page 10/11 Page 10/11 21. Gordon, H.S., et al., “I’m Not Feeling Like I’m Part of the Conversation” Patients’ Perspectives on Communicating in Clinical Video Telehealth Visits. Journal of General Internal Medicine, 2020. 35(6). 21. Gordon, H.S., et al., “I’m Not Feeling Like I’m Part of the Conversation” Patients’ Perspectives on Communicating in Clinical Video Telehealth Visits. Journal of General Internal Medicine, 2020. 35(6). 21. Gordon, H.S., et al., “I’m Not Feeling Like I’m Part of the Conversation” Patients’ Perspectives on Communicating in Clinical Video Telehealth Visits. Journal of General Internal Medicine, 2020. 35(6). 22. Duckett, S., What should primary care look like after the COVID-19 pandemic? Australian Journal of Primary Health, 2020. 26: p. 207-211. g , ( ) 22. Duckett, S., What should primary care look like after the COVID-19 pandemic? Australian Journal of Primary Health, 2020. 26: p. 207-211. 22. Duckett, S., What should primary care look like after the COVID-19 pandemic? Australian Journal of Primary Health, 2020. 26: p. 207-211. 22. Duckett, S., What should primary care look like after the COVID-19 pandemic? Australian Journal of Primary Health, 2020. 26: p. 207-211. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Interviewschedule.docx Interviewschedule.docx Page 11/11
https://openalex.org/W4383102841	https://scipost.org/10.21468/SciPostPhysProc.12.031/pdf	English	null	Characterisation of low background CaWO$_4$ crystals for CRESST-III	SciPost physics proceedings	2,023	cc-by	3,016	1 Physik-Department, Technische Universität München, D-85747 Garching, Germany 2 Max-Planck-Institut für Physik, D-80805 München, Germany 3 Institut für Hochenergiephysik der Österreichischen Akademie der Wissenschaften, A-1050 Wien, Austria 4 Atominstitut, Technische Universität Wien, A-1020 Wien, Austria 5 INFN, Laboratori Nazionali del Gran Sasso, I-67100 Assergi, Italy 6 LIBPhys-UC, Departamento de Fisica, Universidade de Coimbra, P3004 516 Coimbra, Portugal SciPost Phys. Proc. 12, 031 (2023) Characterisation of low background CaWO4 crystals for CRESST-III Angelina Kinast1⋆, G. Angloher2, S. Banik3,4, G. Benato5, A. Bento2,6, A. Bertolini2, R. Breier7, C. Bucci5, J. Burkhart3, L. Canonica2, A. D’Addabbo5, S. Di Lorenzo5, L. Einfalt3,4, A. Erb1,8, F. v. Feilitzsch1, N. Ferreiro Iachellini2, S. Fichtinger3, D. Fuchs2, A. Fuss3,4, A. Garai2, V. M. Ghete3, S. Gerster9, P. Gorla5, P. V. Guillaumon5, S. Gupta3, D. Hauff2, M. Ješkovský7, J. Jochum9, M. Kaznacheeva1, H. Kluck3, H. Kraus10, A. Langenkämper1,2, M. Mancuso2, L. Marini5,11, L. Meyer9, Valentyna Mokina3, A. Nilima2, M. Olmi5, T. Ortmann1, C. Pagliarone5,12, L. Pattavina1,5, F. Petricca2, W. Potzel1, P. Povinec7, F. Pröbst2, F. Pucci2, F. Reindl3,4, J. Rothe1, K. Schäffner2, J. Schieck3,4, D. Schmiedmayer3,4, S. Schönert1, C. Schwertner3,4, M. Stahlberg2, L. Stodolsky2, C. Strandhagen9, R. Strauss1, I. Usherov9, F. Wagner3, M. Willers1 and V. Zema2 ⋆angelina.kinast@tum.de 14th International Conference on Identification of Dark Matter Vienna, Austria, 18-22 July 2022 doi:10.21468/SciPostPhysProc.12 14th International Conference on Identification of Dark Matter Vienna, Austria, 18-22 July 2022 doi:10.21468/SciPostPhysProc.12 1 Introduction CRESST-III (Cryogenic Rare Event Search with Superconducting Thermometers) [1] aims at the direct detection of dark matter (DM) using cryogenic calorimeters. The standard CRESST- III module consists of a scintillating 24 g CaWO4 single crystal as a target. It is operated at ≈10 mK temperature and is equipped with a transition edge sensor (TES) read out by a SQUID (Superconducting QUantum Interference Device) for a precise measurement of the energy deposited by a particle interaction within the crystal. In addition to the CaWO4 crystal, a light detector (also equipped with a TES) is read out in coincidence. This enables discrimination between electromagnetic interactions (background-like events), α-decays (background events, less relative scintillation light) and nuclear recoils (signal-like events, least relative scintillation light) due to the different relative fraction of scintillation light produced. CRESST-III detectors reach thresholds as low as 30.1 eV, allowing a very sensitive measurement of particle recoil energies [1]. One key point for the excellent performance of these detectors is the quality of the target crystals, including a high radiopurity of the CaWO4 material, to minimise backgrounds result- ing from natural decay chains. Especially β-decays can cause events in the region of interest for DM searches. To assure a high quality of the CaWO4 crystals, they have been produced in- house at Technische Universität München (TUM) for many years [2]. In this way, every step of the production is controlled and optimised. The crystal TUM40 operated in CRESST-II showed an excellent performance and a lower background compared to commercially purchased crys- tals operated in the same CRESST run [3].i To further improve the radiopurity, an extensive chemical purification of the raw materials and the CaWO4 powder has been developed at TUM. HPGe screening of the powder shows promising results for an improved radiopurity, however, the sensitivity of this method is limited and only limits on the radiopurity could be stated [4]. From this purified powder, the crystal TUM93 has been produced in 2019. In total three CRESST-III target crystals were cut from the ingot and mounted into CRESST-III modules named TUM93A, TUM93B and TUM93C. The crystal TUM93A was cut from the top of the ingot and is, due to segregation effects during crystal growth, expected to be the most radiopure crystal among the three detector crystals [5]. All modules are currently being operated in the ongoing CRESST-III data-taking campaign started in November 2020. Abstract The CRESST-III experiment aims at the direct detection of dark matter particles via their elastic scattering off nuclei in a scintillating CaWO4 target crystal. For many years CaWO4 crystals have successfully been produced in-house at Technische Universität München with a focus on high radiopurity. To further improve the CaWO4 crystals, an extensive chemical purification of the raw materials has been performed and the crystal TUM93 was produced from this powder. We present results from an α-decay rate analysis per- formed on 344 days of data collected in the ongoing CRESST-III data-taking campaign. The α-decay rate could significantly be reduced. Copyright A. Kinast et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. Received 03-10-2022 Accepted 02-05-2023 Published 04-07-2023 Check for updates doi:10.21468/SciPostPhysProc.12.031 1 Physik-Department, Technische Universität München, D-85747 Garching, Germany 2 Max-Planck-Institut für Physik, D-80805 München, Germany 3 Institut für Hochenergiephysik der Österreichischen Akademie der Wissenschaften, A-1050 Wien, Austria 4 Atominstitut, Technische Universität Wien, A-1020 Wien, Austria 5 INFN, Laboratori Nazionali del Gran Sasso, I-67100 Assergi, Italy 6 LIBPhys-UC, Departamento de Fisica, Universidade de Coimbra, P3004 516 Coimbra, Portugal Copyright A. Kinast et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. Received 03-10-2022 Accepted 02-05-2023 Published 04-07-2023 Check for updates doi:10.21468/SciPostPhysProc.12.031 Copyright A. Kinast et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. 031.1 SciPost Phys. Proc. 12, 031 (2023) 7 Comenius University, Faculty of Mathematics, Physics and Informatics, 84248 Bratislava, Slovakia 8 Walther-Meißner-Institut für Tieftemperaturforschung, D-85748 Garching, Germany 9 Eberhard-Karls-Universität Tübingen, D-72076 Tübingen, Germany 10 Department of Physics, University of Oxford, Oxford OX1 3RH, United Kingdom 11 GSSI-Gran Sasso Science Institute, I-67100 L’Aquila, Italy 12 Dipartimento di Ingegneria Civile e Meccanica, Università degli Studi di Cassino e del Lazio Meridionale, I-03043 Cassino, Italy 1 Introduction A radiopurity analysis focusing on α-decays detected in ≈344 days of this data-taking campaign is presented in this work. For this analysis, a new approach for energy reconstruction has been developed and is presented in the following. 2 Analysis The output of both the phonon detector (PD) and the light detector (LD) are recorded with a continuous data acquisition to enable a dead-time free stream of data which is further pro- cessed offline. In this way, the analysis can be adapted to the specific need of e.g. the low- energy DM analysis or, as in this case, the analysis of α-decays with energies of several MeV. Still, the reconstruction of such highly energetic events with CRESST-III detectors and stan- dard analysis approaches is not possible, due to the optimisation of the detectors to lowest energies. One reason for this is the working principle of the TES used for the signal readout of both the PD and the LD. A TES is a thin W-film operated at a temperature between the superconduct- ing and normal conducting phase (see Figure 1). Energy deposition in the crystal heats the TES (∆T) and results in a resistance change (∆R) proportional to the energy deposition. To max- imise this resistance change, and lower the detector threshold, a steep transition is required. When the energy deposited in the crystal heats the TES completely into its normal conducting phase (like for α-decays), a maximum resistance change and in turn a maximum pulse height is observed which stays constant until the TES cools back into its transition region. In addition, such high energy depositions cause a fast rise in the resistance which cannot be followed by the SQUID electronics, which is losing magnetic flux quanta and changes the absolute baseline voltage of the stream. Figure 2 (left) shows an example of an α-event recorded in the detector TUM93A. The pulse is flat at the top as the TES is in its fully normal conducting state and the baseline level is lower at the end of the pulse compared to the baseline level before the pulse due to the flux quantum loss (FQL). These pulses cannot be reconstructed with standard pulse reconstruction methods as they cannot handle the FQLs. Hence, the new reconstruction method was developed which uses the length of the flat part of the pulse (its saturation time), which is determined by the time the pulse needs to reach 90 % of its maximum voltage. 031.2 031.2 SciPost Phys. Proc. 12, 031 (2023) WP∆T ∆R NormalC. SuperC. Max pulse height Temperature Resistance 0 TC Figure 1: Working principle of a TES. The TES is heated into its transition in the so- called working point (WP). A particle interaction results in a temperature increase ∆T which in turn results in a resistance increase ∆R. The maximum resistance increase is defined by the resistance difference between the normal conducting resistance and the WP resistance. Temperature Figure 1: Working principle of a TES. The TES is heated into its transition in the so- called working point (WP). A particle interaction results in a temperature increase ∆T which in turn results in a resistance increase ∆R. The maximum resistance increase is defined by the resistance difference between the normal conducting resistance and the WP resistance. 2 Analysis The saturation time is indicated by the blue line and is used to reconstruct the energy deposited in the crystal, as it gives a measure of how long the TES needs to come back to its operating temperature. Together with a correction for the SQUID FQLs in which the difference between 031.3 SciPost Phys. Proc. 12, 031 (2023) e−/γ-band α-band saturation time flux quantum loss SciPost Phys. Proc. 12, 031 (2023) e−/γ-band α-band saturation time flux quantum loss Figure 2: Left: Typical event recorded by the PD for an α-decay in the CaWO4 crystal. The pulse has a changing baseline level due to flux quantum losses in the SQUID. In addition, the pulse is flat at the top as the TES is completely normal conducting in this time period. Right: Calibrated scatter plot for the data set of TUM93A. For both, the LD and the PD, the reconstruction was performed using the saturation time. Two bands are visible, the e−/γ-band on the left and the α-band on the right. Figure 2: Left: Typical event recorded by the PD for an α-decay in the CaWO4 crystal. The pulse has a changing baseline level due to flux quantum losses in the SQUID. In addition, the pulse is flat at the top as the TES is completely normal conducting in this time period. Right: Calibrated scatter plot for the data set of TUM93A. For both, the LD and the PD, the reconstruction was performed using the saturation time. Two bands are visible, the e−/γ-band on the left and the α-band on the right. the baseline level before and after the pulse is determined, the energy of α-decay pulses can be reconstructed in both the PD and the LD. In the next step some data selection criteria are applied to the data: Coincidences with the muon veto and the artificial heat pulses sent to the detector for stabilisation and monitoring are excluded. In addition, electronic artefacts like SQUID-resets are removed from the data set and events with too slow a change in resistance are excluded from the data set to prevent the wrong reconstruction of too low energetic pulses. No additional data selection criteria are applied to avoid the possibility of removing α-decay events from the data. The resulting scatter plot of the reconstructed energy in the LD against the reconstructed energy in the PD is shown in Figure 2 (right). 2 Analysis The e−/γ-band, also reconstructed with the saturation time method, is visible as the steep band on the left, as the relative light output is higher for electromagnetic interactions. The α-decay band is nicely separated from the electromagnetic background. The α-spectrum is calibrated using four lines present in the data selected from a wide energy range. As a cross-check the end of the e−/γ-band at 2.6 MeV is used. The 180W decay line at 2.52 MeV, 226Ra at 4.88 MeV, the 210Po surface background line at 5.30 MeV and the 218Po line at 6.11 MeV are fitted by an exponential function as the saturation time has an exponential dependence on the deposited energy. The pulse model on which this assumption is based is published in [6]. The measurement time is corrected for dead times caused by muon veto coincidences and the artificial heat pulses sent to the detector for its stabilisation. 3 Results The calibrated α-spectra for the detectors TUM93A (6.53 kg·d exposure), TUM93B (6.89 kg·d exposure) and TUM93C (6.87 kg·d exposure) are shown in Figure 3. Prominent features are the 180W decay at 2.52 MeV and the two 210Po lines at 5.41 MeV (full energy detected by the crystal) and at 5.30 MeV for decays where the daughter nucleus escapes from the surface of the crystal and does not deposit energy in it. The strong presence of both peaks compared to other energy areas of the spectra hints towards surface contamination of the CaWO4 crystals with 222Rn and with 210Pb, which decayed to 210Po. A background model is currently being developed for a more detailed study of the spectra of all three crystals. 031.4 SciPost Phys. Proc. 12, 031 (2023) 2 3 4 5 6 7 8 9 10 Energy [MeV] 0.0 0.8 1.5 2.3 3.1 3.8 4.6 Counts/20keV/kg/day TUM93A 2 3 4 5 6 7 8 9 10 Energy [MeV] 0.0 0.7 1.5 2.2 2.9 3.6 4.4 Counts/20keV/kg/day TUM93B 0.00 1.46 2.91 4.37 5.82 7.28 Counts/20keV/kg/day TUM93C 2 3 4 5 6 7 8 9 10 Energy [MeV] 0.0 0.5 Po210 ext Po210 int W180 Figure 3: Final α-spectra for all TUM93 detectors. All detectors feature two promi- nent lines at 5.30 MeV and 5.41 MeV. Both result from the decay of 210Po which hints toward surface contamination with 222Rn. At 2.52 MeV the α-decay of 180W is visi- ble. Figure 3: Final α-spectra for all TUM93 detectors. All detectors feature two promi- nent lines at 5.30 MeV and 5.41 MeV. Both result from the decay of 210Po which hints toward surface contamination with 222Rn. At 2.52 MeV the α-decay of 180W is visi- ble. Even though the spectra seem to be dominated by surface contamination, a conservative α-decay rate from natural decay chains in the TUM93 crystals was calculated by summing up all events in the energy region from 3 MeV up to 10 MeV, shown in Table 1. Even though the spectra seem to be dominated by surface contamination, a conservative α-decay rate from natural decay chains in the TUM93 crystals was calculated by summing up all events in the energy region from 3 MeV up to 10 MeV, shown in Table 1. gy g p The rate difference in the three crystals, even though they were cut from the same ingot, has two origins. 3 Results First, during crystal growth impurities are less likely to be built into the crystal lattice compared to the crystal atoms. Hence, the impurity concentration in the melt increases and in turn also along the growth axis in the crystal. This process is called segregation. In addition, the high presence of the 5.30 MeV 210Po line indicates a comparably high surface contamination which can be different for each detector crystal. The highest observed rate in TUM93B could also hint toward a mix-up of the crystals TUM93B and TUM93C during detector mounting. Comparing these conservative limits to the α-activity of e.g. the crystal TUM40, which was studied in detail in [3, 7] with an α-decay rate from natural decay chains of 3.080 mBq kg this yields a minimum impurity reduction factor of >5.97 for TUM93A, >3.18 for TUM93B 031.5 SciPost Phys. Proc. 12, 031 (2023) Table 1: Conservative α-decay rate of isotopes of the three natural decay chains (238U, 235U, 232Th) in an energy range of 3 MeV to 10 MeV. All events are assumed to be of intrinsic origin even though there are hints that the two main contributions are from surface contamination with 210Po. Detector α-Activity µBq kg TUM93A 516 ± 62 TUM93B 919 ± 79 TUM93C 761 ± 76 Detector α-Activity µBq kg TUM93A 516 ± 62 TUM93B 919 ± 79 TUM93C 761 ± 76 and >3.85 for TUM93C. These results show a significant impact of the chemical purification on the α-decay rate in TUM93. The e−/γ-band activity and the activity of single α-decaying isotopes are currently being studied with the help of simulations. and >3.85 for TUM93C. These results show a significant impact of the chemical purification on the α-decay rate in TUM93. The e−/γ-band activity and the activity of single α-decaying isotopes are currently being studied with the help of simulations. Acknowledgements Funding information This work has been funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy - EXC 2094 - 390783311 and through the Sonderforschungsbereich (Collaborative Research Center) SFB1258 ‘Neutrinos and Dark Matter in Astro- and Particle Physics’, by the BMBF 05A20WO1 and 05A20VTA and by the Austrian science fund (FWF): I5420-N, W1252-N27. FW was sup- ported through the Austrian research promotion agency (FFG), project ML4CPD. SG was sup- ported through the FWF project STRONG-DM (FG1). The Bratislava group acknowledges a partial support provided by the Slovak Research and Development Agency (project APVV-15- 0576). [7] A. H. Abdelhameed et al., Erratum to: Geant4-based electromagnetic background model for the CRESST dark matter experiment, Eur. Phys. J. C 79, 987 (2019), doi:10.1140/epjc/s10052-019-7504-y. References [1] A. H. Abdelhameed et al., First results from the CRESST-III low-mass dark matter program, Phys. Rev. D 100, 102002 (2019), doi:10.1103/PhysRevD.100.102002. [2] A. Erb and J.-C. Lanfranchi, Growth of high-purity scintillating CaWO4 single crystals for the low-temperature direct dark matter search experiments CRESST-II and EURECA, CrystEng- Comm 15, 2301 (2013), doi:10.1039/C2CE26554K. [3] R. Strauss et al., Beta/gamma and alpha backgrounds in CRESST-II phase 2, J. Cosmol. Astropart. Phys. 2015, 030 (2015), doi:10.1088/1475-7516/2015/06/030. [4] A. Münster, High-purity CaWO4 single crystals for direct dark matter search with the CRESST experiment, Ph.D. thesis, Technische Universität München (2017). [5] A. Kinast et al., Improving the quality of CaWO4 target crystals for CRESST, J. Low Temp. Phys. 209, 1128 (2022), doi:10.1007/s10909-022-02743-7. [6] F. Pröbst et al., Model for cryogenic particle detectors with superconducting phase transition thermometers, J. Low Temp. Phys. 100, 69 (1995), doi:10.1007/BF00753837. 031.6 SciPost Phys. Proc. 12, 031 (2023) 031.7
https://openalex.org/W3197123215	https://biblio.ugent.be/publication/8738271/file/8738273	English	null	Identification of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming	Immunity	2,021	cc-by	17,433	Identiﬁcation of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming Identiﬁcation of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming Graphical abstract Highlights d KCs are required for in vivo reinvigoration of intrahepatically primed T cells by IL-2 d KCs respond to IL-2 and cross-present hepatocellular Ags d Single-cell RNA-seq identiﬁes two distinct populations of KCs d KC2s have enriched IL-2 sensing machinery and Ag presentation capacity Authors Giorgia De Simone, Francesco Andreata, Camille Bleriot, ..., Luca G. Guidotti, Florent Ginhoux, Matteo Iannacone Correspondence iannacone.matteo@hsr.it In brief De Simone et al. delineate the mechanisms by which hepatocellularly primed HBV-speciﬁc CD8+ T cells acquire antiviral effector functions following IL-2 administration. These mechanisms rely on KCs and, in particular, on a hitherto unidentiﬁed KC subset, referred to as KC2, that is poised to respond to IL-2 and cross-present viral antigens. De Simone et al., 2021, Immunity 54, 2089–2100 September 14, 2021 ª 2021 The Author(s). Published by Elsevier Inc. https://doi.org/10.1016/j.immuni.2021.05.005 ll Authors Giorgia De Simone, Francesco Andreata, Camille Bleriot, ..., Luca G. Guidotti, Florent Ginhoux, Matteo Iannacone p Correspondence iannacone.matteo@hsr.it In brief De Simone et al. delineate the mechanisms by which hepatocellularly primed HBV-speciﬁc CD8+ T cells acquire antiviral effector functions following IL-2 administration. These mechanisms rely on KCs and, in particular, on a hitherto unidentiﬁed KC subset, referred to as KC2, that is poised to respond to IL-2 and cross-present viral antigens. Article Article Arti Identiﬁcation of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming De Simone et al., 2021, Immunity 54, 2089–2100 September 14, 2021 ª 2021 The Author(s). Published by Elsevier Inc. https://doi.org/10.1016/j.immuni.2021.05.005 SUMMARY Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8+ T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-speciﬁc naive CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administra- tion, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selec- tively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity. Article Identiﬁcation of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming Giorgia De Simone,1,2,9 Francesco Andreata,1,9 Camille Bleriot,3,9 Valeria Fumagalli,1,2,9 Chiara Laura,1,2,4 Jose´ M. Garcia-Manteiga,4 Pietro Di Lucia,1 Stefano Gilotto,1,4 Xenia Ficht,1 Federico F. De Ponti,1 Elisa B. Bono,1 Leonardo Giustini,1 Gioia Ambrosi,1 Marta Mainetti,1 Paola Zordan,1 Alexandre P. Be´ ne´ chet,1 Micol Rava` ,1 Svetoslav Chakarov,3 Federica Moalli,1 Marc Bajenoff,5 Luca G. Guidotti,1,2 Florent Ginhoux,3,6,7 and Matteo Iannacone1,2,8,10,11,* 1Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientiﬁc Institute, 20132 Milan, Italy 2Vita-Salute San Raffaele University, 20132 Milan, Italy 3Singapore Immunology Network (SIgN), Agency for Science, Technology & Research (ASTAR), 8A Biomedical Grove, Immunos Building #3-4, Biopolis, Singapore 138648 4Center for Omics Sciences, IRCCS San Raffaele Scientiﬁc Institute, 20132 Milan, Italy 5Aix Marseille University, CNRS, INSERM, CIML, Marseille 13288, France 6Shanghai Institute of Immunology, Shanghai JiaoTong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China 7Translational Immunology Institute, SingHealth Duke-NUS Academic Medical Centre, 169856, Singapore 8Experimental Imaging Centre, IRCCS San Raffaele Scientiﬁc Institute, 20132 Milan, Italy 9These authors contributed equally 10T itt @i l b ll OPEN ACCESS ll OPEN ACCESS Article Identiﬁcation of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming Giorgia De Simone,1,2,9 Francesco Andreata,1,9 Camille Bleriot,3,9 Valeria Fumagalli,1,2,9 Chiara Laura,1,2,4 Jose´ M. Garcia-Manteiga,4 Pietro Di Lucia,1 Stefano Gilotto,1,4 Xenia Ficht,1 Federico F. De Ponti,1 Elisa B. Bono,1 Leonardo Giustini,1 Gioia Ambrosi,1 Marta Mainetti,1 Paola Zordan,1 Alexandre P. Be´ ne´ chet,1 Micol Rava` ,1 Svetoslav Chakarov,3 Federica Moalli,1 Marc Bajenoff,5 Luca G. Guidotti,1,2 Florent Ginhoux,3,6,7 and Matteo Iannacone1,2,8,10,11, 1Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientiﬁc Institute, 20132 Milan, Italy 2Vita-Salute San Raffaele University, 20132 Milan, Italy 3Singapore Immunology Network (SIgN), Agency for Science, Technology & Research (ASTAR), 8A Biomedical Grove, Immunos Building #3-4, Biopolis, Singapore 138648 4Center for Omics Sciences, IRCCS San Raffaele Scientiﬁc Institute, 20132 Milan, Italy 5Ai M ill U i i CNRS INSERM CIML M ill 13288 F 4Center for Omics Sciences, IRCCS San Raffaele Scientiﬁc Institute, 20132 5Aix Marseille University, CNRS, INSERM, CIML, Marseille 13288, France 5Aix Marseille University, CNRS, INSERM, CIML, Marseille 13288, France 6Shanghai Institute of Immunology, Shanghai JiaoTong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China 7Translational Immunology Institute, SingHealth Duke-NUS Academic Medical Centre, 169856, Singapore Translational Immunology Institute, SingHealth Duke NUS Academic Medical Centre, 1698 8Experimental Imaging Centre, IRCCS San Raffaele Scientiﬁc Institute, 20132 Milan, Italy Translational Immunology Institute, SingHealth Duke NUS Academic Medical Centre, 169856, Singapore 8Experimental Imaging Centre, IRCCS San Raffaele Scientiﬁc Institute, 20132 Milan, Italy 9Th th t ib t d ll 8Experimental Imaging Centre, IRCCS San Raffaele Scientiﬁc Institute, 20132 Milan, Italy 9These authors contributed equally 10Twitter: @iannaconelab 11Lead contact 11Lead contact Correspondence: iannacone.matteo@hsr.it https://doi.org/10.1016/j.immuni.2021.05.005 Immunity 54, 2089–2100, September 14, 2021 ª 2021 The Author(s). Published by Elsevier Inc. 2089 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Highlights g g d KCs are required for in vivo reinvigoration of intrahepatically primed T cells by IL-2 d KCs respond to IL-2 and cross-present hepatocellular Ags d Single-cell RNA-seq identiﬁes two distinct populations of KCs d KC2s have enriched IL-2 sensing machinery and Ag presentation capacity De Simone et al., 2021, Immunity 54, 2089–2100 September 14, 2021 ª 2021 The Author(s). Published by Elsevier Inc. https://doi.org/10.1016/j.immuni.2021.05.005 ll ll INTRODUCTION one-third of all blood cells transit slowly every minute (Vollmar and Menger, 2009), allow circulating, intravascular T cells to sense MHC-antigen (Ag) complexes displayed by the non-pro- fessional Ag-presenting hepatocytes (Guidotti et al., 2015; War- ren et al., 2006). Using mouse models of HBV infection, it has been recently shown that hepatocellular priming of virus-speciﬁc naive CD8+ T cells induces local activation and initial vigorous proliferation but eventually leads to the development of dysfunc- tional cells devoid of cytotoxic and antiviral activity (Be´ ne´ chet et al., 2019; Isogawa et al., 2013). The transcriptional signature of these cells does not obviously overlap with that of other known dysfunctional CD8+ T cell states such as exhaustion, and accordingly, CD8+ T cells primed by hepatocytes are not readily responsive to in vivo anti-PD-L1 treatment (Be´ ne´ chet et al., The liver is peculiarly biased toward inducing immune tolerance, as exempliﬁed by the acceptance of liver allografts across com- plete major histocompatibility complex (MHC) mismatch barriers or the propensity of hepatitis B virus (HBV) and other hepato- tropic viruses such hepatitis C virus (HCV) to establish life-long persistent infections (Ficht and Iannacone, 2020; Wong et al., 2015). Liver tolerance involves a complex array of coordinated events that ultimately hinder the effector functions of intrahepatic lymphocytes (Ficht and Iannacone, 2020; Horst et al., 2016; Jenne and Kubes, 2013). For example, the unique anatomy and hemodynamics of the fenestrated and basement mem- brane-less liver capillaries (i.e., sinusoids), through which about Immunity 54, 2089–2100, September 14, 2021 ª 2021 The Author(s). Published by Elsevier Inc. 2089 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A G J P Q R K L M N O H I B C D E F Figure 1. KCs are required for optimal in vivo reinvigoration of intrahepatically primed T cells by IL-2 A) Schematic representation of the experimental setup. Cor93 and Env28 TN (5 3 106) were transferred into C57BL/6 3 BALB/c F1 (WT) or MUP-core 3 F1 (MUP-core) recipients. When indicated, mice were injected with 2.5 3 105 infectious units of non-replicating rLCMV-core/env 4 h prior to TN transfer. S MUP-core mice received clodronate liposomes (CLLs) and/or IL-2/anti-IL-2 complexes (IL-2c) at the indicated time points. INTRODUCTION Livers were collected and a 5 days after T transfer ll OPEN ACCESS Art A G J K L M N O H I B C D E F A D D B C C B E D F I I H G G H K L M N K L M N O O N O K N P Q R Q P R Figure 1. KCs are required for optimal in vivo reinvigoration of intrahepatically primed T cells by IL-2 l in vivo reinvigoration of intrahepatically primed T cells p g p y p y of the experimental setup. Cor93 and Env28 TN (5 3 106) were transferred into C57BL/6 3 BALB/c F1 (WT) or MUP-core 3 BALB en indicated, mice were injected with 2.5 3 105 infectious units of non-replicating rLCMV-core/env 4 h prior to TN transfer. Selecte dronate liposomes (CLLs) and/or IL-2/anti-IL-2 complexes (IL-2c) at the indicated time points. Livers were collected and analyze (G and H) Total numbers (G) and numbers of IFN-g-producing (H) Cor93 and Env28 T cells in the livers of indicated mice. n = 4; p < 0.05, p < 0.01, *p < 0.001, one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with control. Normal distribution was veriﬁed using the Shapiro-Wilk test. (G and H) Total numbers (G) and numbers of IFN-g-producing (H) Cor93 and Env28 T cells in the livers of indicated mice. n = 4; p < 0.05, p < 0.01, p < 0.001, one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with control. Normal distribution was veriﬁed using the Shapiro-Wilk test. (I) Representative confocal immunoﬂuorescence micrographs of liver sections from the indicated mice 5 days after TN transfer. Cor93 T cells were identiﬁed as GFP+ cells and are depicted in green. Env28 T cells were identiﬁed as DsRed+ cells and are depicted in red. Sinusoids were identiﬁed as Lyve-1+ cells and are depicted in gray. Scale bars represent 100 mm. (I) Representative confocal immunoﬂuorescence micrographs of liver sections from the indicated mice 5 days after TN transfer. Cor93 T cells were identiﬁed as GFP+ cells and are depicted in green. Env28 T cells were identiﬁed as DsRed+ cells and are depicted in red. Sinusoids were identiﬁed as Lyve-1+ cells and are depicted in gray. Scale bars represent 100 mm. (legend continued on next page) INTRODUCTION (J) Schematic representation of the experimental setup. MUP-core mice were lethally irradiated and reconstituted with CD11cDTR bone marrow (BM). Eight weeks after BM reconstitution, 1 3 106 Cor93 TN were transferred. Indicated mice were treated with diphtheria toxin (DT) every 48 h starting from 3 days before T cell injection. Indicated mice received IL-2c 1 day after Cor93 T cell transfer. Livers were collected and analyzed 5 days after TN transfer. (K and L) Representative ﬂow cytometry plot (K) and absolute numbers (L) of DCs (identiﬁed as live MHC IIhi CD11c+ cells) from the indicated mice at the time of (J) Schematic representation of the experimental setup. MUP-core mice were lethally irradiated and reconstituted with CD11cDTR bone marrow (BM). Eight weeks after BM reconstitution, 1 3 106 Cor93 TN were transferred. Indicated mice were treated with diphtheria toxin (DT) every 48 h starting from 3 days before T cell injection. Indicated mice received IL-2c 1 day after Cor93 T cell transfer. Livers were collected and analyzed 5 days after TN transfer. (K and L) Representative ﬂow cytometry plot (K) and absolute numbers (L) of DCs (identiﬁed as live, MHC-IIhi, CD11c+ cells) from the indicated mice at the time of Cor93 T cell transfer (PBS, n = 3; DT, n = 4). p < 0.05, one-tailed Mann-Whitney U test. metry plot (K) and absolute numbers (L) of DCs (identiﬁed as live, MHC-IIhi, CD11c+ cells) from the indicated mice at the time of DT, n = 4). p < 0.05, one-tailed Mann-Whitney U test. (legend continued on next page) 2090 Immunity 54, 2089–2100, September 14, 2021 Article ll OPEN ACCESS 2019). The notion that in vivo interleukin-2 (IL-2) administration overcomes this dysfunction (Be´ ne´ chet et al., 2019) not only illus- trates that efﬁcient hepatocellular priming can occur under spe- ciﬁc conditions but also provides the opportunity to identify which cellular and molecular determinants drive immunogenic responses within the tolerogenic liver microenvironment. (Guidotti et al., 1994). These animals, like the HBV replication- competent transgenic mice described below, never develop spontaneous liver pathology, as the hepatocellular expression of the viral gene products occurs non-cytopathically, and endog- enous T cells speciﬁc for these products are profoundly tolerant (Guidotti et al., 1994). INTRODUCTION As controls for proper CD8+ T cell differen- tiation into effector cells, we used wild-type (WT) mice transduced with recombinant, replication-defective lymphocytic choriomeningitis (LCMV)-based vectors (Flatz et al., 2010) tar- geting the HBV core and envelope proteins (rLCMV-core/env) to intrahepatic professional Ag-presenting cells (APCs) (i.e., Kupffer cells [KCs] and hepatic DCs) that are not natural targets of HBV (Be´ ne´ chet et al., 2019). Both groups of mice were in- jected with naive CD8+ TCR transgenic T cells (TN) speciﬁc for epitopes contained within the core and envelope proteins of HBV (Cor93 and Env28 TN, respectively) (Figure 1A) (Isogawa et al., 2013). One day after TN injection, selected MUP-core mice received IL-2 immune complexes (IL-2c) consisting of IL- 2 coupled with non-neutralizing IL-2-speciﬁc monoclonal anti- bodies (S4B6) that enhance the half-life of IL-2 in vivo (Boyman et al., 2006) (Figure 1A). To test whether IL-2c treatment had exclusively a direct effect on TN or whether it required the pres- ence of additional cells, we performed depletion experiments. We initially focused on KCs, as these cells are capable of inducing full effector differentiation of CD8+ T cells upon in vivo rLCMV transduction (Be´ ne´ chet et al., 2019). KCs were depleted through clodronate liposome (CLL) injection 2 days prior to T cell injection (Figure 1A). This treatment effectively depletes KCs while sparing hepatic DCs (Figures 1B–1F) (Be´ ne´ chet et al., 2019; Sitia et al., 2011). Consistent with previously published re- sults (Be´ ne´ chet et al., 2019), Cor93 and Env28 TN transferred to WT mice injected with rLCMV-core/env differentiated into bona ﬁde effector cells that formed tight clusters scattered throughout the liver lobules; in contrast, Cor93 T cells transferred to MUP- core mice generated dysfunctional cells devoid of IFN-g-pro- ducing ability that coalesced around portal tracts (Figures 1G– 1I). IL-2c administration improved the capacity of Ag-speciﬁc Cor93 T cells to expand, differentiate into IFN-g-producing cells and accumulate in clusters scattered throughout the liver lob- ules, but it had no effect on irrelevant Env28 TN (Figures 1G– 1I). Optimal in vivo reinvigoration of intrahepatically primed Cor93 T cells required the presence of KCs, as IL-2c treatment failed to improve T cell expansion, effector differentiation, and in- traparenchymal cluster formation in CLL-treated mice (Figures 1G–1I). INTRODUCTION Similar results were obtained when recombinant IL-2 was used in place of IL-2c and when HBV replication-competent transgenic mice, which express all viral proteins in hepatocytes and secrete enveloped virions containing the HBV particulate core protein into the bloodstream, were used in place of MUP- core recipients (data not shown). Several formulations of IL-2 have been variably used in the past >25 years as a therapy to augment T cell responses against viral or tumor Ags (Blattman et al., 2003; Pol et al., 2020; West et al., 2013), and at the moment of writing, more than 40 clinical trials are evaluating the immune stimulatory potential of this cytokine in different oncological indications (https://www. clinicaltrials.gov). As the IL-2 functional pleiotropy has often driven undesired toxicity in the clinical setting (Pol et al., 2020), deconvoluting the biology responsible for its efﬁcacy may help improve the therapeutic potential of IL-2-based strategies. p p p g The IL-2 receptor consists of a heterocomplex of up to three subunits: a (CD25), b (CD122), and the common g chain (CD132) (Pol et al., 2020). Although each receptor subunit can independently bind IL-2 with low afﬁnity (Kd ~ 108 to 107 M), only the intermediate-afﬁnity bg dimeric (Kd ~ 109 M) and the high-afﬁnity abg trimeric (Kd ~ 1011 M) receptors mediate intra- cellular signal transduction (Pol et al., 2020). In addition to T cells and natural killer (NK) cells, myeloid cells have been reported to express the intermediate-afﬁnity bg receptor, with some den- dritic cell (DC) subtypes displaying the three subunits of the IL- 2 receptor (Bosco et al., 2000; Herr et al., 2014). However, the signiﬁcance of IL-2 receptor expression by myeloid cells in vivo is controversial (Fukao and Koyasu, 2000; Kronin et al., 1998; Liang et al., 2012; Liao et al., 2013; Popov et al., 2008; Raeber et al., 2020). For instance, DCs may supply the a chain in trans (Wuest et al., 2011), thus supporting high-afﬁnity binding of IL- 2 to naive T cells undergoing priming. Other studies have sug- gested that DC production of CD25 quenches IL-2 in the outer T cell area of lymph nodes, thus guiding T cell differentiation (Li et al., 2016). Whether the IL-2-mediated reversal of the T cell dysfunction induced by hepatocellular priming is due to an exclusive effect of this cytokine on T cells or whether myeloid cells are involved is currently unknown. (M) Representative confocal immunoﬂuorescence micrographs of liver sections from the indicated mice 48 h after DT treatment. KCs were identiﬁed as F4/80+ cells and are depicted in red. Sinusoids were identiﬁed as Lyve-1+ cells and are depicted in gray. Scale bars represent 50 mm. (N and O) Representative ﬂow cytometry plot (N) and absolute numbers (O) of KCs (identiﬁed as live, CD45+, TIM4+, F4/80+ cells) from the indicated mice at the time of Cor93 T cell transfer (PBS, n = 3; DT, n = 4). (P and Q) Total numbers (P) and numbers of IFN-g-producing (Q) Cor93 T cells in the livers of the indicated mice. n = 5. (R) Representative confocal immunoﬂuorescence micrographs of liver sections from the indicated mice 5 days after TN transfer. Cor93 T cells were identiﬁed as CD45.1+ cells and are depicted in green. Sinusoids were identiﬁed as Lyve-1+ cells and are depicted in gray. Scale bars represent 100 mm. Data are representative of at least three independent experiments. See also Figure S1. KCs are required for optimal in vivo reinvigoration of intrahepatically primed T cells by IL-2 To shed light on the immune mechanisms underpinning the IL-2- mediated reinvigoration of intrahepatically primed T cells, we initially took advantage of transgenic mice that express a non- secretable version of the particulate HBV core protein under the transcriptional control of the hepatocyte-speciﬁc mouse ma- jor urinary protein (MUP) promoter (hereafter MUP-core mice) Immunity 54, 2089–2100, September 14, 2021 2091 Article C A F I M Q R S T U N O P J K L G H B C D E Figure 2. KCs respond to IL-2 and cross-present hepatocellular Ags (A) Representative ﬂow cytometry plots of CD25 (left panel), CD122 (middle panel), and CD132 (right panel) expression on CD45+ (blue) and F4/80+ (red) cell populations in the livers of C57BL/6 mice. Isotype control is depicted in gray. (B) Mean ﬂuorescent intensity (MFI) of CD25 (left), CD122 (middle), and CD132 (right) expression on live CD45+ (blue) and KCs (red; identiﬁed as live, CD45+, TIM4+ F4/80+ cells) cells in the livers of C57BL/6 mice n 3 A B C D E A B C D E D D A B E G H H F G F I J K L L L K M N O P O P N N M O P P Q Q T U R S T U T U R S T U T U R S Figure 2. KCs respond to IL-2 and cross-present hepatocellular Ags g p p p g (A) Representative ﬂow cytometry plots of CD25 (left panel), CD122 (middle panel), and CD132 (right panel) expression on CD45+ (blue) and F4/80+ (red) cell populations in the livers of C57BL/6 mice. Isotype control is depicted in gray. (B) Mean ﬂuorescent intensity (MFI) of CD25 (left), CD122 (middle), and CD132 (right) expression on live CD45+ (blue) and KCs (red; identiﬁed as live, CD45+, TIM4+, F4/80+ cells) cells in the livers of C57BL/6 mice. n = 3. (C) Schematic representation of the experimental setup. Liver non-parenchymal cells (LNPCs) were isolated from C57BL/6 mice and incubated in vitro for 15 min with increasing doses of rIL-2. pSTAT5 signal was analyzed on CD45+ F4/80+ TIM4+ cells (KCs) or CD31+ CD45 cells (LSECs) using ﬂow cytometry (repre- sentative plot of KCs at the bottom). KCs are required for optimal in vivo reinvigoration of intrahepatically primed T cells by IL-2 Functional enrichment analysis of upregulated genes showed an increased transcrip- tion of genes involved mainly in Ag presentation and proteaso- mal processing, ribosomal RNA processing and splicing, DNA replication and cell cycle, and mitochondrial oxidative meta- bolism (Figure 2H; Figure S2; Table S1). Among the upregulated gene clusters, we focused on the Ag presentation pathway, which includes several macromolecular complexes composed of ubiquitins, chaperones, MHC-I, and proteasome subunits (Figures 2I–2K; Figures S3A–S3E) (Blum et al., 2013). Genes en- coding for these protein families—speciﬁcally MHC-I-related proteins, immunoproteasome subunits, the transcription regu- lator of MHC-I genes Nlrc5 (Kobayashi and van den Elsen, 2012) and the transporter associated with Ag processing 1 (Tap1)—were induced in KCs upon IL-2c treatment (Figures 2I–2K; Figures S3B–S3F). The upregulation of MHC-I and co- stimulatory molecules in KCs isolated from mice treated with IL-2c was conﬁrmed at the protein level (Figure 2L). On the basis of these results, we reasoned that in vivo treatment with IL-2c might increase the cross-presentation ability of KCs. To test KCs respond to IL-2 and cross-present hepatocellular Ags (L) MFI of H2-Kb, CD40, and CD80 expression on KCs (deﬁned as live, CD45+, TIM4+, F4/80+ cells) 48 h after PBS or IL-2c treatment in vivo. n = 3; p < 0.05, one- tailed Mann-Whitney U test. (M) Schematic representation of the experimental setup. HBV replication-competent transgenic mice (HBV Tg) were treated in vivo with PBS or IL-2c. After 48 h, liver non-parenchymal cells (LNPCs) were isolated, and KCs were seeded for 2 h and co-cultured with in vitro-differentiated Cor93 effector T cells (Cor93 TE). After 4 h, T cells were harvested and analyzed using ﬂow cytometry. (M) Schematic representation of the experimental setup. HBV replication-competent transgenic mice (HBV Tg) were treated in vivo with PBS or IL-2c. After 48 h, liver non-parenchymal cells (LNPCs) were isolated, and KCs were seeded for 2 h and co-cultured with in vitro-differentiated Cor93 effector T cells (Cor93 TE). After 4 h, T cells were harvested and analyzed using ﬂow cytometry. h, T cells were harvested and analyzed using ﬂow cytometry. and O) Representative ﬂow cytometry plot (N) and percentage (O) of IFN-g producing Cor93 TEFF cells in the indicated condition ann-Whitney U test. (P) Schematic representation of the experimental setup. C57BL/6 mice were treated in vivo with PBS or IL-2c. After 48 h, LNPCs were isolated, and KCs were puriﬁed by immunomagnetic separation. Puriﬁed KCs were co-cultured with CellTrace violet (CTV)-labeled Cor93 TN. Serum from HBV replication-competent transgenic mice (containing the indicated concentrations of HBeAg) was added to the wells (note that HBeAg contains the Cor93 determinant). After 4 days, Cor93 T cells were harvested and analyzed using ﬂow cytometry. (Q and R) Representative ﬂow cytometry plots (Q) and percentages (R) of proliferating Cor93 T cells at the indicated conditions. p < 0.05 and p < 0.01, one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with every other group within the same Ag dose. n = 3. Normal distribution was veriﬁed using the Shapiro-Wilk test. (S) Schematic representation of the experimental setup. MUP-core mice were lethally irradiated and reconstituted with WT or Tap1/ bone marrow (BM). Eight weeks after BM reconstitution, mice received two injection of clodronate liposomes (CLLs) to remove residual radio-resistant KCs. Two weeks after the last dose of CLL, 5 3 106 Cor93 TN were transferred. Indicated mice received IL-2c 1 day after Cor93 T cell transfer. KCs are required for optimal in vivo reinvigoration of intrahepatically primed T cells by IL-2 (D) F ld h f STAT5 h h l ti t t t ith th i di t d t ti f IL 2 i KC ( d d t ) LSEC (bl d t ) 3 ** 0 001 y g (E) Immunoblot analysis of STAT5 and pSTAT5 in adherent KCs isolated from C57BL/6 mice and incubated in vitro with IL-2c or PBS. p y ( ) q p y y (G) KC sorting strategy. KCs were identiﬁed as live, CD45+, Lineage (CD3, CD19, Ly6G, CD49b), F4/80+, CD64+, MHCIIint, TIM4+ cells (n = 4 per group). (legend continued on next page) (legend continued on next page) 2092 Immunity 54, 2089–2100, September 14, 2021 ll OPEN ACCESS Article To conﬁrm that hepatic DCs are not necessary for the optimal in vivo response to IL-2, we depleted this cell population by diph- theria toxin (DT) injection in MUP-core mice reconstituted with CD11cDTR bone marrow (Figure 1J). This treatment signiﬁcantly decreased the number of hepatic DCs while sparing KCs (Fig- ures 1K–1O). DC depletion did not affect the capacity of IL-2 to promote expansion, effector differentiation, and intraparenchy- mal cluster accumulation of intrahepatically primed Cor93 T cells (Figures 1P–1R). Similarly, other phagocytic cells such as neutrophils and monocytes were found not to be involved in the response to IL-2, as neutrophil depletion (via anti-Ly6G Abs) or combined neutrophil and monocyte depletion (via anti- Gr1 Abs) did not affect the in vivo reinvigoration of intrahepati- cally primed T cells by IL-2 (Figure S1). Taken together, these results indicate that KCs are required for optimal in vivo reinvigo- ration of intrahepatically primed T cells by IL-2. note, the IL-2-dependent fold change in STAT5 phosphorylation observed in KCs was ~10% than that observed in CD4+FoxP3+ splenic T regulatory cells (data not shown). Nevertheless, these data indicate that KCs express a functional IL-2 receptor capable of responding to IL-2 in vitro. To assess the conse- quences of IL-2 treatment on KCs in vivo, we treated C57BL/6 mice with IL-2c and then performed RNA sequencing (RNA- seq) analysis on ﬂow cytometry-sorted KCs 48 h later (Figures 2F and 2G). A total of 4,073 differentially expressed genes (DEGs), 1,515 up- and 2,558 downregulated, were identiﬁed as signiﬁcantly regulated by IL-2c (Table S1). KCs respond to IL-2 and cross-present hepatocellular Ags The x axis represents the log2 fold change of differentially expressed genes (DEGs) upon IL-2c treatment, the y axis the log10(FDR). Only DEGs with FDRs < 0.05 were considered. Genes belonging to speciﬁc biological process are highlighted in different colors (see also Figures S3A–S3E). ) (J) Radar plot of different biological processes. Each dimension of the radar plot is represented as the mean of the transcripts per kilobase million (TPM) of selected genes (see also Figures S3A–S3E), in PBS-treated (blue) and IL-2c-treated (red) samples. Values range from 0 to 350 TPM. plot of different biological processes. Each dimension of the radar plot is represented as the mean of the transcripts per kilob enes (see also Figures S3A–S3E) in PBS-treated (blue) and IL-2c-treated (red) samples Values range from 0 to 350 TPM ) (J) Radar plot of different biological processes. Each dimension of the radar plot is represented as the mean of the transcripts per kilobase million (TPM) of selected genes (see also Figures S3A–S3E), in PBS-treated (blue) and IL-2c-treated (red) samples. Values range from 0 to 350 TPM. (J) Radar plot of different biological processes. Each dimension of the radar plot is represented as the mean of the transcripts per kilobase million (TPM) of selected genes (see also Figures S3A–S3E), in PBS-treated (blue) and IL-2c-treated (red) samples. Values range from 0 to 350 TPM. (K) Heatmap of selected genes linked to Ag presentation that were upregulated in KCs upon IL-2c treatment. Values are Z scores, calculated from scaling by row the log2(TPM) values. ( ) p g p p p p p ( ) selected genes (see also Figures S3A–S3E), in PBS-treated (blue) and IL-2c-treated (red) samples. Values range from 0 to 350 TPM. (K) Heatmap of selected genes linked to Ag presentation that were upregulated in KCs upon IL-2c treatment. Values are Z scores, calculated from scaling by row the log2(TPM) values. (K) Heatmap of selected genes linked to Ag presentation that were upregulated in KCs upon IL-2c treatment. Values are Z scores, calculated from scaling by row the log2(TPM) values. g2( ) (L) MFI of H2-Kb, CD40, and CD80 expression on KCs (deﬁned as live, CD45+, TIM4+, F4/80+ cells) 48 h after PBS or IL-2c treatment in vivo. n = 3; p < 0.05, one- tailed Mann-Whitney U test. KCs respond to IL-2 and cross-present hepatocellular Ags Flow cytometric analyses revealed that a fraction of KCs ex- presses all three subunits of the IL-2 receptor (CD25, CD122, and CD132) (Figures 2A and 2B). We therefore investigated the effect of IL-2 treatment on KCs. To this end, we isolated liver non-parenchymal cells (LNPCs), including KCs, from C57BL/6 mice and stimulated them ex vivo with recombinant IL-2 (Fig- ure 2C). We observed a dose-dependent increase in STAT5 phosphorylation in KCs but not in liver sinusoidal endothelial cells (LSECs) (Figure 2D). Similar results were obtained when IL-2c was used in place of IL-2, and STAT5 phosphorylation in KCs was conﬁrmed by immunoblot analysis (Figure 2E). Of (H) Clustering of top signiﬁcant (EnrichR combined score > 100, false discovery rate [FDR] < 0.05) Gene Ontology biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of processes upregulated in KCs upon in vivo IL-2c treatment. The thermal scale represents the Jaccard similarity coefﬁcient between every gene set pair (blue representing a similarity coefﬁcient of 0 and red a similarity coefﬁcient of 1) (H) Clustering of top signiﬁcant (EnrichR combined score > 100, false discovery rate [FDR] < 0.05) Gene Ontology biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of processes upregulated in KCs upon in vivo IL-2c treatment. The thermal scale represents the Jaccard similarity coefﬁcient between every gene set pair (blue representing a similarity coefﬁcient of 0 and red a similarity coefﬁcient of 1). (H) Clustering of top signiﬁcant (EnrichR combined score > 100, false discovery rate [FDR] < 0.05) Gene Ontology biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of processes upregulated in KCs upon in vivo IL-2c treatment. The thermal scale represents the Jaccard similarity ) Clustering of top signiﬁcant (EnrichR combined score > 100, false discovery rate [FDR] < 0.05) Gene Ontology biological process enes and Genomes (KEGG) pathways of processes upregulated in KCs upon in vivo IL-2c treatment. The thermal scale rep oefﬁcient between every gene set pair (blue representing a similarity coefﬁcient of 0 and red a similarity coefﬁcient of 1). (I) Volcano plot of RNA-seq results. The x axis represents the log2 fold change of differentially expressed genes (DEGs) upon IL-2c treatment, the y axis the log (FDR) Only DEGs with FDRs < 0 05 were considered Genes belonging to speciﬁc biological process are highlighted in different colors (see also Figures (I) Volcano plot of RNA-seq results. Immunity 54, 2089–2100, September 14, 2021 2093 KCs respond to IL-2 and cross-present hepatocellular Ags Livers were collected and analyzed 5 days after Cor93 TN transfer. (T and U) Total numbers (T) and numbers of IFN-g-producing (U) Cor93 T cells in the livers of the indicated mice (MUP-core WT-PBS, n = 3; MUP-core WT-IL-2c, n = 4; MUP-core Tap1/-PBS, n = 4; MUP-core Tap1/-IL-2c, n = 4). p < 0.01 and *p < 0.001, two-way ANOVA with Sidak’s multiple-comparison test. Data are representative of at least three independent experiments. See Figures S2 and S3 and Table S1. Immunity 54, 2089–2100, September 14, 2021 2093 ll OPEN ACCESS Article this possibility, we measured the capacity of in vitro differenti- ated Cor93-speciﬁc effector CD8+ T cells (Cor93 TEFF) to pro- duce IFN-g (as an indirect measure of Ag recognition) upon incu- bation with KCs isolated from control and IL-2c-treated HBV replication-competent transgenic mice (Figure 2M). Consistently with previously published data, baseline KC cross-presentation of the core protein in this experimental system at steady state was negligible (Figures 2N and 2O), despite KCs being constantly exposed to abundant HBV virions in the circulation. Cor93 TN remained dysfunctional even when isolated from the liver of HBV replication-competent transgenic mice previously transferred with highly pathogenic Env28-speciﬁc effector CD8+ T cells (data not shown). This indicates that KC cross-pre- sentation remains insigniﬁcant during acute liver inﬂammation, even though the inﬂammatory conditions potentially favor not only the uptake of HBV virions but also the phagocytosis of damaged hepatocytes containing the particulate HBV core pro- tein. In spite of this, treating HBV replication-competent trans- genic mice with IL-2c slightly but signiﬁcantly increased the cross-presentation capacity of KCs incubated in vitro with Cor93 TEFF cells (Figures 2N and 2O). We also assessed the abil- ity of KCs isolated from IL-2-treated C57BL/6 mice to cross- prime HBV-speciﬁc naive CD8+ T cells exposed to the serum of HBV replication-competent transgenic mice in vitro (Fig- ure 2P). Compared with KCs isolated from PBS-treated mice, KCs exposed to IL-2 in vivo induced a higher proliferation of Cor93 TN in in vitro culture (Figures 2Q and 2R). Finally, to eval- uate the in vivo relevance of our ﬁndings, we took advantage of MUP-core mice, which express only a non-secretable, particu- late form of the HBV core protein and in which KC cross-presen- tation should depend on the uptake of the few hepatocytes that are known to be injured by Cor93 TN transfer (Be´ ne´ chet et al., 2019). KCs respond to IL-2 and cross-present hepatocellular Ags Cells in cluster 3 (n = 12) expressed genes associated with endothelial cells, including Pecam1 (CD31), Clec4g, Lyve1 (Figures 3C and 3D), and Kdr (VGFR2) (Table S2); in addition, their speciﬁc markers were enriched in endothelial cell processes (Figure 3F; Table S3), arguing for contamination of sorted cells with LSECs (Figure 3E). Although both cluster 0 and cluster 1 showed expression of KC markers, they were clearly distinguished by the expression of many genes (Figure 3C). Of note, compared with cells in cluster 0, we found that cells in cluster 1 were en- riched in genes associated with Ag processing, cross-presenta- tion, and IL-2 signaling pathway (Figure 3F; Tables S2 and S3). Among the DEGs, we initially used Mrc1 (CD206) and Lamp2 (CD107b) (Figures S4A and S4B; Table S2) as a ﬁrst approach to identify and ﬂow cytometry-sort the two KC popula- tions. An ad interim bulk RNA-seq analysis of the two popula- tions revealed Esam (ESAM) as highly differentially expressed (Figure S4C). A KC subset with enriched IL-2 sensing machinery and Ag presentation capacity can be identiﬁed On the basis of these data, we designed a panel of markers for use in conventional ﬂow cytometry to identify these KC subpop- ulations and validate the aforementioned high-throughput approach. The CD45+ F4/80+ CD11bint TIM-4+ KC population split into CD206ESAM (hereafter KC1; ~70%–85% of total KCs) and CD206+ESAM+ (hereafter KC2; ~15%–30% of total KCs) cells (Figures 4A and 4B). Imaging analyses conﬁrmed the presence of two distinct KC subpopulations (Figure 4C; Video S1). Importantly, RNA-seq analyses on bulk KC1 and KC2 sorted from C57BL/6 mice conﬁrmed that KC2 are enriched in IL-2 signaling components (IL-2 receptor subunits and mole- cules implicated in intracellular signal transduction) (Figures 4D and 4E; Table S4). Higher expression of the IL-2 receptor sub- units, MHC-I, and co-stimulatory molecules in KC2 was conﬁrmed at the protein level using ﬂow cytometry analysis (Fig- ures 4F–4J). Together, the data suggest that KC2 are better equipped than KC1 to respond to IL-2 and increase their capac- ity to cross-present hepatocellular Ags. Thus, one might predict that IL-2 treatment might render KC2 more sensitive than KC1 to CD8+ T cell-mediated killing. To test this hypothesis, we treated KCs respond to IL-2 and cross-present hepatocellular Ags We generated MUP-core mice whose hematopoietic cells (including KCs) lack Tap1 and therefore cannot express MHC-I and present Ags to CD8+ T cells. This was achieved by injection of either WT or Tap1/ bone marrow into irradiated MUP-core mice, followed by CLL treatment to deplete the residual radio- resistant KCs and allow the complete reconstitution of the entire KC compartment with bone marrow-derived cells (Sitia et al., 2011) (Figure 2S). Cor93 TN injected into MUP-core mice whose hematopoietic cells (including KCs) lacked MHC-I had a much lower response to IL-2c than did Cor93 TN injected into mice car- rying Ag presentation-competent KCs (Figures 2T and 2U), sug- gesting that Cor93 T cells interacted with IL-2-stimulated KCs that cross-presented core protein-derived epitopes after the up- take of damaged hepatocytes. Taken together, these results indicate that optimal reinvigoration of intrahepatically primed CD8+ T cells by IL-2 requires the capacity of KCs to cross-pre- sent HBV Ags, possibly derived from circulating virions and/or damaged hepatocytes. for each cell (n = 169) using the Smart-seq2 pipeline (Picelli et al., 2014). This dataset was analyzed using Seurat (Stuart et al., 2019), and four main cell clusters were identiﬁed and visualized using uniform manifold approximation and projection (UMAP) (Becht et al., 2018) (Figure 3B). Cluster 0 (n = 68) and cluster 1 (n = 59) cells showed higher expression of classical KC-associ- ated gene markers, such as Clec4f, Lyz2, and Csf1r (Figures 3C and 3D; Table S2). Pathway analysis of their respective gene markers yielded immunological pathways and processes typical of macrophages and professional APCs and were thus considered bona ﬁde KCs (Figures 3E and 3F; Table S3). Cells in cluster 2 (n = 30) expressed genes such as Cd34, Cd209c, and Fgd4 but low amounts of macrophage genes (Figures 3C and 3D), while among their speciﬁc markers we found a large number of ribosomal and non-coding genes. They also showed a smaller number of transcripts detected per cell and a higher percentage of mitochondrial genes, indicating a high fraction of apoptotic cells in this population, and hence were excluded from subsequent analyses (Figure 3E and data not shown). Single-cell RNA-seq identiﬁes two distinct populations of KCs among liver-resident macrophages (E) Cell type annotation of the four clusters on the basis of the identiﬁed markers. (F) Pathway analysis of each cluster. Enriched pathways (Huang et al., 2019) are ordered by p value, and the most biologically informative among the top ten are shown. See also Figure S4 Table S2 and Table S3 (A) Sorting strategy for liver macrophages. Liver macrophages are deﬁned as live, CD45+, Lineage (CD3, CD19, Ly6G, CD49b), CD64+, F4/80+ cells. (B) UMAP projection of sorted cells. Each dot corresponds to a single cell, colored according to the unbiased clusters identiﬁed: cluster 0 (red, 68 cells), cluster 1 (green, 59 cells), cluster 2 (blue, 30 cells), and cluster 3 (purple, 12 cells). (C) Heatmap of normalized and scaled expression values of the 2,811 marker genes identifying the four clusters. Genes highlighted on the right are representative of each cluster. Color coding of the bar on the top of the heatmap as in (B). (D) Violin plots showing the normalized expression proﬁle of selected genes differentially expressed in the four clusters. (E) Cell type annotation of the four clusters on the basis of the identiﬁed markers. (F) Pathway analysis of each cluster. Enriched pathways (Huang et al., 2019) are ordered by p value, and the most biologically informative among the top ten are shown. See also Figure S4 Table S2 and Table S3 alized and scaled expression values of the 2,811 marker genes identifying the four clusters. Genes highlighted on the right are coding of the bar on the top of the heatmap as in (B). depleting the residual radio-resistant KCs by CLL to allow the complete reconstitution of the entire KC compartment with bone marrow-derived cells, (3) inducing DTR expression in KC2 by tamoxifen administration, and ﬁnally (4) depleting KC2 by DT injection prior to Cor93 TN transfer followed by IL-2c treatment (Figure 5E). DT treatment caused a ~75% decrease in KC2 (Fig- ures 5G and 5H) and resulted in a lower ability of Cor93 T cells to proliferate and differentiate into cytotoxic effector cells clus- tered throughout the liver lobule in response to IL-2c (Figures 5I–5L). These data indicate that KC2 are required for the optimal reinvigoration of intrahepatically primed T cells by IL-2. HBV replication-competent transgenic mice with IL-2c 24 h after Cor93 TN cell injection and checked the KC1/KC2 ratio 4 days later (Figure 4K). Single-cell RNA-seq identiﬁes two distinct populations of KCs among liver-resident macrophages Consistent with the hypothesis that IL-2 prefer- entially increased the capacity of KC2 to cross-present hepatocellular Ags and thus rendered them more sensitive to CD8+ T cell-mediated killing, we found that KC2 almost completely disappeared in Cor93 T cell-injected HBV transgenic mice treated with IL-2c (Figures 4L–4N). Notably, neither IL-2c treatment alone (in the absence of Cor93 TN cell transfer) nor se- vere liver inﬂammation (induced by Cor93 TEFF) altered the KC1/ KC2 ratio (Figure S5). Immunity 54, 2089–2100, September 14, 2021 2095 Single-cell RNA-seq identiﬁes two distinct populations of KCs among liver-resident macrophages Next, we asked whether the IL-2-responsive KCs represent a distinct subpopulation. To this end, we used high-dimensional single-cell RNA-seq (scRNA-seq) to characterize KC heteroge- neity at steady state. We ﬂow cytometry-sorted live CD45+ Lineage CD64+ F4/80+ liver macrophages from C57BL/6 mice (Figure 3A), isolated RNA, and generated transcriptional proﬁles 2094 Immunity 54, 2089–2100, September 14, 2021 ll OPEN ACCESS C A D F B E C ure 3. Single-cell RNA-seq identiﬁes two distinct populations of KCs among liver-resident macrophages orting strategy for liver macrophages. Liver macrophages are deﬁned as live, CD45+, Lineage (CD3, CD19, Ly6G, CD49b), CD64+, F4/80+ cells. MAP projection of sorted cells. Each dot corresponds to a single cell, colored according to the unbiased clusters identiﬁed: cluster 0 (red, 68 cells), cluster 1 en, 59 cells), cluster 2 (blue, 30 cells), and cluster 3 (purple, 12 cells). eatmap of normalized and scaled expression values of the 2,811 marker genes identifying the four clusters. Genes highlighted on the right are representative ach cluster. Color coding of the bar on the top of the heatmap as in (B). iolin plots showing the normalized expression proﬁle of selected genes differentially expressed in the four clusters. ell type annotation of the four clusters on the basis of the identiﬁed markers. athway analysis of each cluster. Enriched pathways (Huang et al., 2019) are ordered by p value, and the most biologically informative among the top ten shown. also Figure S4 Table S2 and Table S3 B A D C B A D E E F F Figure 3. Single-cell RNA-seq identiﬁes two distinct populations of KCs among liver-resident macrophages Figure 3. Single-cell RNA-seq identiﬁes two distinct populations of KCs among liver-resident macrophages (A) Sorting strategy for liver macrophages. Liver macrophages are deﬁned as live, CD45+, Lineage (CD3, CD19, Ly6G, CD49b), CD64+, F4/80+ cells. (B) UMAP projection of sorted cells. Each dot corresponds to a single cell, colored according to the unbiased clusters identiﬁed: cluster 0 (red, 68 cells), cluster 1 (green, 59 cells), cluster 2 (blue, 30 cells), and cluster 3 (purple, 12 cells). (C) Heatmap of normalized and scaled expression values of the 2,811 marker genes identifying the four clusters. Genes highlighted on the right are representative of each cluster. Color coding of the bar on the top of the heatmap as in (B). (D) Violin plots showing the normalized expression proﬁle of selected genes differentially expressed in the four clusters. Data are representative of at least three independent experiments. See also Figure S5, Table S4, and Video S1. IL-2 Here, we have delineated the mechanisms by which hepatocell- ularly primed HBV-speciﬁc CD8+ T cells acquire antiviral and pathogenic effector functions following the exogenous adminis- tration of IL-2. These mechanisms rely on KCs and, in particular, on a hitherto unidentiﬁed subset of KCs, referred to as KC2, that is poised to respond to IL-2 and cross-present viral Ags con- tained within circulating virions or within hepatocytes. We next sought to generate a model in which KC2 could be selec- tively depleted to assess their role in the cross-presentation of he- patocellular Ags upon in vivo IL-2 treatment. We took advantage of the observation that KC2 (but not KC1) express the endothelial cell marker VE-cadherin (encoded by Cdh5) (Figures 5A–5D) to establish a system allowing inducible depletion of KC2 but not endothelial cells. This was achieved by (1) injecting Cdh5creERT2; Rosa26iDTR bone marrow into irradiated MUP-core mice, (2) The observation that DCs, generally regarded as the main cross-presenting APCs in vivo (Joffre et al., 2012), are Immunity 54, 2089–2100, September 14, 2021 2095 A C D H I J K L M N E G F B Figure 4. Identiﬁcation of a KC subset with enriched IL-2 sensing machinery (A) Representative ﬂow cytometry plot of KC1 and KC2 gating strategy. KCs are identiﬁed as live, CD45+, CD11bint, F4/80+, MHCII+, TIM4+ liver non-parenchymal cells. KC1 are deﬁned as ESAM CD206 KCs. KC2 are deﬁned as ESAM+ CD206+ KCs. (B) Relative representation of KC1 and KC2 percentages in the liver of C57BL/6 mice (n = 15). (C) Representative confocal immunoﬂuorescence micrographs of liver sections from C57BL/6 mice. Sinusoids were identiﬁed as CD38+ cells and are depicted in white. CD206+ cells are depicted in red and F4/80+ cells in green. Scale bars represent 50 or 10 mm (see also Video S1). ll OPEN ACCESS Article A C B ll OPEN ACCESS Article ll OPEN ACCESS Article ll OPEN ACCESS A B A B A C C E G F D G E G D H I J K L M N Figure 4. Identiﬁcation of a KC subset with enriched IL-2 sensing machinery H I J K L M N L M N K L H N M Figure 4. Identiﬁcation of a KC subset with enriched IL-2 sensing machinery Figure 4. IL-2 Identiﬁcation of a KC subset with enriched IL-2 sensing machinery g g y (A) Representative ﬂow cytometry plot of KC1 and KC2 gating strategy. KCs are identiﬁed as live, CD45+, CD11bint, F4/80+, MHCII+, TIM4+ liver non-parenchymal cells. KC1 are deﬁned as ESAM CD206 KCs. KC2 are deﬁned as ESAM+ CD206+ KCs. white. CD206+ cells are depicted in red and F4/80+ cells in green. Scale bars represent 50 or 10 mm (see also Video S1). (D)GSEArelativetotheIL-2pathwayenrichmentinKC2(red)andKC1(blue)samples.Geneswerepre-rankedonthebasisofthelog2foldchangebetweenKC2andKC1. (E) Heatmap representing the relative expression of the IL-2 receptor signaling components in KC1 and KC2 isolated from C57BL/6 mice (n = 3 per group). Values in log2(TPM) were scaled by row across samples (Z score). (F d G) R i ﬂ l (F) d MFI (G) f CD2 CD122 d CD132 i i KC1 KC2 d LSEC (d ﬁ d li CD4 CD31+ (L–N) Ratio between KC1 and KC2 (L) and absolute numbers of KC1 (M) and KC2 (N) in the liver of PBS-treated (blue) or IL-2c-treated (red) mice. n = 4; p < 0.05, one-tailed Mann-Whitney U test. (L–N) Ratio between KC1 and KC2 (L) and absolute numbers of KC1 (M) and KC2 (N) in the liver of PBS-treated (blue) or IL-2c-treated (red) mice. n = 4; p < 0.05, t il d M Whit U t t (L–N) Ratio between KC1 and KC2 (L) and absolute numbers of KC1 (M) and KC2 (N) in the liver of PBS-treated (blue) or IL-2c-treated (red) mice. n = 4; p < 0.05, one-tailed Mann-Whitney U test. Data are representative of at least three independent experiments. See also Figure S5, Table S4, Data are representative of at least three independent experiments. See also Figure S5, Table S4, and Video S1. IL-2 2096 Immunity 54, 2089–2100, September 14, 2021 ll OPEN ACCESS 103 104 105 106 107 108 Cor93 T cells / Liver * 104 105 106 * IFN- + Cor93 T cells / Liver 0 500 1000 1500 sALT (U/L) DT PBS *** DT PBS DT PBS day 5 day 0 PBS DT MUP-core Analyses (day 5) IL-2c +1d Cor93 T N 0 DT -3d / -1d Tamoxifen -1w CLL (x2) -6w Cdh5 creERT2; Rosa26 iDTR BM -10w Irradiation -10w PBS DT X X X X X X X X X X X X X DT PBS 103 104 105 106 107 KCs / Liver DT PBS 0 5 10 15 KC1/KC2 ratio CD206 F4/80 DT PBS * 8% 19% Analyses (day 0) Cdh5 creERT2; Rosa26 tdTomato Tamoxifen -7d tdTomato+cells(%) 0 20 40 60 80 100 KC1 KC2 LSECs tdTomato Mode LSEC KC2 KC1 Lineage CD11b DAPI SSC-A CD45 CD31 LSEC CD64 F4/80 TIM4 MHCII ESAM CD206 KC1 KC2 Among singlets 1 2 3 4 5 6 A E I J K L F G H B C D Figure 5. KC2 are required for the optimal restoration of intrahepatically primed, dysfunctional CD8+ T cells by IL-2 A) Schematic representation of the experimental setup. IL-2 Cdh5 CreERT2; Rosa26 tdTomato mice were treated with tamoxifen, and livers were collected and analyzed days after treatment Analyses (day 0) Cdh5 creERT2; Rosa26 tdTomato Tamoxifen -7d A Lineage CD11b DAPI SSC-A CD45 CD31 LSEC CD64 F4/80 TIM4 MHCII ESAM CD206 KC1 KC2 Among singlets 1 2 3 4 5 6 B Lineage CD11b CD45 CD31 LSEC 2 3 Lineage CD11b DAPI SSC-A CD45 CD31 LSEC Among singlets 1 2 3 B tdTomato Mode LSEC KC2 KC1 C A C B tdTomato+cells(%) 0 20 40 60 80 100 KC1 KC2 LSECs D D CD64 F4/80 TIM4 MHCII ESAM CD206 KC1 KC2 4 5 6 MUP-core Analyses (day 5) IL-2c +1d Cor93 T N 0 DT -3d / -1d Tamoxifen -1w CLL (x2) -6w Cdh5 creERT2; Rosa26 iDTR BM -10w Irradiation -10w PBS DT X X X X X X X X X X X X X E DT PBS 103 104 105 106 107 KCs / Liver DT PBS 0 5 10 15 KC1/KC2 ratio CD206 F4/80 DT PBS * 8% 19% F G H DT PBS 103 104 105 106 107 KCs / Liver CD206 F4/80 DT PBS 8% 19% G F DT PBS 0 5 10 15 KC1/KC2 ratio * H G E F H 1 1 KC1/KC2 ratio H KC1/KC2 ratio 103 104 105 106 107 108 Cor93 T cells / Liver * DT PBS I 104 105 106 * IFN- + Cor93 T cells / Liver DT PBS J 0 500 1000 1500 sALT (U/L) DT PBS *** day 5 day 0 K PBS DT L J I K L DT Figure 5. KC2 are required for the optimal restoration of intrahepatically primed, dysfunctional CD8+ T cells by IL-2 (A) Schematic representation of the experimental setup. Cdh5 CreERT2; Rosa26 tdTomato mice were treated with tamoxifen, and livers were colle 7 days after treatment. (B) Gating strategy for KC1, KC2, and LSECs. (E) Schematic representation of the experimental setup. MUP-core mice were lethally irradiated and reconstituted with Cdh5creERT2; Rosa26iDTR bone marrow (BM). Four weeks later, mice received two injections of clodronate liposomes (CLLs) to remove residual radio-resistant KCs. Nine weeks after BM reconstitution, mice were treated once with 5 mg of tamoxifen by oral gavage. Mice were treated with diphtheria toxin (DT) every 48 h starting 3 days before Cor93 TN injection (1 3 106 cells/mouse). Indicated mice received IL-2c 1 day after Cor93 TN transfer. IL-2 Livers were collected and analyzed 5 days after Cor93 TN transfer. (F) Absolute numbers of total KCs (deﬁned as live, CD45+, TIM4+, F4/80+ cells) in the liver of PBS (blue) or DT (red) treated mice. (G) Representative ﬂow cytometry plots of KC1 (CD206 KCs) and KC2 (CD206+ KCs) populations gated on total KCs (live, CD45+, TIM4+, F4/80+ cells) in the liver of the indicated mice at the time of TN injection. (H) Ratio between KC1 and KC2 in the liver of PBS-treated (blue) or DT-treated (red) mice. n = 3; p < 0.05, one-tailed Mann-Whitney U test. (I and J) Total numbers (I) and numbers (J) of IFN-g-producing Cor93 T cells in the livers of the indicated mice. PBS, n = 5; DT, n = 4. p < 0.05, two-tailed Mann- Whitney U test. (H) Ratio between KC1 and KC2 in the liver of PBS-treated (blue) or DT-treated (red) mice. n = 3; p < 0.05, one-tailed Mann-Whitney U test. (I and J) Total numbers (I) and numbers (J) of IFN-g-producing Cor93 T cells in the livers of the indicated mice. PBS, n = 5; DT, n = 4. p < 0.05, two-tailed Mann- Whitney U test. (K) Amount of ALT in the serum of the indicated mice at the indicated time points PBS n 5; DT n 4 **p < 0 001 two way ANOVA with Sidak’s multiple (L) Representative confocal immunoﬂuorescence micrographs of liver sections from the indicated mice 5 days after Cor93 TN transfer. Cor93 T cells were identiﬁed as CD45.1+ cells and are depicted in green. Sinusoids were identiﬁed as CD38+ cells and are depicted in gray. Scale bars represent 100 mm. Data are representative of two independent experiments (L) Representative confocal immunoﬂuorescence micrographs of liver sections from the indicated mice 5 days after Cor93 TN transfer. Cor93 T cells were identiﬁed as CD45.1+ cells and are depicted in green. Sinusoids were identiﬁed as CD38+ cells and are depicted in gray. Scale bars represent 100 mm. Data are representative of two independent experiments. Data are representative of two independent experiments. Immunity 54, 2089–2100, September 14, 2021 2097 ll OPEN ACCESS Article Article dispensable for the optimal reinvigoration of intrahepatically primed T cells by IL-2 remains to be explored but is consistent with the lack of a functional IL-2 receptor expression by DCs (Raeber et al., 2020). Limitations of study The therapeutic implications of our study rely on the existence of an IL-2-responsive, cross-presenting KC population in humans. Of note, recent publications suggest the existence of a human KC2-like subset that expresses at least some of the human or- thologs of the KC2-speciﬁc genes identiﬁed here (Aizarani et al., 2019; MacParland et al., 2018; Ramachandran et al., 2019; Wu et al., 2020). Future studies are certainly warranted to dissect human KC heterogeneity and function in the healthy as well as in the diseased liver. The location of CD8+ T cell priming during natural HBV infection is unclear. Immunological dogma holds that naive CD8+ T cells initially encounter cognate Ag in secondary lymphoid organs where cross-priming by professional APCs of circulating virions and subviral particles promotes the differentiation into effector cells endowed with liver homing potential. Yet there is no direct experimental evidence indicating that secondary lymphoid or- gans are the priming site for CD8+ T cells in HBV-infected humans or chimpanzees, and the idea behind their involvement has been supported by the assumption that the intrahepatic priming of naive CD8+ T cells (especially in the presence of high Ag load) should promote functional impairment of these cells. On the basis of these considerations, it is generally believed that priming of HBV-speciﬁc naive CD8+ T cells in the liver cannot promote viral clearance but, rather, contribute to the establishment of a persis- tent infection. However, on the basis of the results presented here, it is tempting to speculate that during acute HBV infection, the liver is fully competent to sustain the priming of HBV-speciﬁc effector CD8+ T cells endowed with antiviral activity, provided it occurs in the presence of high local concentrations of IL-2 that in- crease KC2 cross-presentation. A putative source of IL-2 in this scenario might be intrahepatic Ag-speciﬁc effector CD4+ T cells. In support of this hypothesis, which cannot be tested in humans because of the limitations of collecting liver biopsies in acutely infected patients, are the observations that (1) CD4+ T cell depletion in chimpanzees prior to infection precludes effec- tive T cell priming and causes persistent infection with minimal immunopathology (Asabe et al., 2009), and (2) detection of CD8+ T cells in the liver of HBV-infected chimpanzees coincides with the hepatic detection of CD4+ T cells (Guidotti et al., 1999). In parallel to the present study we have used high dimen IL-2 and for other conditions similarly requiring to overcome the tolerogenic potential of the hepatic microenvironment. Such strategies might include liposome or nanoparticle-based formu- lations targeting KC2-expressed surface Ags as well as inte- grase-defective third-generation lentiviral vectors exploiting combinations of transcriptional and post-transcriptional micro- RNA-mediated control (Be´ ne´ chet et al., 2019). The results reported here are noteworthy considering that steady-state KC cross-presentation of HBV Ags is an inefﬁcient process that cannot be increased by liver inﬂammation, hepato- cellular death, or the administration of therapeutic monoclonal antibodies directed against HBsAg leading to the generation of circulating immune complexes (Fumagalli et al., 2020). Our data do not rule out a direct effect of IL-2 on T cells; however, they indicate that optimal in vivo reinvigoration of intrahepatically primed T cells by IL-2 depends on the presence of KC2. The extent to which the reported increase in cross-presentation of hepatocellular Ags by KCs in response to in vivo IL-2 administra- tion is the result of direct IL-2 sensing by KCs or an indirect effect remains to be determined. SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10.1016/j. immuni.2021.05.005. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availabil B RNA puriﬁcation and RNA-seq library preparation B Confocal immunoﬂuorescence histology and histo- chemistry In parallel to the present study, we have used high-dimen- sional single-cell sequencing, mass cytometry, and ﬂow cytometry, coupled with in vivo fate-mapping models to perform an in-depth characterization of KC2 at steady state (Bleriot et al., 2021, this issue of Immunity). These analyses have revealed a speciﬁc metabolic role for KC2 in regulating glucose homeosta- sis and oxidative stress (Bleriot et al., 2021). Future studies should be directed at identifying the signals required for KC2 development and maintenance and explore the potential role of these cells in other diseases affecting the liver. y d QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION 2098 Immunity 54, 2089–2100, September 14, 2021 Article Be´ ne´ chet, A.P., De Simone, G., Di Lucia, P., Cilenti, F., Barbiera, G., Le Bert, N., Fumagalli, V., Lusito, E., Moalli, F., Bianchessi, V., et al. (2019). Dynamics and genomic landscape of CD8+ T cells undergoing hepatic prim- ing. Nature 574, 200–205. of the manuscript; and the members of the Iannacone laboratory for helpful discussions. Flow cytometry was carried out at FRACTAL, a ﬂow cytometry resource and advanced cytometry technical applications laboratory estab- lished by the San Raffaele Scientiﬁc Institute. Confocal immunoﬂuorescence histology was carried out at Alembic, an advanced microscopy laboratory es- tablished by the San Raffaele Scientiﬁc Institute and the Vita-Salute San Raf- faele University. We thank the Singapore Immunology Network (SIgN) immunogenomics platform, part of the SIgN Immunomonitoring platform (sup- ported by ASTAR Biomedical Research Council [BMRC] Industry Alignment Fund [IAF] grant 311006 and BMRC transition funds [H16/99/b0/011]). We would like to acknowledge the PhD program in basic and applied immunology and oncology at Vita-Salute San Raffaele University, as G.D.S., V.F., and C.L. conducted this study as partial fulﬁllment of their PhDs in molecular medicine within that program. M.I. is supported by European Research Council (ERC) Consolidator Grant 725038, ERC Proof of Concept Grant 957502, Italian Asso- ciation for Cancer Research (AIRC) grants 19891 and 22737, Italian Ministry of Health (MoH) grant RF-2018-12365801, Lombardy Foundation for Biomedical Research (FRRB) grant 2015-0010, the European Molecular Biology Organization Young Investigator Program, and a Funded Research Agreement from Gilead Sciences. F.A. is the recipient of a Fondazione Umberto Veronesi postdoctoral fellowship. X.F. is the recipient of iCARE fellowship 23906 from AIRC. F.G. is supported by SIgN core funding, the Singapore National Research Foundation Senior Investigatorship (NRFI) (NRF2016NRF- NRFI001-02), and the European Molecular Biology Organization Young Inves- tigator Program. Blattman, J.N., Grayson, J.M., Wherry, E.J., Kaech, S.M., Smith, K.A., and Ahmed, R. (2003). Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo. Nat. Med. 9, 540–547. Bleriot, C., Barreby, E., Dunsmore, G., Ballaire, R., Chakarov, S., Ficht, X., De Simone, G., Andreata, D., Fumagalli, V., Guo, W., et al. (2021). A subset of Kupffer cells regulates metabolism through the expression of CD36. Immunity 54. https://doi.org/10.1016/j.immuni.2021.08.006. Blum, J.S., Wearsch, P.A., and Cresswell, P. (2013). Pathways of antigen pro- cessing. Annu. Rev. Immunol. 31, 443–473. Bosco, M.C., Curiel, R.E., Zea, A.H., Malabarba, M.G., Ortaldo, J.R., and Espinoza-Delgado, I. (2000). AUTHOR CONTRIBUTIONS G.D.S., F.A., and V.F. designed and performed experiments, analyzed data, performed the statistical analyses, prepared the ﬁgures, and edited the manu- script. C.B. performed the scRNA-seq experiment under the supervision of F.G. P.D.L., X.F., E.B.B., L.G., F.F.D.P., G.A., M.M., P.Z., A.P.B., M.R., S.C., and F.M. performed experiments and analyzed data. C.L., S.G., and J.M.G.-M. performed the analyses on the sequencing data. M.B. provided re- agents and advice for KC2 depletion. L.G.G. and F.G. provided conceptual advice and edited the manuscript. M.I. designed and coordinated the study, provided funding, and wrote the paper. Ficht, X., and Iannacone, M. (2020). Immune surveillance of the liver by T cells. Sci. Immunol. 5, eaba2351. Flatz, L., Hegazy, A.N., Bergthaler, A., Verschoor, A., Claus, C., Fernandez, M., Gattinoni, L., Johnson, S., Kreppel, F., Kochanek, S., et al. (2010). Development of replication-defective lymphocytic choriomeningitis virus vec- tors for the induction of potent CD8+ T cell immunity. Nat. Med. 16, 339–345. Fukao, T., and Koyasu, S. (2000). Expression of functional IL-2 receptors on mature splenic dendritic cells. Eur. J. Immunol. 30, 1453–1457. Fumagalli, V., Di Lucia, P., Venzin, V., Bono, E.B., Jordan, R., Frey, C.R., Delaney, W., Chisari, F.V., Guidotti, L.G., and Iannacone, M. (2020). Serum HBsAg clearance has minimal impact on CD8+ T cell responses in mouse models of HBV infection. J. Exp. Med. 217, 217. DECLARATION OF INTERESTS M.I. participates in advisory boards and consultancies for Gilead Sciences, Roche, Third Rock Ventures, Amgen, and Allovir. L.G.G is a member of the board of directors at Genenta Science and Epsilon Bio and participates in advi- sory boards and consultancies for Gilead Sciences, Roche, and Arbutus Bio- pharma. M.I. and L.G.G. are inventors on patents ﬁled, owned, and managed by San Raffaele Scientiﬁc Institute, Vita-Salute San Raffaele University, and the Telethon Foundation on technology related to work discussed in this manuscript (WO2020/016434, WO2020/016427, WO2020/030781, WO2020/ 234483, European Union [EU] patent applications 19211249.8 and 20156716.1, and U.K. patent application 1907493.9). F.G. is a member of the Immunity advisory board. Guidotti, L.G., Martinez, V., Loh, Y.T., Rogler, C.E., and Chisari, F.V. (1994). Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice. J. Virol. 68, 5469–5475. Guidotti, L.G., Matzke, B., Schaller, H., and Chisari, F.V. (1995). High-level hepatitis B virus replication in transgenic mice. J. Virol. 69, 6158–6169. Guidotti, L.G., Rochford, R., Chung, J., Shapiro, M., Purcell, R., and Chisari, F.V. (1999). Viral clearance without destruction of infected cells during acute HBV infection. Science 284, 825–829. Guidotti, L.G., Inverso, D., Sironi, L., Di Lucia, P., Fioravanti, J., Ganzer, L., Fiocchi, A., Vacca, M., Aiolﬁ, R., Sammicheli, S., et al. (2015). Immunosurveillance of the liver by intravascular effector CD8(+) T cells. Cell 161, 486–500. Received: December 4, 2020 Revised: March 13, 2021 Accepted: May 5, 2021 Published: August 31, 2021 Revised: March 13, 2021 Accepted: May 5, 2021 Herr, F., Lemoine, R., Gouilleux, F., Meley, D., Kazma, I., Heraud, A., Velge- Roussel, F., Baron, C., and Lebranchu, Y. (2014). IL-2 phosphorylates STAT5 to drive IFN-g production and activation of human dendritic cells. J. Immunol. 192, 5660–5670. Published: August 31, 2021 Article IL-2 signaling in human monocytes involves the phosphorylation and activation of p59hck. J. Immunol. 164, 4575–4585. Boyman, O., Kovar, M., Rubinstein, M.P., Surh, C.D., and Sprent, J. (2006). Selective stimulation of T cell subsets with antibody-cytokine immune com- plexes. Science 311, 1924–1927. De Giovanni, M., Cutillo, V., Giladi, A., Sala, E., Maganuco, C.G., Medaglia, C., Di Lucia, P., Bono, E., Cristofani, C., Consolo, E., et al. (2020). Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells. Nat. Immunol. 21, 321–330. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. ACKNOWLEDGMENTS We envision that strategies aimed at targeting IL-2 to KC2 should be considered for the treatment of chronic HBV infection We thank M. Freschi, M. Raso, and A. Fiocchi for technical support; M. Silva for secretarial assistance; D. Pinschewer for rLCMV; R. Ostuni for critical reading 2098 Immunity 54, 2089–2100, September 14, 2021 ll OPEN ACCESS ll OPEN ACCESS Article Immunity 54, 2089–2100, September 14, 2021 2099 REFERENCES Joffre, O.P., Segura, E., Savina, A., and Amigorena, S. (2012). Cross-presen- tation by dendritic cells. Nat. Rev. Immunol. 12, 557–569. Kobayashi, K.S., and van den Elsen, P.J. (2012). NLRC5: a key regulator of MHC class I-dependent immune responses. Nat. Rev. Immunol. 12, 813–820. Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140. Kronin, V., Vremec, D., and Shortman, K. (1998). Does the IL-2 receptor alpha chain induced on dendritic cells have a biological function? Int. Immunol. 10, 237–240. Sitia, G., Iannacone, M., Aiolﬁ, R., Isogawa, M., van Rooijen, N., Scozzesi, C., Bianchi, M.E., von Andrian, U.H., Chisari, F.V., and Guidotti, L.G. (2011). Kupffer cells hasten resolution of liver immunopathology in mouse models of viral hepatitis. PLoS Pathog. 7, e1002061. Kuleshov, M.V., Jones, M.R., Rouillard, A.D., Fernandez, N.F., Duan, Q., Wang, Z., Koplev, S., Jenkins, S.L., Jagodnik, K.M., Lachmann, A., et al. (2016). Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 44 (W1), W90–W97. Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck, W.M., 3rd, Hao, Y., Stoeckius, M., Smibert, P., and Satija, R. (2019). Comprehensive Integration of Single-Cell Data. Cell 177, 1888–1902.e21. Li, B., and Dewey, C.N. (2011). RSEM: accurate transcript quantiﬁcation from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323. Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., and Mesirov, J.P. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression proﬁles. Proc. Natl. Acad. Sci. U S A 102, 15545–15550. Li, J., Lu, E., Yi, T., and Cyster, J.G. (2016). EBI2 augments Tfh cell fate by pro- moting interaction with IL-2-quenching dendritic cells. Nature 533, 110–114. Liang, D., Zuo, A., Shao, H., Born, W.K., O’Brien, R.L., Kaplan, H.J., and Sun, D. (2012). Role of CD25+ dendritic cells in the generation of Th17 autoreactive T cells in autoimmune experimental uveitis. J. Immunol. 188, 5785–5791. Vollmar, B., and Menger, M.D. (2009). The hepatic microcirculation: mecha- nistic contributions and therapeutic targets in liver injury and repair. Physiol. Rev. 89, 1269–1339. Liao, W., Lin, J.-X., and Leonard, W.J. (2013). Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 38, 13–25. Warren, A., Le Couteur, D.G., Fraser, R., Bowen, D.G., McCaughan, G.W., and Bertolino, P. (2006). REFERENCES Aizarani, N., Saviano, A., Sagar, Mailly, L., Durand, S., Herman, J.S., Pessaux, P., Baumert, T.F., and Gr€un, D. (2019). A human liver cell atlas reveals hetero- geneity and epithelial progenitors. Nature 572, 199–204. Horst, A.K., Neumann, K., Diehl, L., and Tiegs, G. (2016). Modulation of liver tolerance by conventional and nonconventional antigen-presenting cells and regulatory immune cells. Cell. Mol. Immunol. 13, 277–292. Asabe, S., Wieland, S.F., Chattopadhyay, P.K., Roederer, M., Engle, R.E., Purcell, R.H., and Chisari, F.V. (2009). The size of the viral inoculum contributes to the outcome of hepatitis B virus infection. J. Virol. 83, 9652–9662. Huang, R., Grishagin, I., Wang, Y., Zhao, T., Greene, J., Obenauer, J.C., Ngan, D., Nguyen, D.-T., Guha, R., Jadhav, A., Southall, N., Simeonov, A., and Austin, C.P. (2019). The NCATS BioPlanet – An Integrated Platform for Exploring the Universe of Cellular Signaling Pathways for. Toxicology, Systems Biology, and Chemical Genomics. Front Pharmacol. https://doi.org/10.3389/fphar. 2019.00445. Becht, E., McInnes, L., Healy, J., Dutertre, C.-A., Kwok, I.W.H., Ng, L.G., Ginhoux, F., and Newell, E.W. (2018). Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. 37, 38–44. Immunity 54, 2089–2100, September 14, 2021 2099 ll OPEN ACCESS Article Raeber, M.E., Rosalia, R.A., Schmid, D., Karakus, U., and Boyman, O. (2020). Interleukin-2 signals converge in a lymphoid-dendritic cell pathway that pro- motes anticancer immunity. Sci. Transl. Med. 12, eaba5464. Iannacone, M., Sitia, G., Isogawa, M., Marchese, P., Castro, M.G., Lowenstein, P.R., Chisari, F.V., Ruggeri, Z.M., and Guidotti, L.G. (2005). Platelets mediate Iannacone, M., Sitia, G., Isogawa, M., Marchese, P., Castro, M.G., Lowenstein, P.R., Chisari, F.V., Ruggeri, Z.M., and Guidotti, L.G. (2005). Platelets mediate cytotoxic T lymphocyte-induced liver damage. Nat. Med. 11, 1167–1169. cytotoxic T lymphocyte-induced liver damage. Nat. Med. 11, 1167–1169. Isogawa, M., Chung, J., Murata, Y., Kakimi, K., and Chisari, F.V. (2013). CD40 activation rescues antiviral CD8+ T cells from PD-1-mediated exhaustion. PLoS Pathog. 9, e1003490. Ramachandran, P., Dobie, R., Wilson-Kanamori, J.R., Dora, E.F., Henderson, B.E.P., Luu, N.T., Portman, J.R., Matchett, K.P., Brice, M., Marwick, J.A., et al. (2019). Resolving the ﬁbrotic niche of human liver cirrhosis at single-cell level. Nature 575, 512–518. Jenne, C.N., and Kubes, P. (2013). Immune surveillance by the liver. Nat. Immunol. 14, 996–1006. Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., and Smyth, G.K. (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47. REFERENCES T lymphocytes interact with hepatocytes through fenes- trations in murine liver sinusoidal endothelial cells. Hepatology 44, 1182–1190. Liao, Y., Smyth, G.K., and Shi, W. (2019). The R package Rsubread is easier, faster, cheaper and better for alignment and quantiﬁcation of RNA sequencing reads. Nucleic Acids Res. 47, e47. Liberzon, A., Birger, C., Thorvaldsdo´ ttir, H., Ghandi, M., Mesirov, J.P., and Tamayo, P. (2015). The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 1, 417–425. West, E.E., Jin, H.-T., Rasheed, A.-U., Penaloza-Macmaster, P., Ha, S.-J., Tan, W.G., Youngblood, B., Freeman, G.J., Smith, K.A., and Ahmed, R. (2013). PD-L1 blockade synergizes with IL-2 therapy in reinvigorating ex- hausted T cells. J. Clin. Invest. 123, 2604–2615. MacParland, S.A., Liu, J.C., Ma, X.-Z., Innes, B.T., Bartczak, A.M., Gage, B.K., Manuel, J., Khuu, N., Echeverri, J., Linares, I., et al. (2018). Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage popula- tions. Nat. Commun. 9, 4383. Wong, Y.C., Tay, S.S., McCaughan, G.W., Bowen, D.G., and Bertolino, P. (2015). Immune outcomes in the liver: Is CD8 T cell fate determined by the envi- ronment? J. Hepatol. 63, 1005–1014. Picelli, S., Faridani, O.R., Bjo¨ rklund, A˚ .K., Winberg, G., Sagasser, S., and Sandberg, R. (2014). Full-length RNA-seq from single cells using Smart- seq2. Nat. Protoc. 9, 171–181. Wu, X., Hollingshead, N., Roberto, J., Knupp, A., Kenerson, H., Chen, A., Strickland, I., Horton, H., Yeung, R., Soysa, R., and Crispe, I.N. (2020). Human liver macrophage subsets deﬁned by CD32. Front. Immunol. 11, 2108. Pol, J.G., Caudana, P., Paillet, J., Piaggio, E., and Kroemer, G. (2020). Effects of interleukin-2 in immunostimulation and immunosuppressionImmunomodulatory effects of IL-2. J. Exp. Med. 217, 2261. Wuest, S.C., Edwan, J.H., Martin, J.F., Han, S., Perry, J.S.A., Cartagena, C.M., Matsuura, E., Maric, D., Waldmann, T.A., and Bielekova, B. (2011). A role for interleukin-2 trans-presentation in dendritic cell-mediated T cell activation in humans, as revealed by daclizumab therapy. Nat. Med. 17, 604–609. Popov, A., Driesen, J., Abdullah, Z., Wickenhauser, C., Beyer, M., Debey- Pascher, S., Saric, T., Kummer, S., Takikawa, O., Domann, E., et al. (2008). Infection of myeloid dendritic cells with Listeria monocytogenes leads to the suppression of T cell function by multiple inhibitory mechanisms. J. Immunol. 181, 4976–4988. Zordan, P., Cominelli, M., Cascino, F., Tratta, E., Poliani, P.L., and Galli, R. (2018). Tuberous sclerosis complex-associated CNS abnormalities depend on hyperactivation of mTORC1 and Akt. J. Clin. Invest. 128, 1688–1706. 2100 Immunity 54, 2089–2100, September 14, 2021 REFERENCES 2100 Immunity 54, 2089–2100, September 14, 2021 ll OPEN ACCESS ll OPEN ACCESS STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies PE-CF594 anti-mouse CD3e BD Biosciences BD Biosciences Cat# 562286; RRID: AB_11153307 eFluor 450 anti-mouse CD4 eBioscience eBioscienceCat# 48-0042-82; RRID: AB_468865 PB rat anti-mouse CD8a BD Biosciences BD Biosciences Cat# 558106; RRID: AB_397029 BV650 anti-mouse/human CD11b BioLegend BioLegend Cat# 101239; RRID: AB_11125575 PE-CF594 rat anti-mouse CD19 BD Biosciences BD Biosciences Cat# 562291; RRID: AB_11154223 PE/Cyanine7 anti-mouse CD25 BioLegend BioLegend Cat# 102015; RRID: AB_312864 BV605 anti-mouse CD31 BioLegend BioLegend Cat# 102427; RRID: AB_2563982 BUV395 anti-mouse CD45 BD Biosciences BD Biosciences Cat# 564279; RRID: AB_2651134 BV711 anti-mouse CD64 BioLegend BioLegend Cat# 139311; RRID: AB_2563846 APC/Cyanine7 anti-mouse F4/80 BioLegend BioLegend Cat# 123117; RRID: AB_893489 AF700 anti-mouse I-A/I-E BioLegend BioLegend Cat# 107621; RRID: AB_493726 PE/cyanine7 anti-mouse Tim-4 BioLegend BioLegend Cat# 130010; RRID: AB_2565719 FITC TIM-4 Biorbyt BiorbytCat# orb103599 AF647 anti-mouse CD69 BioLegend BioLegend Cat# 104517; RRID: AB_492848 APC/cyanine7 anti-mouse CD45.1 BioLegend BioLegend Cat# 110715; RRID: AB_313504 AF647 anti-Mouse IFN-g BD Biosciences BD Biosciences Cat# 557735; RRID: AB_396843 PE anti-mouse CD11c BioLegend BioLegend Cat# 117308; RRID: AB_313777 PE/cyanine7 anti-mouse I-Ab BioLegend BioLegend Cat# 116420; RRID: AB_10575296 AF647 anti-Stat5 (pY694) BD Biosciences BD Biosciences Cat# 612599; RRID: AB_399882 PE-cyanine7 anti-mouse/rat foxp3 Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# 25-5773-82; RRID: AB_891552 PE anti-mouse CD122 BioLegend BioLegend Cat# 123210; RRID: AB_940617 PE anti-mouse CD132 BioLegend BioLegend Cat# 132306; RRID: AB_2280163 APC rat anti-mouse CD40 BD Biosciences BD Biosciences Cat# 558695; RRID: AB_1645224 PE hamster anti-mouse CD80 BD Biosciences BD Biosciences Cat# 553769; RRID: AB_395039 BV650 mouse anti-mouse H-2Kb BD Biosciences BD Biosciences Cat# 742861; RRID: AB_2741103 PE anti-mouse ESAM BioLegend BioLegend Cat# 136203; RRID: AB_1953300 AF647 anti-mouse CD206 BioLegend BioLegend Cat# 141712; RRID: AB_10900420 PE-CF594 rat anti-mouse Ly-6G BD Biosciences BD Biosciences Cat# 562700; RRID: AB_2737730 PE anti-mouse Ly-6C antibody BioLegend BioLegend Cat# 128008; RRID: AB_1186132) PE-CF594 rat anti-mouse CD49b BD Biosciences BD Biosciences Cat# 562453; RRID: AB_11153857 PE anti-mouse CD107b Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# 12-5989-82; RRID: AB_466103 AF488 anti-mouse F4/80 BioLegend BioLegend Cat# 123120; RRID: AB_893479 APC anti-mouse CD206 BioLegend BioLegend Cat# 141708; RRID: AB_10900231 AF594 anti-mouse CD38 BioLegend BioLegend Cat# 102725; RRID: AB_2566435 AF647 anti-mouse CD45.1 BioLegend BioLegend Cat# 110720; RRID: AB_313491 Puriﬁed anti-mouse CD38 BioLegend BioLegend Cat# 102702; RRID: AB_312923 F4/80 monoclonal antibody Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# MF48000; RRID: AB_10376289 InVivoMAb anti-mouse CD16/CD32 Bio X Cell Bio X Cell Cat# BE0307; RRID:AB_2736987 LYVE-1 antibody Novus Biological Novus Biological Cat# NB600- 1008; RRID:AB_10000497 Immunity 54, 2089–2100.e1–e8, September 14, 2021 e1 ll OPEN ACCESS Article Continued REAGENT or RESOURCE SOURCE IDENTIFIER Alexa ﬂuor 488, chicken anti-rabbit IgG (H+L) cross-adsorbed secondary antibody Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# A-21441; RRID: AB_2535859 Alexa ﬂuor 568, goat anti-rat IgG (H+L) cross- adsorbed secondary antibody Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# A-11077; RRID: AB_2534121 Alexa ﬂuor 488, chicken antirRat IgG (H+L) cross- adsorbed secondary asntibody Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# A-21470; RRID: AB_2535873 Rabbit mAb anti-stat5 Cell Signaling Technology Cell Signaling Technology Cat# 94205; RRID: AB_2737403 XP Rabbit mAb anti-phospho-Stat5 (Tyr694) Cell Signaling Technology Cell Signaling Technology Cat# 4322; RRID: AB_10544692 Peroxidase AfﬁniPure Goat Anti-Rabbit IgG (H+L) Jackson ImmunoResearch Labs Jackson ImmunoResearch Labs Cat# 111-035-003; RRID: AB_2313567 InVivoMAb anti-mouse IL-2 Bio X Cell Bio X Cell Cat# BE0043-1; RRID: AB_1107705 InVivoMab anti-mouse Ly6G Bio X Cell Bio X Cell Cat# BE0075-1; RRID: AB_1107721 InVivoMab anti-mouse Ly6G/Ly6C Bio X Cell Bio X Cell Cat# BE0075; RRID: AB_10312146 Recombinant viral vectors rLCMV-core/env (Be´ ne´ chet et al., 2019) N/A Chemicals, peptides, and recombinant proteins Recombinant mouse IL-2 protein R and D Systems R and D Systems Cat# 402-ML-500/CF Clodronate liposomes Liposoma Liposoma Cat# C-025 Diphtheria toxin Millipore Millipore Cat# 322326 Tamoxifen Sigma Sigma Cat# T5648-5G Corn oil Sigma Sigma Cat# C8267-500ML Viobility 405/520 ﬁxable dye Miltenyi Biotec Miltenyi Cat# 130-109-816 LIVE/DEAD ﬁxable far-red dye Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc, Cat# L34973 DAPI Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# D1306; RRID:AB_2629482 Phosﬂow perm buffer III BD Biosciences BD Biosciences Cat# 558050; RRID: AB_2869118 O.C.T. REFERENCES Bio Optica Bio Optica Cat# 05-9801 FluorSave reagent Millipore Millipore Cat# 345789 Critical commercial assays Foxp3 / transcription factor staining buffer set Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc, Cat# 00-5523-00 CellTrace violet cell proliferation kit Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc, Cat# C34571 Anti-F4/80 MicroBeads UltraPure, mouse Miltenyi Biotec Miltenyi Biotec Cat# 130-110-443, RRID: AB_2858241 EasySep mouse naive CD8+ T cell isolation kit Stem Cell technologies Stem Cell technologiesCat# 19858 Nextera XT DNA library preparation kit Illumina Illumina Cat# FC-131-1024 DNA high sensitivity reagent kit Perkin Elmer Labchip Perkin Elmer Labchip Cat# CLS760672 Arcturus picoPure RNA isolation kit Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# KIT0204 ERCC RNA spike-In mix Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# 4456740 ReliaPrep RNA miniprep system Promega Promega Cat# PRZ6012 TURBO DNA-free kit Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# AM1907 Qubit RNA HS assay kit Thermo Fisher Scientiﬁc Thermo Fisher Scientiﬁc Cat# Q32852 NovaSeq reagent kits Illumina Illumina Cat# 20028312 Clarity Western ECL substrate kit Bio-Rad Bio-Rad Cat# 1705060S Deposited data Bulk RNaseq data This manuscript NCBI Gene Expression Omnibus (GEO) accession GEO: GSE152211 Single-cell RNaseq data This manuscript NCBI Gene Expression Omnibus (GEO) accession GEO: GSE168989 (Continued on next page) e2 Immunity 54, 2089–2100.e1–e8, September 14, 2021 ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: organisms/strains Mouse: C57BL/6 Charles River C57BL/6 colony Mouse: BALB/c Charles River BALB/c colony Mouse: CBy.PL(B6)-Thya/ScrJ The Jackson Laboratory Stock No: 005443 Mouse: C57BL/6-Tg(CAG-EGFP)1Osb/J The Jackson Laboratory Stock No: 003291 Mouse: B6.Cg-Gt(ROSA)26Sortm14(CAG- tdTomato)Hze/J The Jackson Laboratory Stock No: 007914 Mouse: B6.Cg-Tg(CAG-DsRedMST)1Nagy/J The Jackson Laboratory Stock No: 006051 Mouse: B6.129S2-Tap1tm1Arp/J The Jackson Laboratory Stock No: 002458 Mouse: B6.FVB-1700016L2RikTg(Itgax-DTR/ EGFP)57Lan/J The Jackson Laboratory Stock No: 004509 Mouse: C57BL/6- Gt(ROSA)26Sortm1(HBEGF)Awai/J The Jackson Laboratory Stock No: 007900 Mouse: Tg(Cdh5-cre/ERT2)1Rha The Jackson Laboratory MGI:3848982 Mouse: MUP-core 50 [MC50] (Guidotti et al., 1994) Internal colony Mouse: lineage 1.3.32 (Guidotti et al., 1995) Internal colony Mouse: lineage BC10.3 (Isogawa et al., 2013) Internal colony Mouse: lineage 6C2.36 (Isogawa et al., 2013) Internal colony Software and algorithms FlowJo V10 FlowJo https://www.ﬂowjo.com/ RSEM tool (Li and Dewey, 2011) https://deweylab.github.io/RSEM/ Seurat (v3.2.2) (Stuart et al., 2019) https://satijalab.org/seurat/ Enrichr (Kuleshov et al., 2016) https://maayanlab.cloud/Enrichr/ featureCounts (Liao et al., 2019) https://www.rdocumentation.org/packages/ Rsubread/versions/1.22.2/topics/featureCounts edgeR (Robinson et al., 2010) http://bioconductor.org/packages/release/bioc/ html/edgeR.html STAR aligner (Dobin et al., 2013) https://github.com/alexdobin/STAR LIMMA R package (Ritchie et al., 2015) https://bioconductor.org/packages/release/bioc/ html/limma.html pheatmap R Raivo Kolde https://www.rdocumentation.org/packages/ pheatmap/versions/1.0.12/topics/pheatmap fmsb R Minato Nakazawa https://cran.r-project.org/web/packages/fmsb/ index.html Cytoscape Cytoscape https://cytoscape.org GseaPreranked (Subramanian et al., 2005) https://gsea-msigdb.github.io/gseapreranked- gpmodule/v6/index.html homologene R package Ogan Mancarci, Leon French https://cran.r-project.org/web/packages/ homologene/index.html Prism 9 GraphPad software https://www.graphpad.com/scientiﬁc-software/prism BD FACSDiva V8 BD Biosciences https://www.bdbiosciences.com/us/instruments/ research/software/ﬂow-cytometry-acquisition/bd- facsdiva-software/m/111112/overview CytExpert Beckman Coulter https://www.beckman.com/ﬂow-cytometry/ instruments/cytoﬂex/software Leica Application Suite X (LAS X) Leica Microsystem https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/ Imaris bitplane Imaris https://imaris.oxinst.com/products/imaris-for-cell- biologists?gclid=Cj0KCQiAgomBBhDXARIs AFNyUqOQMD64vZvZMyBoHWFOYRm_ ZPxHWLb_tWDl0pGjii8ZVNDkW- UNtRgaAnhfEALw_wcB Fiji-Imagej Imagej https://imagej net/Fiji/Downloads Continued REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: organisms/strains Mouse: C57BL/6 Charles River C57BL/6 colony Mouse: BALB/c Charles River BALB/c colony Mouse: CBy.PL(B6)-Thya/ScrJ The Jackson Laboratory Stock No: 005443 Mouse: C57BL/6-Tg(CAG-EGFP)1Osb/J The Jackson Laboratory Stock No: 003291 Mouse: B6.Cg-Gt(ROSA)26Sortm14(CAG- tdTomato)Hze/J The Jackson Laboratory Stock No: 007914 Mouse: B6.Cg-Tg(CAG-DsRedMST)1Nagy/J The Jackson Laboratory Stock No: 006051 Mouse: B6.129S2-Tap1tm1Arp/J The Jackson Laboratory Stock No: 002458 Mouse: B6.FVB-1700016L2RikTg(Itgax-DTR/ EGFP)57Lan/J The Jackson Laboratory Stock No: 004509 Mouse: C57BL/6- Gt(ROSA)26Sortm1(HBEGF)Awai/J The Jackson Laboratory Stock No: 007900 Mouse: Tg(Cdh5-cre/ERT2)1Rha The Jackson Laboratory MGI:3848982 Mouse: MUP-core 50 [MC50] (Guidotti et al., 1994) Internal colony Mouse: lineage 1.3.32 (Guidotti et al., 1995) Internal colony Mouse: lineage BC10.3 (Isogawa et al., 2013) Internal colony Mouse: lineage 6C2.36 (Isogawa et al., 2013) Internal colony Software and algorithms FlowJo V10 FlowJo https://www.ﬂowjo.com/ RSEM tool (Li and Dewey, 2011) https://deweylab.github.io/RSEM/ Seurat (v3.2.2) (Stuart et al., 2019) https://satijalab.org/seurat/ Enrichr (Kuleshov et al., 2016) https://maayanlab.cloud/Enrichr/ featureCounts (Liao et al., 2019) https://www.rdocumentation.org/packages/ Rsubread/versions/1.22.2/topics/featureCounts edgeR (Robinson et al., 2010) http://bioconductor.org/packages/release/bioc/ html/edgeR.html STAR aligner (Dobin et al., 2013) https://github.com/alexdobin/STAR LIMMA R package (Ritchie et al., 2015) https://bioconductor.org/packages/release/bioc/ html/limma.html pheatmap R Raivo Kolde https://www.rdocumentation.org/packages/ pheatmap/versions/1.0.12/topics/pheatmap fmsb R Minato Nakazawa https://cran.r-project.org/web/packages/fmsb/ index.html Cytoscape Cytoscape https://cytoscape.org GseaPreranked (Subramanian et al., 2005) https://gsea-msigdb.github.io/gseapreranked- gpmodule/v6/index.html homologene R package Ogan Mancarci, Leon French https://cran.r-project.org/web/packages/ homologene/index.html Prism 9 GraphPad software https://www.graphpad.com/scientiﬁc-software/prism BD FACSDiva V8 BD Biosciences https://www.bdbiosciences.com/us/instruments/ research/software/ﬂow-cytometry-acquisition/bd- facsdiva-software/m/111112/overview CytExpert Beckman Coulter https://www.beckman.com/ﬂow-cytometry/ instruments/cytoﬂex/software Leica Application Suite X (LAS X) Leica Microsystem https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/ Imaris bitplane Imaris https://imaris.oxinst.com/products/imaris-for-cell- biologists?gclid=Cj0KCQiAgomBBhDXARIs AFNyUqOQMD64vZvZMyBoHWFOYRm_ ZPxHWLb_tWDl0pGjii8ZVNDkW- UNtR A hfEAL B Fiji-Imagej (Continued on next page) ll OPEN ACCESS ll OPEN ACCESS Article Continued REAGENT or RESOURCE SOURCE IDENTIFIER Other FACS CANTO II BD Bioscience N/A CytoFLEX LX Beckman Coulter N/A FACSAria Fusion BD Bioscience N/A Illumina HiSeq 4000 system Illumina N/A Agilent Bioanalyser Agilent N/A UVItec Eppendorf N/A SP5 or SP8 confocal microscope Leica Microsystem N/A Vet abcTM scil N/A Data and code availability Data and code availability Bulk RNA-seq data generated during this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GEO: GSE152211. Single Cell RNA-seq data generated during this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GEO: GSE168989. Bulk RNA-seq data generated during this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GEO: GSE152211. Single Cell RNA-seq data generated during this study have been deposited in the Gene Expression Omnibus (GEO) with the Bulk RNA-seq data generated during this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GEO: GSE152211. Single Cell RNA-seq data generated during this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GEO: GSE168989. Single Cell RNA-seq data generated during this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GEO: GSE168989. Single Cell RNA-seq data generated during this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GEO: GSE168989. Lead contact Lead contact Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Matteo Iannacone (iannacone.matteo@hsr.it). Lead contact Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Matteo Iannacone (iannacone.matteo@hsr.it). Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Matteo Iannacone (iannacone.matteo@hsr.it). Mice Mice C57BL/6, CD45.1 (inbred C57BL/6), BALB/c, Thy1.1 (CBy.PL(B6)-Thya/ScrJ), b-actin-GFP [C57BL/6-Tg(CAG-EGFP)1Osb/J], Ai14(RCL-tdT)-D [B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J ], b-actin-DsRed [B6.Cg-Tg(CAG-DsRedMST)1Nagy/J], Tap1/ (B6.129S2-Tap1tm1Arp/J), CD11cDTR [B6.FVB-1700016L2RikTg(Itgax-DTR/EGFP)57Lan/J], ROSA26iDTR [C57BL/6-Gt(ROSA) 26Sortm1(HBEGF)Awai/J], Cdh5CreERT2 [Tg(Cdh5-cre/ERT2)1Rha] mice were purchased from Charles River or The Jackson Labo- ratory. MUP-core transgenic mice (lineage MUP-core 50 [MC50], inbred C57BL/6, H-2b), that express the HBV core protein in 100% of the hepatocytes under the transcriptional control of the mouse major urinary protein (MUP) promoter, have been previously described (Guidotti et al., 1994). HBV replication-competent transgenic mice (lineage 1.3.32, inbred C57BL/6, H-2b), that express all of the HBV Ags and replicate HBV in the liver at high viral copies without any evidence of cytopathology, have been previously described (Guidotti et al., 1995). In indicated experiments, MUP-core and HBV replication-competent transgenic mice were used as C57BL/6 x BALB/c H-2bxd F1 hybrids. Cor93 TCR transgenic mice (lineage BC10.3, inbred CD45.1), in which > 98% of the splenic CD8+ T cells recognize a Kb-restricted epitope located between residues 93-100 in the HBV core protein (MGLKFRQL), have been previously described (Isogawa et al., 2013). Env28 TCR transgenic mice (lineage 6C2.36, inbred Thy1.1 BALB/c), in which ~83% of the splenic CD8+ T cells recognize a Ld-restricted epitope located between residues 28–39 of HBsAg (IPQSLDSWWTSL), have been previously described (Isogawa et al., 2013). For imaging experiments Cor93 transgenic mice were bred against b-actin-GFP, while Env28 transgenic mice were bred against b-actin-DsRed mice (inbred BALB/c). Bone marrow (BM) chimeras were generated by irra- diation of MUP-core or C57BL/6 mice with one dose of 900 rad and reconstitution with the indicated BM; mice were allowed to recon- stitute for at least 8 weeks before experimental manipulations. Mice were housed under speciﬁc pathogen-free conditions and entered experiments at 8-10 weeks of age. In all experiments, mice were matched for age, sex and (for the 1.3.32 animals) serum HBeAg concentration before experimental manipulations. All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientiﬁc Institute and are compliant with all relevant ethical regulations. C57BL/6, CD45.1 (inbred C57BL/6), BALB/c, Thy1.1 (CBy.PL(B6)-Thya/ScrJ), b-actin-GFP [C57BL/6-Tg(CAG-EGFP)1Osb/J], Ai14(RCL-tdT)-D [B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J ], b-actin-DsRed [B6.Cg-Tg(CAG-DsRedMST)1Nagy/J], Tap1/ (B6.129S2-Tap1tm1Arp/J), CD11cDTR [B6.FVB-1700016L2RikTg(Itgax-DTR/EGFP)57Lan/J], ROSA26iDTR [C57BL/6-Gt(ROSA) 26S t 1(HBEGF)A i/J] Cdh5CreERT2 [T (Cdh5 /ERT2)1Rh ] i h d f Ch l Ri Th J k L b Materials availability This study did not generate new unique reagents This study did not generate new unique reagents. METHOD DETAILS Naive T cell isolation, adoptive transfer and in vivo treatments Naive T cell isolation, adoptive transfer and in vivo treatments Mice were adoptively transferred with 5 3 106 or 1 3 106 HBV-speciﬁc naive CD8+ TCR transgenic T cells isolated from the spleens of Cor93 and/or Env28 TCR transgenic mice, as described (Be´ ne´ chet et al., 2019). IL-2/anti-IL-2 complexes (IL-2c) were prepared by incubating 1.5 mg of rIL-2 (R&D Systems) with 50 mg anti-IL-2 mAb (clone S4B6-1, BioXcell) per mouse, as previously described (Boy- man et al., 2006). Mice were injected with IL-2c intraperitoneally (i.p.) one day after T cell transfer, unless otherwise indicated. In indi- cated experiments, naive CD8+ T cells from the spleens of Cor93 TCR transgenic mice were differentiated in vitro for 7-9 days into effector cells prior to adoptive transfer (1 3 107 cells), or in vitro co-culture, as described (Be´ ne´ chet et al., 2019; Guidotti et al., 2015). In indicated experiments, Kupffer cells (KCs) were depleted by intravenous injection of 200 mL of clodronate-containing liposomes (Liposoma) 2 days prior to T cell injection, as described (Be´ ne´ chet et al., 2019), unless otherwise indicated. In indicated experiments, mice were injected i.p. with 200 mg of anti-Ly6G depleting antibody (clone 1A8, BioXcell) one day before and one day after T cell transfer. In indicated experiments, mice were injected intravenously (i.v.) with 200 mg of anti-Gr1 depleting antibody (clone RB6- 8C5, BioXcell) every 48h starting from 3 days before T cell transfer. In indicated experiments, C57BL/6 or MUP-core mice were lethally irradiated and reconstituted for at least 8 weeks with BM from CD11c-DTR mice; dendritic cells were subsequently depleted by injecting i.p. 20 ng per gram of mouse of diphtheria toxin (Millipore) every 48h starting from 3 days before T cell transfer. In indi- cated experiments, MUP-core mice were lethally irradiated and reconstituted for at least 8 weeks with BM from C57BL/6 or Tap1/ mice. To achieve full reconstitution of Kupffer cells from donor-derived BM, mice were injected with 200 mL of clodronate-containing liposomes 28 and 31 days after BM injection. In indicated experiments, MUP-core mice were lethally irradiated and reconstituted for at least 8 weeks with BM from Cdh5CreERT2; Rosa26iDTR; Rosa26tdTomato; CX3CR1GFP mice. To achieve full reconstitution of Kupffer cells from donor-derived BM, mice were injected with 200 mL of clodronate-containing liposomes 28 and 31 days after BM injection. Viruses and viral vectors Replication-incompetent LCMV-based vectors encoding HBV core and envelope proteins (rLCMV-core/env) were generated, grown and titrated as previously described (Be´ ne´ chet et al., 2019). Mice were injected intravenously (i.v.) with 2.5 3 105 infectious units of rLCMV vector 4h before CD8+ T cell injection. All infectious work was performed in designated BSL-2 or BSL-3 workspaces, in accor- dance with institutional guidelines. e4 Immunity 54, 2089–2100.e1–e8, September 14, 2021 ll OPEN ACCESS Article Cell isolation and ﬂow cytometry y y Single-cell suspensions of liver, spleen and blood were generated as described (Be´ ne´ chet et al., 2019). Kupffer cell isolation was performed as described (Be´ ne´ chet et al., 2019). All ﬂow cytometry stainings of surface-expressed and intracellular molecules were performed as described (De Giovanni et al., 2020). Cell viability was assessed by staining with Viobility 405/520 ﬁxable dye (Miltenyi, #130-109-816), LIVE/DEAD Fixable Far-Red dye (Invitrogen, # L34973) or DAPI (Invitrogen, # D1306). Abs used included: anti-CD3 (clone: 145-2C11, Cat#562286, BD Biosciences), anti-CD4 (clone: RM4-5, Cat #48-0042-82, eBioscience), anti-CD8a (clone: 53-6.7, Cat# 558106, BD Biosciences), anti-CD11b (clone: M1/70, Cat#101239), anti-CD19 (clone: 1D3, Cat#562291 BD Bio- sciences), anti-CD25 (clone: PC61, Cat#102015), anti-CD31 (clone: 390, Cat#102427), anti-CD45 (clone: 30-F11, Cat#564279 BD Biosciences), anti-CD64 (clone: X54-5/7.1, Cat#139311), anti-F4/80 (clone: BM8, Cat#123117), anti-I-A/I-E (clone: M5/114.15.2, Cat#107622), anti-TIM4 (clone: RTM4-54 Cat#130010), anti-TIM4 (polyclonal, Cat#orb103599 Biorbyt), anti-CD69 (clone: H1.2F3, Cat# 104517), anti-CD45.1 (clone: A20, Cat#110716), anti-IFN-g (clone: XMG1.2, Cat# 557735 BD Biosciences), anti-CD11c (clone: N418, Cat# 117308), anti-I-Ab (clone: AF6-120.1, Cat# 116420), anti-Stat5 pY694 (clone: 47, Cat# 612599 BD Biosciences), anti- Foxp3 (clone FJK-16 s, Cat#25-5773-82 Thermoﬁsher), anti-CD122 (clone TM-B1 Cat#123210), anti-CD132 (clone TUgm2 Cat#132306), anti-CD40 (clone 3/23 Cat#558695 BD Biosciences), anti-CD80 (clone 1610A1 Cat#553769 BD Biosciences), anti- H2-Kb (clone AF6-88.5 Cat#742861 BD Biosciences), anti-ESAM (clone 1G8/ESAM, Cat#136203), anti-CD206 (clone C068C2, Cat#141712), anti-Ly6G (clone 1A8, Cat #562700 BD Biosciences), anti-Ly6C (clone HK1.4, Cat# 128008), anti-CD49b (clone DX5, Cat#562453 BD Biosciences), anti CD107b (clone M3/84, Cat #12-5989-82 eBioscience). All Abs were purchased from Bio- Legend, unless otherwise indicated. Recombinant dimeric H-2Ld:Ig and H-2Kb:Ig fusion proteins (BD Biosciences) complexed with peptides derived from HBsAg (Env28-39) or from HBcAg (Cor93-100), respectively, were prepared according to the manufac- turer’s instructions. Dimer staining was performed as described (Iannacone et al., 2005). Flow cytometry staining for phosphorylated STAT5 was performed using Phosﬂow Perm Buffer III (BD Bioscience), following the manufacturer’s instructions. Flow cytometry staining for Foxp3 was performed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience), following the manufacturer’s instructions. In indicated experiments, cells were stained with CellTraceTM Violet cell proliferation kit (CTV, Invitrogen), following man- ufacturer’s instructions. All ﬂow cytometry analyses were performed in FACS buffer containing PBS with 2 mM EDTA and 2% FBS on a FACS CANTO II (BD Bioscience) or CytoFLEX LX (Beckman Coulter) and analyzed with FlowJo software (Treestar). METHOD DETAILS To induce the expression of the Cre recombinase, mice were treated with 5 mg of Tamoxifen (Sigma) by oral gavage in 200 mL of corn oil one week before further manipulations. KC2 were depleted subsequently by injecting i.p. 20 ng per gram of mouse of diphtheria toxin (Millipore) 3 days and 1 day prior to T cell transfer. For the experiment described in Figure 2, KCs were sorted from liver non-parenchymal cells as live, lineage negative (CD3-, CD19-, Ly6G-, CD49b-), CD45+, CD11bint, F4/80+, CD64+, MHCII+, TIM4+ cells. For the experiment described in Figure 3, single cells were sorted from liver non-parenchymal cells as live, CD45+, lineage negative (CD3-, CD19-, Ly6G-, CD49b-), F4/80+, CD64+ cells. For the experiment described in Figure 4, KCs were sorted from liver non-parenchymal cells as live, CD45+, CD11bint, F4/80+, MHCII+, TIM4+ cells. Among total KCs, KC1 were sorted as CD206- ESAM- cells and KC2 as CD206+, ESAM+ cells. Total KCs, KC1 and KC2 were ﬂow cytometry-sorted with a 100 mm nozzle at 4C on a FACSAria Fusion (BD) cell sorter in a buffer containing PBS with 2% FBS. Cells were always at least 98% pure (data not shown). In indicated experiments, F4/80+ cells were puriﬁed from liver Immunity 54, 2089–2100.e1–e8, September 14, 2021 e5 Cell puriﬁcation For the experiment described in Figure 2, KCs were sorted from liver non-parenchymal cells as live, lineage negative (CD3-, CD19-, Ly6G-, CD49b-), CD45+, CD11bint, F4/80+, CD64+, MHCII+, TIM4+ cells. For the experiment described in Figure 3, single cells were sorted from liver non-parenchymal cells as live, CD45+, lineage negative (CD3-, CD19-, Ly6G-, CD49b-), F4/80+, CD64+ cells. For the experiment described in Figure 4, KCs were sorted from liver non-parenchymal cells as live, CD45+, CD11bint, F4/80+, MHCII+, TIM4+ cells. Among total KCs, KC1 were sorted as CD206- ESAM- cells and KC2 as CD206+, ESAM+ cells. Total KCs, KC1 and KC2 were ﬂow cytometry-sorted with a 100 mm nozzle at 4C on a FACSAria Fusion (BD) cell sorter in a buffer containing PBS with 2% FBS. Cells were always at least 98% pure (data not shown). In indicated experiments, F4/80+ cells were puriﬁed from liver ll OPEN ACCESS Article Article non-parenchymal cells by positive immunomagnetic separation (Miltenyi Biotec, #130-110-443), according to the manufacturer’s in- structions. In indicated experiments, CD8+ T cells were puriﬁed from splenocytes using EasySepTM kit (StemCell # 19858), according to the manufacturer’s instructions. non-parenchymal cells by positive immunomagnetic separation (Miltenyi Biotec, #130-110-443), according to the manufacturer’s in- structions. In indicated experiments, CD8+ T cells were puriﬁed from splenocytes using EasySepTM kit (StemCell # 19858), according to the manufacturer’s instructions. RNA puriﬁcation and RNA-seq library preparation Bulk RNA-seq on CD206-CD107b- and CD206+CD107b+ cells (shown in Figure S4): between 20,000 and 50,000 cells were ﬂow cy- tometry-sorted using CD206 (Mrc1) and CD107b (Lamp2) to identify CD206-, CD107b- and CD206+, CD107b+ cells. Total RNA was extracted using Arcturus PicoPure. RNA Isolation kit (Arcturus. Thermo Fisher Scientiﬁc, Waltham, MA, USA) according to manufac- turer’s protocol. All Mouse RNAs were analyzed on Agilent Bioanalyser (Agilent, Santa Clara, CA, USA) for quality assessment with RNA Integrity Number (RIN) range from 5.8 to 6.7 and median of RIN 6.4. cDNA libraries were prepared using 2 ng of total RNA and 1ul of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion. Thermo Fisher Scientiﬁc, Waltham, MA, USA) using the Smart-Seq v2 protocol (Picelli et al., 2014) with the following modiﬁcations: i) addition of 20 mM TSO; ii) use of 200 pg cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA). All samples were subjected to an in- dexed paired-end sequencing run of 2x151 cycles on an Illumina HiSeq 4000 system (Illumina) (25 samples/lane) p q g y q y ( ) ( p ) Bulk RNA-seq experiment on total KCs (shown in Figure 2) and bulk RNA-seq experiment on sorted KC1 and KC2 (shown in Fig- ure 4): ﬂow cytometry-sorted KCs, KC1 and KC2 were lysed in ReliaPrep RNA Cell Miniprep System (Promega #Z6011) and total RNA was isolated following manual extraction. DNA digestion was performed with TURBO DNA-free Kit (Invitrogen #AM1907). RNA was quantiﬁed with Qubit RNA HS Assay Kit (Invitrogen # Q32852) and analysis of its integrity was assessed with Agilent RNA 6000 Pico Kit (Agilent #5067-1513) on a Bioanalyser instrument. 6 RNA samples of sorted KC1 and KC2, were processed with the ‘‘SMART-seq Ultra Low Input 48’’ library protocol in order to obtain 30.0M clusters of fragments of 1x100nt of length through NovaSeq 6000 SP Reagent Kit (100 cycles). RNA-seq bioinformatics analysis Bulk RNA-seq experiment on CD206-, CD107b- and CD206+, CD107b+ cells (shown in Figure S4): raw reads were obtained and map- ped to the mouse genome build GRCm38. Gene counts were generated using featureCounts (part of the R subread package) (Liao et al., 2019) with GENCODE version M9 annotations. Differential Expression Analysis genes (DEGs) and MA plots were performed using the R package edgeR (Robinson et al., 2010). Bulk RNA-seq experiment on total KCs (shown in Figure 2) and on sorted KC1 and KC2 (shown in Figure 4): raw reads were aligned to mouse genome build GRCm38 using STAR aligner (Dobin et al., 2013). Read counts per gene were then calculated using featur- eCounts based on GENCODE gene annotation version M16. Read counts were subject to log2 TPM (transcript per million) normal- ization to account for transcript length and library size. Only genes with a TPM value higher than 1 in at least 4 (for the total KC experiment in Figure 2) or 3 (for the KC2 versus KC1) sam- ples were considered for following analysis. Differentially Expressed Genes (DEGs) between groups treated with IL-2c and PBS were identiﬁed by generating a linear model using LIMMA R package (Ritchie et al., 2015). Only DEGs with an adjusted P value < 0.05 (using Benjamini Hochberg correction method) were selected for further analysis. For the ﬁnal KC2 versus KC1 comparison an additional \| logFC\| > 1 ﬁlter was applied. Single-cell RNA-seq Single cells were sorted on a 96-well plate and cDNA libraries were generated using the Smart-seq v2 protocol (Picelli et al., 2014) with the following modiﬁcations: i) 1mg/ml BSA Lysis buffer (Ambion Thermo Fisher Scientiﬁc, Waltham, MA, USA); ii) use of 200 pg cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA). All samples were subjected to an indexed paired-end sequencing run of 2x151 cycles on an Illumina HiSeq 4000 system (Illumina, San Diego, CA, USA) (298 samples/lane). The RSEM tool (Li and Dewey, 2011) was used to perform Transcript Per Million (TPM) normalization starting from FASTQ ﬁles. Single cell data analysis was performed using Seurat (v3.2.2) (Stuart et al., 2019). 169 cells were obtained after applying a ﬁlter to the TMP matrix of at least 200 genes expressed per cell and only genes expressed in at least 3 cells were retained. TPM expression was further normalized and scaled using the SCTransform function, and Umap reduction was then applied on ﬁrst 12 Principal Com- ponents after running PCA. Unbiased clustering was made using the FindClusters function in Seurat with default parameters and a resolution value of 1. Speciﬁc markers for the different unbiased clusters were found using the function FindAllmarkers or FindMarkers in Seurat with default parameters and were then used for functional enrichment analysis with the online tool Enrichr (Kuleshov et al., 2016). The plots showing normalized expression values with a color scale on top of Umap plots (on Figure S4) and the Violin plots of spe- ciﬁc genes were produced with FeaturePlot and VlnPlot Seurat functions, respectively. Radar plots visualization Radar plots were generated using the fmsb R package. Different sets of genes were selected based on literature analysis, deﬁning different biological processes. For each category, the mean TPM expression for each gene within samples (separately for control and treated samples) was calculated. Next, the mean between all the genes belonging to a category was calculated and used as the value to represent the dimension in the radar plot. Functional enrichment analysis y Bulk RNA-seq analysis of the experiment described in Figure 2: of the 4073 signiﬁcant (FDR < 0.05) identiﬁed DEGs between control (PBS) and treated (IL-2c) samples, 1515 were upregulated and 2558 were downregulated. Those were subject to a functional e6 Immunity 54, 2089–2100.e1–e8, September 14, 2021 Article ll OPEN ACCESS enrichment analysis using the EnrichR R package (Kuleshov et al., 2016). Both the up- and the downregulated DEGs were checked for any biological signature enrichment in both the Gene Ontology Biological Process Database and the Kyoto Encyclopedia of Genes and Genomes for Mouse . After merging the results for the two databases, 858 signiﬁcant (FDR < 0.05) Terms were identiﬁed, of which 428 were derived from the upregulated DEGs and 430 from the downregulated ones. In order to select the top enriched terms, only those with a high Combined Score (-log(p value) * Odds Ratio) were considered. Based on the distribution of the Com- bined Score in the upregulated terms and in the downregulated ones, a threshold of 100 was chosen for the former, while a threshold of 30 for the latter. enrichment analysis using the EnrichR R package (Kuleshov et al., 2016). Both the up- and the downregulated DEGs were checked for any biological signature enrichment in both the Gene Ontology Biological Process Database and the Kyoto Encyclopedia of Genes and Genomes for Mouse . After merging the results for the two databases, 858 signiﬁcant (FDR < 0.05) Terms were identiﬁed, of which 428 were derived from the upregulated DEGs and 430 from the downregulated ones. In order to select the top enriched terms, only those with a high Combined Score (-log(p value) * Odds Ratio) were considered. Based on the distribution of the Com- bined Score in the upregulated terms and in the downregulated ones, a threshold of 100 was chosen for the former, while a threshold of 30 for the latter. Immunoblot analysis Immunoblot on plated KCs was performed as described (Zordan et al., 2018). Primary Abs include anti-STAT5 and anti-pSTAT5 (Tyr694) (rabbit; Cell Signaling Technology #8215) and b-actin (polyclonal; Abcam ab228001). As secondary Ab horseradish perox- idase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, Cat# 111-035-003) was used. Reactive proteins were visualized using a Clarity Western ECL substrate kit (Bio-Rad), and exposure was performed using UVItec (Cambridge MINI HD, Eppendorf). Images were acquired by NineAlliance software. Clustering of upregulated terms For visualization and analysis, both upregulated and downregulated terms were subject to a clustering algorithm, in order to identify the most prominent biological signatures. Brieﬂy, a Jaccard Index Similarity score was calculated for each pair set of terms, based on the DEGs annotated for each term, using an in-house developed script. Next, terms were clustered using a hierarchical clustering method, using as distance measure the Pearson correlation between the calculated Jaccard Index Similarity scores. An arbitrary number of clusters was selected and manually annotated based on the terms present. To visualize the result, the pheatmap R pack- age was used. Gene set enrichment analysis Gene Set Enrichment Analysis (GSEA) from bulk RNA-seq of KC1 and KC2 (Figure 4) was performed using the GseaPreranked Java tool (Subramanian et al., 2005) using pre-ranked Log2 fold changes between KC2 and KC1 populations in expressed genes. HALL- MARK_IL2_STAT5_SIGNALING Gene Set contained in MsigDB (Broad Institute) (Liberzon et al., 2015), Version 6. Since the gene set is based on human genes, mouse orthologs in humans were identiﬁed using the homologene R package (https://cran.r-project.org/ web/packages/homologene/index.html). Network plot visualization Network plot (Figure S3F) was built using Cytoscape software (V 3.8.0 for MacOS). Brieﬂy, starting from EnrichR tables (Table S1), a matrix deﬁning every pair of term-gene was generated, and used as a node list input for Cytoscape. Confocal immunoﬂuorescence histology and histochemistry Confocal microscopy analysis of livers was performed as described (Guidotti et al., 2015). For confocal images of KC1 and KC2, C57BL/6 mice were injected i.v. with 2 mg of anti-F4/80 Alexa Fluor 488 (BioLegend, #123120) and 2 mg of anti-CD206 APC (Bio- Legend, #141708) 10 minutes before harvesting the liver. The liver was ﬁxed overnight in PBS with 4% paraformaldehyde and sub- sequently incubated for 24h in PBS with 30% sucrose. Next, liver lobes were embedded in O.C.T (Killik Bio-Optica) and cut at 14C into 60 mm thick sections with a cryostat. Sections were blocked for 15 min with blocking buffer (PBS, 0.5% BSA, 0.3 % Triton) and stained for 1h at room temperature (RT) with anti-CD38 Alexa Fluor 594 (BioLegend, #102725) in wash/stain buffer (PBS, 0.2% BSA, 0.1% triton). Sections were then washed twice for 5 min, stained with DAPI (Sigma) for 5 min, washed again and mounted for imaging with FluorSave Reagent (Millipore). For additional confocal imaging, the following primary Abs were used for staining: anti-CD45.1 AF647 (110720, BioLegend), anti-F4/80 (BM8, Invitrogen), anti-Lyve-1 (NB600-1008, Novus Biological), anti-CD38 (102702, Bio- Legend). The following secondary Abs were used for staining: Alexa Fluor 488-, Alexa Fluor 514-, Alexa Fluor 568-conjugated anti-rabbit or anti-rat IgG (Life Technologies). Image acquisition was performed with a 63x oil-immersion or 20x objective on an SP5 or SP8 confocal microscope (Leica Microsystem). To minimize ﬂuorophore spectral spillover, the Leica sequential laser excita- tion and detection modality was used. Where necessary to compensate for uneven slide illumination, ﬂuorescent intensity of layers was normalized using Imaris normalize Layers tool. Where necessary, autoﬂuorescence was ﬁltered from the image by channel sub- traction of a deep red autoﬂuorescent channel from APC signal with the Imaris Channel Arithmetics tool. Immunity 54, 2089–2100.e1–e8, September 14, 2021 e7 ll OPEN ACCESS Article Article Biochemical analyses The extent of hepatocellular injury was monitored by measuring serum alanine aminotransferase (sALT) activity at multiple time points after treatment, as previously described (Guidotti et al., 2015). Serum HBeAg was measured by enzyme-linked immunosorbent as- says (ELISA), as previously described (Guidotti et al., 2015). Blood cell counts were measured by Vet abc (scil). e8 Immunity 54, 2089–2100.e1–e8, September 14, 2021 QUANTIFICATION AND STATISTICAL ANALYSIS Results are expressed as mean ± s.e.m. All statistical analyses were performed in Prism (GraphPad Software), and details are pro- vided in the ﬁgure legends. Normality of data distribution was tested in all graphs with a Shapiro-Wilk or D’Agostino & Pearson normality test and parametric tests were chosen only when normality could be conﬁrmed for each dataset. One-tailed test were cho- sen over two-tailed test when basic biology dictates that the change between the control and treatment group can only occur into one direction (e.g., in cell depletion experiments, where the number of cells will be decreased in the treatment versus the control group). Comparisons are not statistically signiﬁcant unless indicated.
https://openalex.org/W2325744899	https://www.sjweh.fi/download.php?abstract_id=1382&file_nro=1	English	null	Effectiveness of and compliance to preventive measures against the occupational transmission of human immunodeficiency virus.	Scandinavian journal of work, environment & health	1,994	cc-by	7,268	Downloaded from www.sjweh.fi on October 24, 2024 Downloaded from www.sjweh.fi on October 24, 2024 Scand J Work Environ Health 1994;20(6):393-400 https://doi.org/10.5271/sjweh.1382 Issue date: 01 Dec 1994 by Elise Roy, MD,1 Pierre Robillard, MD2 by Elise Roy, MD,1 Pierre Robillard, MD2 ROY E, ROBILLARD P. Effectiveness of and compliance to preventive measures against the occu- pational transmission of human immunodeficiency virus. Scand J Work Environ Health 1994;20: 393-400. To prevent the occupational transmission of human immunodeficiency virus (HIV) and other blood- borne infections in health care settings, guidelines have been issued as universal precautions and body substance isolation. Patient testing has also been advocated. The literature on the compliance to and effectiveness of these measures was reviewed and analyzed to establish the state of knowledge and make appropriate recommendations to improve guidelines. It showed that workers' compliance to rec- ommended measures is relatively poor. The effectiveness and cost-effectiveness of universal precau- tions and body substance isolation remain to be demonstrated. Testing patients for HIV infection and other bloodborne pathogens does not appear to be a more appropriate solution. Focus should be placed on preventing parenteral exposures and applying risk assessment methods to identify health care set- tings and procedures at higher risk. These measures would allow safer medical devices to he targeted and would ensure that financial resources would be available to implement appropriate preventive mea- sures. KEY TERMS - body substance isolation, compliance, cost-effectiveness, health care worker, hepatitis B, hepatitis C, risk assessment, universal precautions. As of June 1993, 174 cases of occupational hum an immunodeficiency virus (HIV) infection had been re- ported among health care workers worldwide (1- 12). Of these, 63 are documented seroconversions, meaning that the health care worker had a negative, postexposure HIV antibody test result and subse- quently tested positi ve for HIV. The other I I I cas- es are con sidered to be probable occupational infec- tion s, as seroconversion was not documented and no other risk factors could be identified. The risk of oc- cupational HIV infection following parenteral expo- sure to contaminated blood is estimated to be 0.3% (13). The risk of transmission resulting from expo- sure to mucous membranes or nonintact skin lesions is con sidered to be much lower, around 0.09% (14). atit is C of occupational origin hav e als o been re- ported (15-20) and the risk of transmi ssion follow- ing a parenteral exposure to contaminated blood, al- though not precisely quantified, could be as high as 10% (21). Reprint requests to: Dr E Roy, Centre for AIDS Studies, Montreal Public Health Unit, Montreal General Hospital, 1616Rene Levesque Blvd West, 3'" Floor, Montreal, Can- ada, H3H IP8. Effectiveness of and compliance to preventive measures against the occupational transmission of human immunodeficiency virus. by Roy E, Robillard P Affiliation: Centre for AIDS Studies, Montreal General Hospital, Canada. This article in PubMed: www.ncbi.nlm.nih.gov/pubmed/7701284 REVIEWS Scand J Work Environ Health 1994;20:393-400 I Centre for AIDS Studies, Montreal Public Health Unit, Montreal General Hospital, Montreal, Canada. Direction of Public Health of Montreal, Occupational and Environmental Health Unit, Montreal, Canada. Universal precautions Universal precautions were proposed by the Centers for Disease Control and Prevention to prevent blood- borne infections in health care settings (24, 25). The level of exposure to body fluids constitutes the main determinant of occupational risk of infection. Add- ed to this concept is the principle of universality of risk, all patients being assumed to be infected. Guidelines were issued regarding work practices, personal protective equipment (barriers), cleaning practices, disinfection and decontamination, and the manipulation and disposal of needl es and sharp ob- jects. These measures aim to prevent exposure to specific body fluid s such as blood, semen, vaginal secretions, and any body fluid visibly contaminated with blood, as well as cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, pericardial fluid , and amniotic fluid . Saliva is excluded except in the context of dental and oral procedures. In practical term s, the guidelines recommend the use of individual protective equipment, such as gloves, masks, protective eyewear, apro ns, and gowns, to avoid exposure by direct contact or by splashes. To prevent injuries caused by needles or other sharp objects, needle recapping is proscribed and sharp objects must be disposed of in puncture-resistant containers. As to the age of clientele, those treating children had a lower utilization rate (29, 32) than those treat- ing patients 15--44 years of age, who had the high- est rate (29). As to the type of procedure carr ied out, the rate of glove use among nurses varied from 70% for phle- botomy to 92% for arterial punctures (36). The use of barrier methods was 17% for major procedures, 56% for minor ones, 19% when the patient was bleeding, and 44% when the patient was not bleed- ing (29). The lack of easy access to protective meth- ods in emergency situations could explain these sur- prising results, as sugges ted by a study showing that supplying protective equipment in readily available kits in strateg ic locations of emergency rooms had increased the use of universal precautions from 16 to 62% in a Florida hospital (37). Compliance to the adequate handling and disposal of needles is generally higher than with barrier meth- ods. A study showe d that the manipulation of nee- dles by emergency room residents was adequate in 97% of the cases at an American hospital (37). by Elise Roy, MD,1 Pierre Robillard, MD2 Faced with these very serious infections, measures have been proposed to avoid transmission to health care workers, namely, universal precautions and body substance isol ation. These two sys tems are based On the principle that all patients are considered to be in- fecti ous. This notion of universality constitutes a major change in the app roach to preventing the trans- mission of bloodborne pathogens in health care set- ting s that, until recently, was more selective, rely- ing on the patient's presumed or establi shed diagno- sis. There is resistance to this change among health care workers, and some even suggest that the pres- ence of infections should be acti vel y sou ght out in all patients since knowledge that a patient is infec ted would allow health care workers to protect them- selves better (22, 23). For example, in certain cas- es, a therapeutic decision could be changed when a patient is known to be infected so that alternatives that arc safer for health care personnel could be pre- scribed. Oth er bloodborn e pathogens are also at the origin of occupational infections, for example, the hepati- tis B virus, for which the risk of transmi ssion fol- lowing percutaneous exposure is estimated to be up to 100 times greater than that of HIV. Cases of hep- Thi s article examines the uni versal and selective approaches by reviewing the state of knowledge con- cerning worker compliance, and the effectiveness of both of these appro aches in regard to the occupa- tion al transmission of HIV in the workplace. 393 Scand J Work Environ Health 1994, vol 20, no 6 Universal measures find ings, the rate of glove use and the use of other barrier methods varied according to several param- eters, mainly the profession, the clientele, and the type of procedure. Physicians (surgeo ns, specialists, generalists, residents, and medical students) used gloves much less than nurses, in a proportion vary- ing from 10 to 40% for the former and 40 to 75% for the latter (28-33). Swedish dentists with private practices used gloves during only 54% of their pro- cedures, while those in public practice did so in 77% of the cases (34). Paramedics only rarely complied to the systematic use of barrier methods, the percent- age of utili zation varying from 8 to 37% (29, 35). Universal precautions How- ever, despi te recommendations about needle recap- ping, it was observed that, in four Michi gan hospi- tals, 8 to 51% of the needles found in containers had been recapped before disposal (38). The situation seems even worse for paramedics, who recapped needles in 78% of the cases (35), and for physicians who did so in a proportion varying from 50 to 60% (31). Compliance to universal precautions With respect to the effective- ness of universal precautions, a study targeted a co- hort of health care workers participating in the United States National Institutes of Health (NIH) surveil- lance program of occupational exposures to HIV (39). Workers had to answer a questionnaire on cu- taneous exposures sustained during the previous year. The analyses compared exposures before and after the implementation of universal precautions. The average annual number of exposures per worker dropped from 35.8 to 18.1 for blood (P::;O.OOI) and from 77.8 to 40.8 for other body fluids (P::;O.OOl). Cost and cost-effectiveness studies. An American study looked at the annual costs of implementing universal precaution s in a 900-bed university hospi- tal (51). The conclusion was an annual increase in costs of USD 350 900. However neither the effec- tiveness nor the benefits of universal precautions were evaluated. In a Canadian study the cost-effec- tiveness ratio of universal precautions in a 450-bed hospital was found to be CAD 128 862 000 per case of occupational HIV infection prevented , according to the authors' best-case estimate (52). It was con- cluded that universal precautions were not cost-ef- fective and that priority should be put on the preven- tion of parenteral exposures, notably through a bet- ter design of materials and techniques used. Another study gathered data on physicians' expo- sures in hospital medical care units before and after the implementation of universal precautions. Every- day they had to answer a questionnaire on cutane- ous and parenteral exposures sustained during the day (40). A global reduction in exposures of 5.07 to 2.66 per patient-month per physician, associated with an increase in aborted exposures from 3.41 to 5.90, was found . As for accidental needle sticks, the rate fell from 0.39 to 0.15, but this difference was not sta- tistically significant (P =0.123). The methodologi- cal differences between these two studies and the absence of a reference group make it difficult to draw conclusions on the effectiveness of universal precau- tions. g q Despite the scarcity of studies on costs, universal precautions seem to constitute an expensive solution with to which prevent the occupational transmi ssion of HIV. However direct and indirect costs of expo- sure to body fluids were ignored in the studies. Compliance to universal precautions Compliance to universal precautions The degree of compliance by health care workers to some or all universal precaution measures was stud ied in different clinical settings. In a study of 806 observations made of 24 health care workers in emergency rooms, dental clinics , and plastic surgery clinics, 31.3 % were compliant, 40.7% were partial- ly compliant, and 28% were noncompliant (26). Use of puncture-resistant containers for the disposal of sharp objects was satisfactory, but needle recapping was still very prevalent and com pliance to hand washing very low. Another study combining two methods, questionnaire and observation, found that universal precautions were followed in only 19% of the 1441 procedures observed (27). Compliance ac- cording to profession was 27% for nurses, 5% for laboratory technic ians, and 3% for physicians. Com- pliance to procedures of safe pipetting (97%) and the handling of needles (72%) was good but dropped for hand washing (28%) and the wearing of gloves (35%). In 81% of the cases , physicians did not ma- nipulate needles in compliance with recommenda- tions. Discomfort, interference with procedures, for- getfulness, and lack of safety materials were the rea- sons most often listed for noncompl iance to univer- sal precautions. Most of these studies were conducted with a ques- tionnaire administered to workers, but one study us- ing two methods, questionnaire and direct observa- tion, showed that workers tended to overestimate their compliance to universal precautions (27). None- theless , the tendencies drawn from all the aforemen- tioned studies are similar and show that compliance to recommendations relating to needle manipulation is higher than that relating to the use of barrier meth- ods, that nurses are more compliant than physicians, and that access to, and the ease of use of, protective equipment well integrated into regular task perform- ance tended to increase compliance. Furthermore, the notion of universality is still not well integrated since the potential risk presented by the patient is taken into consideration by health care workers in their decision to use preventive measures. p Most studies evaluated only some of the measures proposed by universal precautions, particularly bar- rier methods such as use of gloves. According to their 394 Scand J Work Environ Health 1994, vol 20, no 6 is complementary to the guidelines of the Centers for Disease Control and Prevention on universal precau- tions. Effectiveness and cost ofuniversal precautions Effectiveness studies. Compliance to universal precautions These costs could be substantial, for example, those related to (i) reporting exposure, (ii) a 6- to 12-month post- exposure follow-up of workers possibly admini s- tering immunoglobulin or zidovudine, (iii) the loss of productivity associated with these visits, and (iv) the postexposure stress of workers awaiting their HIV antibody test results. Otherwise, preventing other bloodborne infections (hepatitis B and C) was not taken into account although it constituted another important parameter in the cost-effectiveness ratio equation. Indeed Stock et al found that universal pre- cautions present a more interesting cost-effectiveness ratio when the risk of hepatiti s B is considered , al- though vaccination is certainly the most effective preventive means for this infection (52). Other authors have attempted to evaluate the ef- fectiveness of certain components of universal pre- cautions. For rigid containers, there was no reduc- tion in the total number of needlesticks nor in the practice of recapping in association with their imple- mentation (41--44). Two of the studies (41,42) showed however a reduction in needlesticks in rela- tion to needle disposal, and one (45) reported a glo- bal reduction of all needlesticks, as well as those re- lated to recapping among nurses. Other studies have dealt with the effectiveness of using some barrier methods , such as gowns and sur- gical sheets (46--48). It was concluded that the ef- fectiveness in preventing blood contact varied ac- cording to the type of material, its impermeability, and wear and tear. Body substance isolation The expressi on body substance isolation describes a system of prevention of nosocomial infecti ons, in- cluding the transmission of occupational infections (53). Like universal precautions, this system is ap- plied to all patients, regardless of their diagnosis. A major difference is that body substance isolation tar- gets all body fluids and moist body substances. It in- cludes six components (54), the following four of which are aimed at bloodbome infections: (i) gloves must be worn for all expected contact with a body substance, (ii) other barriers such as masks, glasses, gowns, and aprons must be worn when contamina- tion of clothing or skin or splashe s are anticipated, (iii) reusable items must be securely contained to pre- vent leakage, (iv) needles and other sharp objects must be disposed of in puncture-resistant containers; needles should not be recapped. There are few other difference s with universal precautions, for example, Even if barrier methods such as gloves succeed- ed in reducing the number of cutaneous exposures (39, 40), their role in preventing the transmission of bloodborne pathogens among health care workers is not clear since needlesticks and cuts, which are not prevented by the use of gloves, present the greatest risk for the transmission of bloodborne infections. Recent studies have been conducted with the pur- pose of evaluating new medical devices designed for preventing accidental needlesticks, such as self- resheathing needles, new connecting devices for in- travenous lines or blunt suture needles. The results are promising and would allow immediate causes of exposures to be eliminated (ie, unnecessary needles or ill-conceived devices) (49, 50). Such an approach 395 Scand J Work Environ Health 1994, vol 20, no 6 and organ donati ons, for which HIV antibody test- ing is systematic. Upon nearing the end of the first decade of the HIV epidemic, however, what some call "HIV exceptionalism" could be challenged and therefore lead to less stringent rules on informed con- sent (65). with body substance isolation, the health care worker is called upon to judge the pertinence of using bar- rier methods such as wearing gloves during phlebot- omy, a practice which is optional if the worker is experienced and does not deem it necessary (55). Some have found that body substance isolation is easier to implement than universal precautions (56), but few studies have evaluated its application and effectiveness, particularly concerning bloodborne infections. Selective measures As an alternative to universal approaches, identify- ing patients infected with HIV has been proposed so that supplemental preventive measures can be taken with these patients and their speci mens (59-6 I). This proposition is in line with labeling infectious specimens to advise the worker to take appropriate preventive measures. Antibody testing for human immunodeficiency virus HIV-infected person s can be identified with a sim- ple serological test. Current tests for HIV antibod- ies, including two immunoenzymatic tests (ELISA) followed by a more specific confirmation test, are very reliable (62). The high sensitivity and specifi- city of the test sequence allow virtual identification of all individuals (except those who are in the post- infection period) with a very low proportion of false positive results. The importance of quality control assuring the high specificity of confirmation tests must be emphasized because a lowering of specifi- city could actually compromise the predictive value of a positive result in populations in which the prev- alence of infection is low. Cost-effectiveness studies. The cost-effectiveness ra- tio of a testing program aimed at patients mainly var- ies as a function of the prevalence and incidence of infection and of the preventive measures taken after testing. According to an American study, the cost of a mandatory testing program for all patients in a 350- bed public hospital would surpass the benefits, and such a program would constitute a waste of resources (68). In 1989 in the Province of Quebec, it was esti- mated that a testing program for patients admitted to acute care hospitals would cost more than 18 mil- lion Canadian dollars (around 13 million US dollars) per year for 800 000 admissions (69). This calcula- tion reflects only a part of the real costs of such an operation since it only took into account direct costs related to the testing and counseling of hospitalized patients, excludin g all those in ambulatory care or out-of-hospital settings. Other direct costs, such as those related to the infrastructure necessary to an- swer the incremental demand for tests, those caused by the repetition of tests, or indirect expenditures such as traveling costs or worktime lost by the in- dividuals being tested, were not included in the cal- culation. Despite the good perform ance of the test, there is a delay for obtaining the result that could limit its widespread use, especially in emergency situations. Effectiveness and cost ofa testing program fo r patients Effectiveness studies. The effectiveness of patient testing in preventing occupational HIV infection has not been widel y studied. Some authors have evalu- ated the association between knowing the serologi- cal status of the patient and the rate of accidental ex- posure to blood among operating room personnel. In one study, no association was found between the per- ceived or known status of a patient and the rate of exposures (66). A similar tendency was observed in another study (67). Apart from the identification of infected patients and the labeling of their specimens, other means of reducing the risk of HIV infection have been pro- posed, for example, therapeutic substitution (propos- ing alternative treatments to the more conventional ones) or reference to a more-experienced profes- sional. To our knowledge, these "preventive meth- ods" have not been studied from the point of view of effectiveness. Body substance isolation In one study, an increase of 60 to 80% in adequate glove use was observed after body sub- stance isolation was implemented, but adequate hand washing was not prevalent (30 to 45%) (57). In a sur- vey, despite adequate training and knowledge of the principles of body substance isolation, nearly half of the respondents frequently using needles recapped them occasionally or often. It can be concluded that, as for universal precautions, worker compliance to body substance isolation is not optimal (58). Selective measures The arrival on the market of rapid and effective tests with sensitivity and specificity approaching those of conventional techniques (63,64) could be a solution to this problem. The utilization of these tests as di- agnostic tools is not currently authorized in all coun- tries. Another aspect that cannot go unmentioned is that all persons undergoing an HIV antibody test must freely give their informed consent and receive ap- propriate counseling. The only exception to this rule of informed consent is in the case of blood, semen, With this available evidence, systematically test- ing all patients appears to be an extremely expen- sive solution to the risk of occupational HIV trans- mission in health care settings. 396 Scand J Work Environ Health 1994, vol 20, no 6 years after the Centers for Disease Control and Pre- vention's first recommendations on HIV preventive measures in health care settings, the situation is still confused. Anecdotal reports suggest that prevention practices in health care settings are often mixed , uni- versal precautions havin g been partially imple- mented, whereas some professionals test patients who "look infected." An alternate solution to universal testing could be selective testing of certain populations, for example, of individual s in clinical settings in which a high preval ence of HlV is expected. Thus one author found that testin g clients 15 to 54 years of age (12% of the patients admitted) upon admission to hospi- tals where the annual rate of AIDS diagnosis is I for 1000 discharges permitted the identification of two- thirds of infected persons (70). We would therefore expect that a testing program directed toward a clientele selected according to certain criteria would allow HlV-infected patients to be detected more ef- fectively and would cost less than if all patients were tested. One study looked at this question in regard to various prevalence scenarios by estimating the cost per patient tested and the cost per occupational in- fection prevented (71). The authors found that the additional costs related to testing hospitalized pa- tient s in the United State s would amount to USD 4 billion, or USD 23 000 per infected person detected if the seroprevalence rate had been I%. This cost would have been brought down to USD 14000 per patient if the prevalence had been 10%, and it would have increased to USD 217 000 ifit had been 0.05%. Selective measures The additional costs for every HIV occupational in- fection prevented would be USD 751 000 (range USD 186 000 to USD 133 trillion). The author con- cluded that identifying infected patients with the goal of preventing occupational HlV infecti ons would not be justified. In the same way, the Centers for Dis- ease Control and Prevention do not recommend sub- stituting the use of universal precautions with a testing program for patien ts (72). Otherwise the literature shows that universal pre- cautions are not very concrete, as regards innumer- able work situations within health care settings. This situation has brought on major differences in their interpretation and appli cation, as shown in a study in which, although 66.4 % of Canadian hospitals stat- ed having implemented universal precautions, only 5.6% had applied all the minimal measures required by the Centers for Disease Control and Prevention (78). Moreover, the effectiveness of universal pre- cautions in preventing bloodborne infections has not been demonstrated. Certain methodological weak- nesses of evaluation studies, like the absen ce of a control group and poor external validity, have pre- vented such a demonstration. Many health care workers do not comply with uni- versal precauti ons in that they interfere with their work, are expensive, and are not very effective (29, 79, 80). On one hand , some fail to comply system- atically because they feel that they are at low risk and prefer to apply recommended measures only when they suspect a patient of being infected. On the other hand, some do not trust universal precautions and consider that testing is a more effective way to prevent HlV transmission. In fact, testing can be somewhat reassuring since it allows the hazard to be identified, necessary and perhaps extraordinary mea- sures to be taken, and immediate knowledge of what to do in case of exposure. Testing also fits in with the tradition of infection prevention based on diag- nosis. Finally, an HIV antibody testing program for pa- tients would be difficult to justify without inclu ding one for hepatitis Band C (73- 74). Selective measures Morbidity asso- ciated with hepatitis B, where I% of adult cases de- velop a fulminant form and 10% a chronic form, with a risk of developing cirrhosis or liver cancer, would ju stify testing for hepatiti s B as much as for HlV an- tibodies, at least in the absence of vaccination for all health care professionals exposed to body fluids. Fur- therm ore, a significant chronic long-term morbidity associ ated with hepatitis C virus infection has been shown (75). However, as shown in the literature, HIV antibody testing of patients is not a more cost-effective solu- tion than universal precautions, and it surely does not make sense without concurrent testing for other bloodborne infections. Moreover it is impossible to test every patient each time they consult, and the magnitude of occupational risk does not justify such expenses. while the epidemic is spreading and serv- ices to infected people are insufficient. Furthermore, such a program would considerably reduce available resources for protec tive equipment, and therefore render obsol ete the efforts allocated to testin g. Discussion HIV transmission in health care settings is still a matter of concern for health care workers. For some of them , the risk of infecti on is relatively high, no- tably in areas where the prevalence of HIV infection is elevated. For others, although the actual risk is low, it is perceived as being high, which can some- times induce undesirable behavior, such as denying access to care for infected patients (76, 77). This worry, whether justified or not, has divided health care workers between advocates of universal and se- lective preventive approaches. Consequently, seve n Clearly, universality of preventive measures should be maintained. It is however imperative to improve the effectiveness of these measures, par- ticularly in the prevention of parenteral exposures, by developing safer devices and by makin g preven- tion measures "user friendly." It is also imperative to improve the cost-effectiveness of the prevention program by targeting health care settings where risk of infection is higher. 397 Scand J Work Environ Health 1994, vol20,no6 10. Ippolito G, Puro V, De Carli G, the Italian Study Group on Occupational risk of HIV infection. Rates of HIV seroconversions by type of exposure: an up- date of the Italian multicentric study. In: Institute for Clinical and Experimental Virology of the Free Uni- versity of Berlin. IXth international conference on AIDS; 1993 June 6-11. Berlin: Institute for Clinical and Experimental Virology of the Free University of Berlin, 1993;2:730. The prerequisites for risk assessment are the knowledge of local epidemiology of the bloodbome infections included in the prevention program and knowledge of the extent of exposure to body fluids and its associated factors. As for the first prerequi- site, information on infection rates in the general population is usually available through surveillance systems or prevalence studies monitoring the epidem- ic. As for the second, however, the epidemiology of blood and body fluid exposures is not well known mainly because of the wide variability of the infor- mation gathered by hospitals and the small size of the individual data bases not allowing in-depth anal- yses. Therefore there is a need to develop a stand- ardized system of surveillance of exposures on a wide enough scale to allow the factors associated with these exposures to be identified. This step would permit more effective strategies and devices for pre- venting parenteral exposures to be developed, and, consequently, it should improve compliance. II. References I. Anonymous. Needlestick transmission of HTLV-III from a patient infected in Africa. Lancet 1984;2: 1376-7. 17. Mayo-Smith MF. Type non-A, non-B and type B hep- atitis transmitted by a single needlestick. Am J Infect Control 1987;15:266-7. 2. Neisson-Vemant C, Arfi S, Mathez D, Leibowitch J, Monplaisir N. Needlestick HIV seroconversion in a nurse [letter]. Lancet 1986;2:814. ; 18. Seeff LB. Hepatitis C from a needlestick injury. Ann Intern Med 1991;115:411. 19. Ahtone J, Francis D, Bradley D, Maynard J. Non-A, non-B hepatitis in a nurse after percutaneous needle exposure [letter]. Lancet 1980;1:1142. 3. Gioannini P, Sinicco A, Cariti G, Lucchini A, Paggi G, Giachino O. HIV infection acquired by a nurse. Eur J Epidemiol 1988;4:119-26. p 20. Lim WL, Yeoh EK. Occupationally acquired hepati- tis C virus infection. Lancet 1992;339:304-5. p 4. Ponce de Leon RS, Sanchez-Mejorada G, Zaidi-Jack- obson M. AIDS in a blood bank technician in Mexi- co City. Infect Control Hosp Epidemiol 1988;9:101- 2. 21. Mitsui T, Iwano K, Masuko K, Yamazaki C, Okamo- to H, Tsuda F, et al. Hepatitis C virus infection in med- ical personnel after needlestick accident. Hepatology 1992;16:1109-14. 5. Looke DFM, Grove DI. Failed prophylactic zidovu- dine after needlestick injury [letter]. Lancet 1990;338: 884. 22. Howard RJ. Human immunodeficiency virus testing and the risk to the surgeon of acquiring HIV. Gyne- col Obstet 1990;171:22-6. 6. Health and Welfare Canada. A case of HIV infection possibly transmitted in an occupational setting - On- tario. Can Commun Dis Rep 1992;18:102-3. 23. Gerberding JL. Does knowledge of human immuno- deficiency virus infection decrease the frequency of occupational exposure to blood? Am J Med 1991;91 suppl 3B:308S-1 IS. 7. Tait DR, Pudifin DJ, Gathiram V, Windson 1M. HIV seroconversions in health care workers, Nathal, South Africa. Presented at the VIII International Conference on AIDS, Amsterdam, July 1992. pp 24. Centers for Disease Control. Recommendations for prevention of HIV transmission in health-care settings. MMWR 1987;36 suppI2S;IS-18S. , , y 8. Dunn DT, Newell ML, Ades AE, Peckham CS. Risk of human immunodeficiency virus type I transmission through breastfeeding. Lancet 1992;340:585-8. pp 25. Centers for Disease Control. Guidelines for prevention of transmission of human immunodeficiency virus and hepatitis B virus to health-care and public-safety work- ers. MMWR 1989;38. g g 9. Ciesielski C, Metler R, Hammett T, Ward J. Occupa- tionally acquired HIV infection - United States. Discussion Lot F, Abiteboul D, Bouvet E, Laporte A. Surveillance of occupationally acquired HIV infections in France. In: Institute for Clinical and Experimental Virology of the Free University of Berlin. IXth international con- ference on AIDS; 1993 June 6-11. Berlin: Institute for Clinical and Experimental Virology of the Free University of Berlin, 1993;2:723. 12. Perez L, Fitch K, de Andres R, Najera R. HIV sero- conversions following occupational exposure in Eu- ropean healthcare workers. In: Institute for Clinical and Experimental Virology of the Free University of Ber- lin, IXth international conference on AIDS; 1993 June 6-11. Berlin: Institute for Clinical and Experimen- tal Virology of the Free University of Berlin, 1993;2: 724. 13. Henderson DK, Fahey BJ, Willy M, Schimitt JM, Carey K, Koziol DE, et al. Risk for occupational trans- mission of human immunodeficiency virus type I (HIV-I) associated with clinical exposures: a prospec- tive evaluation. Ann Intern Med 1990;113:740-6. Such a prevention program, based on risk assess- ment, is deemed necessary in most countries in the context of the financial restrictions imposed upon health care systems worldwide. Other elements, like local legislation and the already implemented preven- tive measures, must be accounted for in the planning process, but the magnitude of the risk of bloodborne infections for health care workers seems to us to be the main driving force in the implementation of pre- ventive measures. 14. Ippolito G, Puro V. De Carli G, Italian Study Group on Occupational Risk of HIV Infection. The risk of occupational human immunodeficiency virus infection in health care workers: Italian multicentric study. Arch Intern Med 1993;153:1451-8. 15. Kiyosawa K, Sodeyama T, Tanaka E, Nakano Y, Fu- rata S, Nishioka K, et al. Hepatitis C in hospital em- ployees with needlestick injuries. Ann Intern Med 1991;115:367-9. 16. Schlipkoter U. Transmission of hepatitis C virus (HCV) from a haemodialysis patient to a medical staff member. Scand J Infect Dis 1990;22:757-8. References Poor compliance with uni- versal precautions: a universal phenomenon? Pediatr Emerg Care 1990;6:183-5. g 33. Baraff LJ, Talan DA. Compliance with universal pre- cautions in a university hospital emergency depart- ment. Ann Emerg Med 1989;18:654-7. p 51. Doebbeling BN, Wenzel RP. The direct costs of uni- versal precautions in a teaching hospital. JAMA 1990; 264:2083-7. g ; 34. Hellgren K. Use of gloves among dentists in Sweden . Swed DentJ 1990;14:137-42. 52. Stock SR, Gafni A, Bloch RF. Universal precautions to prevent HIV transmission to health care workers: an economic analysis. Can Med Assoc J 1990;142: 937-6. 35. Smyser MS, Bryce J, Joseph JG. AIDS-related knowl- edge , attitudes and precautionary behaviors among emergency medical professionals. Public Health Rep 1990;I05:496-503. 53. Garner JS. Universal precautions and isolat ion sys- tems. In: Bennett JV, Brachman PS, editors. Hospital infections. Boston, MA: Little, Brown, 1992:241-2. ; 36. Kaczmarek RG, Moore RM, McCrohan J, Arrow- smith-Lowe JT, Caquelin C, Reynolds C, et al. Glove use by health care workers : results of a tristate inves- tigation. Am J Infect Control 1991;19:228-32. 54. Lynch P, Jackson MM, Cummings MJ, Stamm WE. Rethinking the role of isolation practices in the pre- vention of nosocomial infections. Ann Intern Med 1987;107 :243-6. g 37. Hammond JS, Eckes JM, Gomez GA, Cunningham DN. HIV, trauma, and infection control : universal pre- cautions are universally ignored. J Trauma 1990;30: 555-61 . ; 55. Jackson MM, Lynch P, McPherson DC, Cummings MJ, Greenawalt NC. Why not treat all body substances as infectious. Am J Nurs 1987;87:1137-9. 38. Becker MH, Janz NK, Band J, Bartley J, Snyder MB, Gaynes RP. Noncompliance with universal precautions policy: why do physici ans and nurses recap needles ? Am J Infect Control 1990;18:232-9. 56. Gerberding JL. Reducing occupational risk of HIV. Hosp Pract 199I;26: 103(10):115-8. p 57. Lynch P, Cummings MJ, Roberts PL, Herriot MJ, Yat- es B, Stamm WE. Implementing and evaluating a sys- tem of generic infection precautions: body substance isolation. Am J Infect Control 1990;18: 1-12. 39. Fahey BJ, Koziol DE, Banks SM, Henderson DK. Fre- quency of non parenteral occupational exposures to blood and body fluids before and after universal pre- cautions training. Am J Med 1990;90:145-153. 58. Troya SH, Jackson MM, Lovrich-Kerr M, McPherson DC. A survey of nurses' knowledge, opinions, and reported uses of the body substance isolation system. Am J Infect Control 1991;19:268-76. g 40. References In: Institute for Clinical and Experimental Virology of the Free University of Berlin. IXth international confer- ence on AIDS; 1993 June 6-11. Berlin: Institute for Clinical and Experimental Virology of the Free Uni- versity of Berlin, 1993;I:93. ; 26. Dajczman E, Dascal A, Orenstein P, Frank H. Survey of infection control precautions: a comparison to rec- ommended guidelines. Can J Infect Control 1992;7: 7-12. 27. Naccache H, Fortin C, Croteau A, Godin G. Univer- 398 Scand J Work Environ Health 1994, vol 20, no 6 sal precautions in a teaching hospital: compliance and perception. AIDS Pat Care 1993;7:134-7. national forum on AIDS, hepatitis, and other blood- borne diseases, 1992 Mar29-Aprl. Atlanta, GA: Symedco , 1992:46. p p 28. Brattebo G, Wisborg T, Sjursen H. Health workers and the human immunodeficiency virus: knowledge, igno- rance and behavior. Public Health 1990;I04:123-30. 47. y , 47. Telford G, Quebbeman EJ. In-use evaluation of sur- gical gowns: a prospective trial. In: Centers for Dis- ease Control. 5th nation al forum on AIDS, hepatitis, and other blood-borne diseases, 1992 Mar29-Aprl. Atlanta, GA: Symedco, 1992:46. 29. Kelen GD, DiGiovanna TA, Celentano DD, Kalainov D, Bisson L, Junkins E, et al. Adherence to universal (barrier) precautions during interventions on critical- ly ill and injured emergency department patients. J Acquired Immune Defic Syndrome 1990;3:987-94. y 48. Lovitt SA, Smith JW, Muzik AC, Pearce PR, Nichols RL. Isolation gown s: a false sense of security? In: Centers for Disease Control. 5th national forum on AIDS, hepatitis, and other blood-borne diseases, 1992 Mar29-Aprl. Atlanta, GA: Symedco, 1992:47. 30. Talan DS, Baraff LJ. Effect of education on the use of universal precautions in a university hospital emer- gency department. Ann Emerg Med 1990;19:1322- 6. p y 49. Jagger J, Pearson RD. Do universal precautions reduce needlestick injuries? JAMA 1991;266:3:359-60. 31. Elford J, Cockcroft A. Compulsory HIV antibody test- ing, universal precautions and the perceived risk of HIV: a survey among medical students and consult- ant staff at a London teaching hospital. AIDS Care 1991;3:151-8. 50. Chiarello LA, Nagin DS, Kowal ski E. Evalu ation of needlestick prevention devices in ten New York state (NYS) hospitals. In: US Department of Health and Human Services, US Department of Labor. A national conference on prevention of device-mediated blood- borne infections, 1992 Aug 17-19. Washington, DC: US Department of Health and Human Servi ces, US Department of Labor, 1992:30. 32. Ros SP, Cabrera-Ros BL. 65. Bayer R. Public health policy and the AIDS epidem- ic, an end to HlY exceptionalism? N Engl J Med 1991; References N Engl J Med 1991; 399 Scand J Work Environ Health 1994, vol 20, no 6 324:1500-2. 74. Kelen GO, Green GB, Purcell RH, Chan OW, Qaqish BF, Sivertson KT, et al. Hepatitis B and hepatitis C in emergency department patients. N Engl J Med 1992; 326: 1399--404. 66. Gerberding JL, Littel C, Tarkington A, Brown A, Schecter WP. Risk of expos ure of surgical personnel to patients' blood during surgery at San Francisco Gen- eral HospitaL N Engl J Med 1990;322:1788- 93. 75. Kaklamani E, Trichopoulos D, Tzonou A, Zavitsanos X, Koumantaki Y, Hatzakis A, et al. Hepatitis B and C viruses and their interaction in the origin of hepa- tocellular carcinoma. JAMA 199 1;265: 1974---6. p g 67. Tokars JI, Bell DM, Culver DH, Marcus R, Mendel- son MH, Sloan EP. Percutaneous injuries during sur- gical procedures. JAMA 1992:267:2899-904. g p 68. La Croix S, Russo G. Costs and benefits of mandato- ry HIV testing for healthcare workers and hospital pa- tients. In: Harvard University, Dutch Foundation AIDS Conference, editors. VIII international conference on AIDSIIII STD world congress; 1992 Jul 19-24. Am- sterdam: CONGREX Holland BV, 1992:D463. E 76. Yagi S, Yagi A, Takahashi T, Kikushi S. How AIDS is perceived by nurses and health care workers in Daini Hospital, Nippon Medical School. In: Institute for Clinical and Experimental Virology of the Free Uni- versity of Berlin. IXth International Conference on AIDS ; 1993 June 6-1 1. Berlin: Institute for Clinical and Experimental Virology of the Free University of Berlin, 1993;2:1891. 69. Roy E, Desjardins J, Lauzon D. Virus de l'immuno- deficience humaine et retrait preventif de la travail- leuse enceinte ou qui allaite. Quebec: Comite provin- cial en sante au travail du Quebec, 1989. 77. Bresolin L, Rinaldi R, Hemmin g J, Gans J. A nation- al survey of 4000 physicians. In: Intern ationa l Insti- tute for Clinical and Experimental Virology of the Free University of Berlin . IXth international conference on AIDS; 1993 June 6-11 . Berlin: Institute for Clinical and Experimental Virolo gy of the Free University of Berlin , 1993;2:894. 70. Janssen RS, St-Louis ME, Satten GA, Critchley SE, Petersen LR, Stafford RS, et al. HIV infection among patients in US acute care hospitals, strategies for coun- sellin g and testing of hospital patients. N Engl J Med 1992:327;445--52. 71. References Wong ES, Stotka JL, Chinchili VM, Williams DS, Stu- art CG, Markowitz SM. Are universal precautions ef- fective in reducing the number of occupational expo- sures among health care workers? JAMA [991 ;265: 1123-1128. p y Am J Infect Control 1991;19:268-76. 59. Board of Trustees, American Medical Association . Prevention and control of acquired immunodeficien- cy syndrome. JAMA 1987;258:2097-103. 41. Ribner BS, Landry MN, Gholson GL, Linden LA. Im- pact of a rigid, puncture resistant container system upon needlestick injuries. Infect Control 1987;8:63- 66. y y ; 60. Rhame FS, Maki DG. The case for wider use of test- ing for lIlY infection . N Engl J Med 1989;320:1248- 54. 42. Krasinsky K. LaCouture R, Holzman RS. Effect of changing needle dispo sal systems on needle puncture injuries. Infect Control 1987;8:59-62. 6 1. Angell M. A dual approach to the AIDS epidemic. N Engl J Med 1991;324:1498-500. g ; 62. Canadian Task Forc e on the Periodi c Health Exami- nation . Periodic health examination, 1992 update : 3. HIY antibody screening. Can Med Assoc J 1992;147: 867-76. j 43. Edmond M, Khakoo R, McTaggart Bonny, Solomon R. Effect of bedside needle dispo sal units on needle recapping frequency and needlestick injury . Infect Control Hosp Epidemiol 1988;9:114-6. 63. Urassa WK, Bredberg-Raden U, Mbena E, Palsson K, Minja E, Lema RA, et al. Altern ative confirmatory strategies in HIY-I antibody testing. J Acquired Im- mune Dcfic Syndrome 1992;5:170-6. p p ; 44. Smith DA, Eisenstein HC, Esrig C, Godbold J. Con- stant incidence rates of needle-stick injury paradoxi- cally suggest modest preventi ve effect of sharps dis- posal system . J Occup Med 1992;34:546-51. y 64. Behets F, Bishagara K, Disasi A, Likin S, Ryder RW, Brown C, et al. Diagnosis of HIY infect ion with in- strument-free assays as an alternative to the ELISA and Western Blot testing strategy: an evaluation in Cen- tral Africa. J Acquired Immune Defic Syndrome 1992: 5:878-82. p y p ; 45. Linnemann CC, Cannon C, DeRonde M, Lanphe ar B. Effect of educational programs, rigid sharps contain- ers, and universal precautions on reported needlestick injuries in healthcare workers. Infect Control Hosp EpidemioI1991;12:214-9. p ; 46. Nichols RL. Barrier efficiency of surgical drapes and isolation gowns. In: Centers for Disease Control. 5th 65. Bayer R. Public health policy and the AIDS epidem- ic, an end to HlY exceptionalism? Received for publication: 2 Febru ary 1994 References Lurie P, Avins AL, Phillips KA, Kahn JG, Lowe RA, Franks P, et aL Routine HIV testing of inpatients: costs, benefits, and problem s. In: Institute for Clini- cal and Experimental Virology of the Free Universi ty of Berlin. IXth international conference on AIDS; 1993 June 6-11. Berlin : Institute for Clinical and Experi- mental Virology of the Free University of Berlin, 1993;1:97. 78. Birnbaum 0 , Schulzer M, Mathias RG, Kelly M, Chow AW. Adoption of guidelines for universal precautions and body substance isolation in canadian acute-care hospitals. Infect Control Hosp Epidemiol 1990;I I: 465-72. 79. Courington KR, Patterson SL, Howard RJ. Universal precautions are not universally followed. Arch Surg 1991;126:93- 6. 80. Gruber M, Beavers FE, Johnson B, Brackett M, Lopez T, Feldman MJ, Ventura M. The relationship between knowledge about acquired immunodeficiency syn- drome and the implementation of universal precautions by registered nurses. Clin Nurse Spec 1989;3:182- 5. 72. Centers for Disease Control and Prevention. Recom- mendations for HIV testing services for inpatients and outpatients in acute-care hospital settings. MMWR 1993;42:1---6. 73. Gordin FM, Gibert C, Hawley HP, Willoughby A. Prevalence of human immunodeficiency virus and hep- atitis B virus in unselected hospital admissions: im- plications for mandatory testing and universal precau- tions. J Infect Dis 1990:161;14-7. Received for publication: 2 Febru ary 1994 400
https://openalex.org/W2725401397	https://europepmc.org/articles/pmc5502857?pdf=render	English	null	Draft Genome Sequence of Enterococcus faecalis ATCC BAA-2128	Genome announcements	2,017	cc-by	1,368	Draft Genome Sequence of Enterococcus faecalis ATCC BAA-2128 Catherine Putonti,a,b,c,d Laurynas Kalesinskas,a,b Evan Cudone,a Kathleen C. Engelbrecht,e David W. Koenig,e Alan J. Wolfed Catherine Putonti,a,b,c,d Laurynas Kalesinskas,a,b Evan Cudone,a Kathleen C. Engelbrecht,e David W. Koenig,e Alan J. Wolfed Catherine Putonti,a,b,c,d Laurynas Kalesinskas,a,b Evan Cudone,a Kathleen C. Engelbrecht,e David W. Koenig,e Alan J. Wolfed Bioinformatics Program, Loyola University Chicago, Chicago, Illinois, USAa; Department of Biology, Loyola University Chicago, Chicago, Illinois, USAb; Department of Computer Science, Loyola University Chicago, Chicago, Illinois, USAc; Department of Microbiology and Immunology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, Maywood, Illinois, USAd; Corporate Research and Engineering, Kimberly-Clark Corporation, Neenah, Wisconsin, USAe Bioinformatics Program, Loyola University Chicago, Chicago, Illinois, USAa; Department of Biology, Loyola University Chicago, Chicago, Illinois, USAb; Department of Computer Science, Loyola University Chicago, Chicago, Illinois, USAc; Department of Microbiology and Immunology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, Maywood, Illinois, USAd; Corporate Research and Engineering, Kimberly-Clark Corporation, Neenah, Wisconsin, USAe ABSTRACT While a part of the native gut microﬂora, the Gram-positive bacterium Enterococcus faecalis can lead to serious infections elsewhere in the body. The draft genome of E. faecalis strain ATCC BAA-2128, isolated from piglet feces, was exam- ined. This draft genome consists of 42 contigs, 12 of which exhibit homology to an- notated plasmids. Received 14 May 2017 Accepted 16 May 2017 Published 6 July 2017 Citation Putonti C, Kalesinskas L, Cudone E, Engelbrecht KC, Koenig DW, Wolfe AJ. 2017. Draft genome sequence of Enterococcus faecalis ATCC BAA-2128. Genome Announc 5:e00575-17. https://doi.org/10.1128/genomeA .00575-17. Copyright © 2017 Putonti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Catherine Putonti, cputonti@luc.edu. A s part of an attempt to generate complete genomes for strains in the ATCC collection, we report the genome sequence and annotation of Enterococcus faecalis ATCC BAA-2128, isolated from piglet feces. Isolated as part of a study assessing the performance beneﬁts of diets supplemented with copper, zinc, or antibiotics, the E. faecalis ATCC BAA-2128 strain is resistant to copper, erythromycin, and tetracycline (1). Copyright © 2017 Putonti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Catherine Putonti, cputonti@luc.edu. The strain was purchased from ATCC and grown on 5% sheep blood agar (BD BBL prepared plated media) under 5% CO2 at 35°C for 48 h. PROKARYOTES crossm PROKARYOTES crossm genomea.asm.org 1 Volume 5 Issue 27 e00575-17 Received 14 May 2017 Accepted 16 May 2017 Published 6 July 2017 Citation Putonti C, Kalesinskas L, Cudone E, Engelbrecht KC, Koenig DW, Wolfe AJ. 2017. Draft genome sequence of Enterococcus faecalis ATCC BAA-2128. Genome Announc 5:e00575-17. https://doi.org/10.1128/genomeA .00575-17. Copyright © 2017 Putonti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Catherine Putonti, cputonti@luc.edu. genomea.asm.org 1 Draft Genome Sequence of Enterococcus faecalis ATCC BAA-2128 To extract genomic DNA, the cells were resuspended in 0.5 mL of DNA extraction buffer (20 mM Tris-Cl, 2 mM EDTA, 1.2% Triton X-100, pH 8), followed by the addition of 50 L of lysozyme (20 mg/mL), 30 L of mutanolysin, and 5 L of RNase (10 mg/mL). After incubation at 37°C for 1 h, 80 L of 10% SDS and 20 L of proteinase K were added and incubated for 2 h at 55°C. Then, 210 L of 6M NaCl and 700 L of phenol-chloroform were added. After a 30-min incubation with rotation, the solutions were centrifuged at 13,500 rpm for 10 min, and the aqueous phase was extracted. An equivalent volume of isopropanol was added, and the solution was centrifuged at 13,500 rpm for 10 min after a 10-min incubation. The supernatant was decanted, and the DNA pellet was precipitated using 600 L of 70% ethanol. Following ethanol evaporation, the DNA pellet was resuspended in Tris-EDTA (TE) and stored at 20°C. Genomic DNA was diluted in water to a concentration of 0.2 ng/L, as measured by a ﬂuorometric-based method (Life Technologies, Inc.), and 5 L were used to obtain a total of 1 ng of input DNA for library preparation (Nextera XT DNA Library preparation kit). The library was sequenced on the MiSeq sequencer (Illumina) using the MiSeq version 2 reagent kit (500 cycles), producing 1,751,626 paired-end reads. Reads were trimmed, removing adapter sequences and phiX contaminants, using BBDuk from the BBMap package (http://sourceforge.net/projects/bbmap). Trimmed reads were assem- bled using SPAdes version 3.5 (2), followed by scaffolding with SSPACE (3). The draft genome consists of 42 contigs (N50 232,593 bp) with an average coverage of 265.7. The genome size was 2,964,736 bp with an observed GC content of 37.41%. BLASTn queries to the NR/NT database revealed that 12 of these contigs show greatest homology to plasmid sequences of other E. faecalis strains and enterococci, including three putative complete plasmids that are 21, 31, and 35 kbp in length. Annotations were produced using the software tool Peasant (4). Six rRNAs, 52 tRNAs, and 2,852 Volume 5 Issue 27 e00575-17 genomea.asm.org 1 Putonti et al. protein-coding sequences were detected. Two conﬁrmed and two putative clustered regularly interspaced short palindromic repeat sequences (CRISPRs) were found (5). CRISPR spacer sequences were then queried via BLASTn to viral sequences within the NR/NR database. 3. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. https:// doi.org/10.1093/bioinformatics/btq683. g q 4. Brenner J, Kalesinskas L, Putonti C. 2017. Exploring the diversity of Bacillus whole genome sequencing projects using Peasant, the prokaryotic as- sembly and annotation tool. bioRxiv 132084. https://doi.org/10.1101/ 132084. Draft Genome Sequence of Enterococcus faecalis ATCC BAA-2128 While not all spacer sequences produced hits, BLASTn did identify spacer sequence homology to the genomes of four phages: VPE25, vB_EfaS_IME196, SAP5, and IME-EF4. Accession number(s). The draft whole-genome project for E. faecalis ATCC BAA-2128 has been deposited at DDBJ/EMBL/GenBank under accession number NAQY00000000. Raw sequence reads were deposited at DDBJ/EMBL/GenBank under accession number SRR5363781. 5. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–W57. https://doi.org/10.1093/nar/gkm360. ACKNOWLEDGMENTS We acknowledge Gina Kuffel and Michael Zilliox for sequencing this genome and David Paulucki’s assistance in culturing the strain. This work was supported by Kimberly-Clark Worldwide, Inc. 2. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome as- sembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021. Volume 5 Issue 27 e00575-17 1. Amachawadi RG, Shelton NW, Jacob ME, Shi X, Narayanan SK, Zurek L, Dritz SS, Nelssen JL, Tokach MD, Nagaraja TG. 2010. Occurrence of tcrB, a transferable copper resistance gene, in fecal enterococci of swine. Foodborne Pathog Dis 7:1089–1097. https://doi.org/10.1089/fpd.2010 .0540. 3. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. https:// doi.org/10.1093/bioinformatics/btq683. 4. Brenner J, Kalesinskas L, Putonti C. 2017. Exploring the diversity of Bacillus whole genome sequencing projects using Peasant, the prokaryotic as- sembly and annotation tool. bioRxiv 132084. https://doi.org/10.1101/ 132084. 5. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–W57. https://doi.org/10.1093/nar/gkm360. REFERENCES 1. Amachawadi RG, Shelton NW, Jacob ME, Shi X, Narayanan SK, Zurek L, Dritz SS, Nelssen JL, Tokach MD, Nagaraja TG. 2010. Occurrence of tcrB, a transferable copper resistance gene, in fecal enterococci of swine. Foodborne Pathog Dis 7:1089–1097. https://doi.org/10.1089/fpd.2010 .0540. 1. Amachawadi RG, Shelton NW, Jacob ME, Shi X, Narayanan SK, Zurek L, Dritz SS, Nelssen JL, Tokach MD, Nagaraja TG. 2010. Occurrence of tcrB, a transferable copper resistance gene, in fecal enterococci of swine. Foodborne Pathog Dis 7:1089–1097. https://doi.org/10.1089/fpd.2010 .0540. 2. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome as- sembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021. Volume 5 Issue 27 e00575-17 genomea.asm.org 2
https://openalex.org/W2910263849	http://revistatdh.org/index.php/Revista-TDH/article/download/6/4	Portuguese	null	O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana	Revista Jurídica Trabalho e Desenvolvimento Humano	2,018	cc-by	8,037	RESUMO Este ensaio objetiva refletir sobre as doenças psicossomáticas no ambiente de trabalho. As doenças psicossomáticas, dentre elas a depressão, são os grandes males que acometem a nossa sociedade atualmente. A depressão se caracteriza pela perda ou diminuição de interesse e prazer pela vida, gerando angústia e prostração, sintoma da melancolia permanente. Essa doença pode ser desenvolvida por diversas razões, mas na sociedade atual ela vem sendo percebida e notada com maior frequência e não raro ligadas ao trabalho. As doenças psiquiátricas são ignoradas por muitas empresas, que não reconhecem a gravidade e as enxergam apenas como uma situação emocional passageira ou como falta de disposição pessoal para o trabalho, utilizando-se da conhecida prática de atribuir culpa à própria vitima. O presente ensaio objetiva a análise da possibilidade de se caracterizar a depressão como doença do trabalho, procurando delimitar até que ponto a enfermidade e o trabalho repercutem um sobre o outro, numa relação de causa e efeito, ressaltando os principais aspectos referentes aos conhecimentos relativos à depressão, em especial no ambiente de trabalho. Assim, o método empregado é o dialético dedutivo, a partir da análise documental de dados que informam a gravidade do problema, o seu tratamento no âmbito legislativo, doutrinário e jurisprudencial, diante da realidade social instalada pela nova geração conhecida como tecnológica e informacional, na qual valores humanos pretéritos passam a ter menor relevo, em especial frente à realidade do capital. Essa liquidez de entendimento social, em especial pelo empregador, lança grande desafio para a proteção do adoecido e a efetivação das suas garantias fundamentais no meio ambiente de trabalho. PALAVRAS-CHAVE: Depressão. Meio ambiente de trabalho. Transtorno psicológico. Proteção legal ao trabalhador. Doença do Trabalho. KEYWORDS: Depression. Work environment. Psychological disorder. Legal protection to the worker. Work Sickness. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana The environment of work and health of the worker: challenges to the realization of human dignity El medio ambiente del trabajo y la salud del trabajador: desafios a la efectividad de la dignidad humana Leonardo Tavares Lima¹ Vitória Mattos Gonçalves² Jair Aparecido Cardoso³ ABSTRACT This essay aims to reflect on psychosomatic diseases in the work environment. Psychosomatic illnesses, among them depression, are the great evils that affect our society today. Depression is characterized by the loss or diminution of interest and pleasure in life, generating anguish and prostration, a symptom of permanent melancholy. This disease can be developed for a number of reasons, but in today's society it has been perceived and noticed more frequently and often associated with work. Psychiatric illnesses are ignored by many companies, who do not recognize their gravity and see them only as a transient emotional situation or as a lack of personal disposition for work, using the well-known practice of blaming the victim his or herself. This essay aims at analyzing the possibility of characterizing depression as a work disease, trying to delimit the extent to which illness and work affect one another, in a relation of cause and effect, highlighting the main aspects related to the existing knowledge on depression, especially in the work environment. Thus, the method employed is the deductive dialectic, based on the analysis of data that inform the seriousness of the problem, its treatment in the legal, doctrinal and jurisprudential contexts, in light of the social reality brought forth by the new generation known as technological and informational, in which human values from the past have become less important, especially in relation to the reality of capital. This liquidity of social understanding, especially by the employer, launches a great challenge for the protection of the sick person and for the realization of their fundamental guarantees in the working environment. KEYWORDS: Depression. Work environment. Psychological disorder. Legal protection to the worker. Work Sickness. 58 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região RESUMEN Este ensayo tiene como objetivo reflexionar sobre las enfermedades psicosomáticas en el ambiente de trabajo. Las enfermedades psicosomáticas, entre ellas la depresión, son los grandes males que acometen nuestra sociedad actualmente. La depresión se caracteriza por la pérdida o disminución de interés y placer por la vida, generando angustia y postración, síntoma de la melancolía permanente. Esta enfermedad puede ser desarrollada por diversas razones, pero en la sociedad actual ella viene siendo percibida y notada con mayor frecuencia y no raras veces vinculadas al trabajo. Las enfermedades psiquiátricas son ignoradas por muchas empresas, que no reconocen la gravedad y las ven sólo como una situación emocional pasajera o como falta de disposición personal para el trabajo, utilizando la conocida práctica de atribuir culpa a la propia víctima. El presente ensayo objetiva el análisis de la posibilidad de caracterizar la depresión como enfermedad del trabajo, buscando delimitar hasta qué punto la enfermedad y el trabajo repercuten uno sobre el otro, en una relación de causa y efecto, resaltando los principales aspectos referentes a los conocimientos relativos a la depresión, especialmente en el ambiente de trabajo. Así, el método empleado es el dialéctico deductivo, a partir del análisis documental de datos que informan la gravedad del problema, su tratamiento en el ámbito legislativo, doctrinal y jurisprudencial, ante la realidad social instalada por la nueva generación conocida como tecnológica e informacional, en la cual los valores humanos pretéritos pasan a tener menor relieve, en especial frente a la realidad del capital. Esta liquidez de entendimiento social, en especial por el empleador, plantea un gran desafío para la protección del enfermo y la efectividad de sus garantías fundamentales en el medio ambiente de trabajo. PALABRAS-CLAVE: Depresión. Medio ambiente de trabajo. Trastorno psicológico. Protección legal al trabajador. Enfermedad del Trabajo. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 INTRODUÇÃO: A EVOLUÇÃO E A DOENÇA SOCIAL A sociedade evolui e com ela as suas complexidades. Vivemos numa época em que as evoluções parecem estar em verdadeiro ritmo olímpico, barreiras culturais são quebradas a cada instante, e a sociedade parece que quer quebrar o seu próprio recorde de celeridade, causando verdadeiro estresse social. Não raro essas tensões sociais requerem atenção, pois trazem reflexos sociais diversos. Assim como o direito criou fases para marcar a sua evolução - direitos de primeira, segunda e terceira geração (ou dimensão) -, assim também a evolução social e econômica seguiu os mesmos passos, causando reflexos na evolução do trabalho. A evolução social com a sua modernidade e pós-modernidade e do trabalho com a suas revoluções industriais - da primeira, da segunda e da terceira revolução. A exemplo do direito, no qual já se fala em direito da quarta geração, para proteção da biodiversidade, na área econômica e social já se fala na quarta revolução industrial, a qual, por meio da revolução tecnológica e informacional, quebra paradigmas com relação a cultura social pretérita, criando novas realidades. A sociedade evolui e com ela as suas complexidades. Vivemos numa época em que as evoluções parecem estar em verdadeiro ritmo olímpico, barreiras culturais são quebradas a cada instante, e a sociedade parece que quer quebrar o seu próprio recorde de celeridade, causando verdadeiro estresse social. Não raro essas tensões sociais requerem atenção, pois trazem reflexos sociais diversos. Assim como o direito criou fases para marcar a sua evolução - direitos de primeira, segunda e terceira geração (ou dimensão) -, assim também a evolução social e econômica seguiu os mesmos passos, causando reflexos na evolução do trabalho. A evolução social com a sua modernidade e pós-modernidade e do trabalho com a suas revoluções industriais - da primeira, da segunda e da terceira revolução. A exemplo do direito, no qual já se fala em direito da quarta geração, para proteção da biodiversidade, na área econômica e social já se fala na quarta revolução industrial, a qual, por meio da revolução tecnológica e informacional, quebra paradigmas com relação a cultura social pretérita, criando novas realidades. 59 59 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região Essa nova situação, impulsionada pelo próprio desenvolvimento econômico e social, parece criar outras realidades em que outras áreas do saber ainda estão assimilando para melhor compreender esses desafios sociais. A área da saúde é um exemplo claro desta situação. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 INTRODUÇÃO: A EVOLUÇÃO E A DOENÇA SOCIAL Na proporção em que avança a medicina, começam a existir também novas doenças, e essas doenças demoram a ser classificadas pelo Código Internacional das Doenças (CID), e após este reconhecimento pela área da saúde, passam por outro momento, um limbo administrativo, no qual a ciência da saúde passa a reconhecer a doença, mas o estado demora para classificá-la na lista das doenças, em especial na lista das doenças ocupacionais, para proteção do trabalhador afetado por ela. Exemplo claro disso é da lesão por esforço repetitivo (LER). Antes de seu reconhecimento, o trabalhador acometido por tal enfermidade era classificado, muitas vezes, por profissional não afeto ao trabalho, de indolente, dentre outros adjetivos negativos que lhes era imputado. Modernamente já se reconhece os efeitos dessa lesão e a gravidade desse fato para a saúde humana, classificando-a em lesão pelo esforço repetitivo decorrentes das mais diversas atividades, tais como do lazer, do esporte e do trabalho. No caso da lesão por esforço repetitivo decorrente do trabalho, passou a ser classificada como Distúrbios Osteomusculares Relacionados ao Trabalho – DORT – ou ler-dort. Segundo a Agência Europeia para a Segurança e a Saúde no Trabalho – FACTS (2000) diversos países (Estados-Membros) desenvolveram, nas décadas de 1960 e 1970 do século XX, políticas públicas para enfrentamento ao problema das Lesões por esforços repetitivos” (Repetitive Strain Injuries - RSI), o que nos convence de que a situação emanou, por parte do poder público, considerável tempo para a maturação e reconhecimento de tal problema em diversos países ligados a União Europeia. Em que pese a gravidade do problema, no Brasil, somente em 1987 esta doença foi reconhecida pela previdência social, embora sua gênese possa estar radicada no inicio da primeira revolução industrial (BRASIL, 2000). A exemplo desta doença, muitas outras poderiam ser citadas, todavia, neste ensaio, dedicaremos a atenção para as doenças emocionais, ou psicossomáticas. Assim como a lesão por esforço repetitivo que recebeu na sua origem diversos adjetivos negativos, os quais normalmente eram atribuídos ao seu detentor, vitimizando duplamente o adoecido, as doenças psicossomáticas parecem estar trilhando o mesmo caminho. 60 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região 60 O mesmo vem ocorrendo com os portadores das doenças psicossomáticas, em suas diversas modalidades, muitas delas relacionadas ao trabalho. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 INTRODUÇÃO: A EVOLUÇÃO E A DOENÇA SOCIAL O caso é tão grave que também vem ganhando notoriedade internacional, sendo conhecida como síndrome de burnout, ou doença decorrente do estresse excessivo e crônico, provocado por sobrecarga ou excesso de trabalho (GEOVANA, s.d.). Como explica a médica Nicole Geovana (s.d.), o nome "burnout" vem do inglês e significa literalmente "queimar até o fim". Trata-se, portanto, de um esgotamento físico e mental decorrente de uma vida profissional desgastante e sobrecarregada. É nesta mesma fonte que colhemos a assertiva de que esta patologia pode evoluir para transtornos mentais ou físicos, ou os dois ao mesmo tempo. A depressão é um exemplo típico deste tipo de enfermidade, e o local de trabalho parece ser terreno fértil e propício para sua criação e propagação. As áreas da saúde classificam o termo “doença” como desordem no funcionamento de algum órgão, da psique ou de todo o conjunto, podendo apresentar sintomas. Ressalta- se, portanto, dentro da classificação, a inclusão da psique, isto é, o que se relaciona às doenças mentais, e não apenas às físicas. A depressão é uma doença que vem se intensificando com o passar dos anos, principalmente em culturas ocidentais. Isso se dá em razão de ela estar em um contexto de valorização excessiva da individualidade, ou seja, o mérito por conquistas é todo do indivíduo, assim como a culpa pelos fracassos, impedindo que possa haver qualquer compartilhamento de sentimentos com outras pessoas ou com a própria família, vez que todos padecem do mesmo mal social. A sensação de isolamento ocasionada pela depressão também é um fator de risco para outros tipos de doenças, ou seja, uma doença mental mostra-se capaz de afetar a saúde física do indivíduo. Assim como o trabalho é uma ação individual, mas de reflexo coletivo, a doença emocional, embora na maioria das vezes silenciosa, acomete também o individuo trabalhador, mas o seu reflexo é suportado por toda a sociedade, que arcará, além do reflexo coletivo pelo adoecimento social – psicopatia coletiva -, com os custos por ela ocasionado e demais repercussões. 61 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região 61 61 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 1 O EQUILÍBRIO DO MEIO AMBIENTE DO TRABALHO O meio ambiente de trabalho é o local no qual o trabalhador passa a maior parte de sua vida e constrói suas relações e experiências pessoais e interpessoais. Assim, o desequilíbrio desse local afetará diretamente a qualidade de vida do trabalhador nele inserido. O modo de produção capitalista promove o mau uso da força de trabalho, porque valoriza o trabalhador somente pelas extensões de seus braços, somente pelas suas forças físicas, e não como individuo que tem seus direitos fundamentais consagrados pela constituição, como ser humano que é. O homem é o centro das relações sociais, e por esta razão o trabalho tem que ser para o homem e não o homem para o trabalho, e esta assertiva não decorre de um apelo semântico e não pode ser tida como uma frase de efeito. Relegar este fato é o primeiro passo às demais inversões sociais de valores. E, quando valores sociais se invertem, notadamente, os motivados pelo capital, o homem animaliza-se em ambos os sentidos, na condição de explorado e na condição de explorador. Não são raras as ocasiões em que o empregado passa por tratamentos que ferem sua dignidade. Essas situações podem acarretar prejuízos graves, muitos deles definitivos, à sua saúde física e psíquica, além de afetar, consequentemente seu convívio familiar e social, sua autoestima pessoal e profissional, com reflexo direto no seu direito existencial. Se a luta é por um trabalho digno, neste contexto deve estar inserido também um descanso digno e um lazer restaurador. É um direito humano também fundamental. O trabalho digno deve envolver um descanso digno, um tratamento digno, um respeito ao trabalhador enquanto pessoa humana, enfim, ele não pode ser apenas um ser vivente em relação com os demais seres viventes em um mesmo local, um confinamento, aprisionado pelo capital, como se fosse uma sentença irrecorrível pelo simples delito da necessidade do trabalho. Não é outro o entendimento de Ercílio Denny, ao gizar: Para ser humano no sentido completo ou autêntico da palavra, não basta existir de fato. É preciso possuir um conjunto unitário de convicções e de valores que definam a existência humana em linha de princípio: o seu escopo e o seu significado. Além disso, tais princípios devem ser LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1 O EQUILÍBRIO DO MEIO AMBIENTE DO TRABALHO 1, n. 1, p. 57-73, 2018 62 62 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região evidentes. Estes não só não devem estar expostos a contínuas discussões, mas devem também indicar ao homem a sua posição no mundo observável. Porque o homem pensa a si mesmo por meio da mediação com o mundo observável (DENNY, 2003, p. 320). Nesse sentido, Amauri Mascaro Nascimento (2017, p. 491) propõe uma noção mais ampla de meio ambiente do trabalho, que abarque os aspectos organizacionais do meio ambiente, a fim de analisar todos os fatores que podem afetar a saúde mental do trabalhador, dentre eles as jornadas de trabalho, horas extras, intervalos, dentre outros elementos que podem impactar no equilíbrio labor-ambiental. Portanto, o objetivo de definir o meio ambiente do trabalho como equilibrado ultrapassa a percepção de um ambiente físico adequado dentro de normas de higiene e segurança do trabalhador, há de se relevar também a preocupação com a sua saúde física e mental. Por esta razão a salubridade do ambiente deve considerar o aspecto físico, mas também o aspecto não físico, que aqui designamos de emocional, para valorizar o trabalhador no ambiente em que vive a maior parte da sua vida. Por esta razão, não basta fornecer o equipamento de proteção individual (EPI), é necessário que se construa e forneça um equipamento de proteção emocional (EPE), que ora defendemos, e que será visto alhures. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA Leonardo T ; GONÇALVES Vitória M ; CARDOSO Jair A O meio ambiente do trabalho e a saúde do trabalhador: desafios à 2 DEPRESSÃO COMO DOENÇA DO TRABALHO O individualismo excessivo em sociedades ocidentais toma protagonismo no ambiente de trabalho, local repleto de competitividade. Quando deprimidas, as pessoas encaram-se a partir da visão negativa de tudo a seu redor, como incapazes de venderem o seu trabalho ou de, com ele, produzirem insuficientemente. O presente ensaio se propõe a analisar o andamento processual e normativo dos órgãos regulamentadores sobre a caracterização da depressão como doença do trabalho para que se possa efetivar, cada vez mais, uma resposta adequada às demandas judiciais submetidas à apreciação da Justiça do Trabalho. Além disso, conseguir correlacionar os conhecimentos técnicos dentro da esfera legal para determinar a responsabilização do 63 63 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região 63 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região empregador e possíveis soluções para os conflitos no ambiente de trabalho. empregador e possíveis soluções para os conflitos no ambiente de trabalho. Além da importância do assunto para o Direito, ele também é importante ao ambiente de trabalho e a economia nacional. O contraditório neste aspecto é que as pessoas tendem a ficar depressivas pelo excesso de cobrança sobre a sua produtividade, dentre outros fatos que evidentemente contribuem também para tal situação. Ocorre, entretanto, que quando a pessoa está deprimida a sua produtividade tende a cair. Essa queda é tão notória que pode chegar a superar a perda de produtividade por absenteísmo, além de contribuir em desfavor da qualidade de seu trabalho. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 3 ABSENTEÍSMO, PRESENTEÍSMO E DESCONEXÃO Ao tocar no tema do absenteísmo, surge a necessidade de se falar sobre o seu conceito e de outros que, com ele, se relacionam: o presenteísmo e a desconexão. Estes fenômenos ocorrem no meio ambiente do trabalho. Absenteísmo ou absentismo, no latim, absens é ato ou efeito de se abster, deixar de fazer alguma coisa para a qual estava obrigada, tem o sentido também de estar fora, afastado, ausente. No campo trabalhista o absenteísmo é a ausência do trabalhador no emprego. Ele pode possuir diversas causas e pode existir em diversas formas: absenteísmo amparado por lei – ausência legal ao trabalho -, absenteísmo compulsório - impedimentos de ordem disciplinar-, absenteísmo por doença – também amparado em lei - e absenteísmo voluntário, ou falta injustificada – a ausência do trabalho em decorrência de ato volitivo unilateral de iniciativa do empregado, não amparado por lei para fins de abono. Sasaki (2013) revela que nem todas as doenças levam ao absenteísmo, visto que, em casos como a depressão, muitos funcionários podem seguir trabalhando. Neste caso, trata- se do presenteísmo. O presenteísmo é o oposto do absenteísmo, é a presença física do empregado no local de trabalho, mesmo não estando saudável física ou psicologicamente. Dessa forma, eles são incapazes de cumprirem integralmente suas obrigações trabalhistas. Esta doença, ainda em construção de uma hipótese diagnóstica, pode ser causado por fatores relacionados ao trabalho, como a elevada pressão em decorrência do controle sobre suas funções, o relacionamento com os colegas, e a estipulação de prazos exagerados, e por fatores pessoais, como a situação financeira e a vida em família. (Hasen e Andersen, 2008). 64 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região 64 O custo pela ausência física do empregado é fácil de ser calculada, pois o empregador valendo-se do seu padrão salarial e em face do período de sua ausência ao trabalho, e utilizando-se da fórmula incidente sobre a consequente interrupção ou suspensão do contrato de trabalho - dependendo da forma de afastamento -, calcula o valor a ser contabilizado, consciente de que em ambos os casos existem custos administrativos. No caso do presenteísmo, entretanto, é difícil dimensionar os custos do dano causado à organização do trabalho, pois a queda de produtividade do empregado pode superar a perda de produtividade por absenteísmo, além de contribuir para o aumento das possibilidades de acidentes no local de trabalho. 3 ABSENTEÍSMO, PRESENTEÍSMO E DESCONEXÃO Além do fator produtividade, há que ser considerado a qualidade final do produto, a qual também poderá ser comprometida. O prejuízo, nesse sentido, vai além do financeiro, atingindo, por esta razão também o social, pois em ambos os casos a sociedade também arcará com tal custo, pois em algum momento enfrentará a sorte de suportar mais um trabalhador adoecido, que deverá ser tratado pelo sistema único de saúde. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 4 O EPE COMO INSTRUMENTO DE PRECAUÇÃO, PREVENÇÃO E PROTEÇÃO DA SAÚDE FÍSICA E EMOCIONAL DO EMPREGADO Inspirado no equipamento de proteção individual (EPI), dada a incidência das doenças psicossomáticas originada no ambiente de trabalho, defendemos a necessidade da criação do equipamento de proteção emocional (EPE) para o empregado, não previsto, evidentemente nas NRs da portaria 3124/78. Utilizando-se de uma linguagem metafórica, entendemos que na era da quarta revolução industrial, o fornecimento de um equipamento físico para proteção da saúde do empregado hodiernamente não mais condiz com a realidade do meio ambiente saudável, diante da necessidade de também se preocupar com a proteção integral do empregado no ambiente do trabalho. Esse novo “instrumento” também deve ser fornecido ao empregado, como se um EPI fosse. Assim como o EPI foi criado para a prevenção das ocorrências físicas, defendemos a necessidade da criação de um EPE para prevenção de ocorrências emocionais. Assim como defendemos a responsabilidade objetiva do empregador pelo acidente do trabalho, entendemos que o adoecimento decorrente do trabalho deve seguir a mesma sorte. 65 65 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região O leitor deve estar se perguntando agora, como resolvemos isso. Este é o ponto alto desta reflexão. Não se objetiva aqui a solução a este sério e delicado problema, mas de levantar a necessidade de sua conscientização diante da alta incidência dos adoecimentos psicossomáticos nos mais diversos ambientes de trabalho e por suas mais diversas formas. Por esta razão a solução não deve decorrer de resposta única, formatada com frases de efeito, mas deve ser construída de forma processual, sendo a primeira etapa a conscientização da sua necessidade, para posterior criação desta ferramenta de harmonização ambiental. Cada ambiente deve criar um instrumento próprio de dialogo adequado àquele local, na busca do seu equilíbrio. Eis a razão para a criação de um equipamento de proteção individual, mas como afirmado algures, de reflexo coletivo, interno e externo a empresa. Esta é uma forma de garantia da dignidade do trabalhador, a fim de que o ambiente de trabalho permaneça saudável, e possa contribuir para a saúde física e mental do empregado. Para criação deste instrumento, o conceito de proteção deve englobar também os conceitos de precaução e prevenção, nos aspectos físicos e emocionais. Esta é uma questão importante porque os princípios são à base de todo ordenamento jurídico. 4 O EPE COMO INSTRUMENTO DE PRECAUÇÃO, PREVENÇÃO E PROTEÇÃO DA SAÚDE FÍSICA E EMOCIONAL DO EMPREGADO Nesse diapasão, o princípio protetor deve envolver a proteção à saúde física e mental do trabalhador, no seu aspecto curativo, mas em especial no preventivo. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 5 O EQUIPAMENTO DE PROTEÇÃO EMOCIONAL – EPE Dada à responsabilidade do empregador para com a saúde, física e mental, de seus funcionários e a gravidade da questão, defendemos que o EPE também reúna os conceitos de prevenção e de precaução, anteriormente apresentados. A medicina age em dois âmbitos: preventivo e curativo, ambos devendo ser considerados pelo EPE. Dessa forma, sustenta-se essas duas vertentes para estabelecerem soluções a serem aplicadas pelo empregador no ambiente de trabalho. No âmbito preventivo, o empregador deve adotar medidas para eliminar o stress no ambiente de trabalho. Os empregadores devem contar com profissionais capazes, para fomentar a busca do um ambiente saudável, diagnosticando as diversas situações que 66 66 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região possam trazer desiquilíbrio ao ambiente do trabalho, tendente a influenciar na vida do trabalhador, com a possibilidade de seu adoecimento. O trabalhador, nesse sentido, deve ser tratado como ser humano, não poderá ser tratado como vil instrumento de trabalho, que poderá ser descartado a qualquer momento a própria sorte, como se fosse uma peça inanimada. Como acima destacado, para Ercílio Denny não baste existir de fato, o ser humano precisa justificar sua existência para sí e para os outros que com ele vive em sociedade, e onde cria suas relações. Ele precisa ter a garantia da autoestima e da efetivação da sua existência, e para isso ele precisa se livrar das amarras, libertar-se, para assim garantir a efetivação dos seus Direitos Fundamentais. Não é outro e entendimento de Thomas Fleiner (2003, p. 11), que nos traz sua contribuição: Encontrando-me em uma bela e ensolarada praia italiana, decidi-me livremente a trabalhar neste livro, em vez de sonhar, banhar-me, beber um café ou mesmo assistir à televisão. O que isso tem a ver com a dignidade humana? Muito! Diferentemente de todos os outros seres vivos, o homem determina, pelo menos em parte, a causa de suas próprias ações. […] Quando essa liberdade é invadida por outrem, o homem tem a sua dignidade violada. A dignidade humana pressupõe o respeito ao âmbito da liberdade que as pessoas necessitam para formar suas opiniões e, de acordo com estas, determinar suas ações. Ademais, é mister dar ao homem a possibilidade de desenvolver-se segundo seus projetos de vida. Qualquer medida coercitiva que prejudique essencialmente a sua liberdade de decisão se constitui num ataque contra a dignidade humana. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 5 O EQUIPAMENTO DE PROTEÇÃO EMOCIONAL – EPE Para Fleiner a dignidade humana tem como alicerce a liberdade, pois a violação a esse direito viola a sua dignidade, e isso é importante para que o homem possa desenvolver o seu próprio projeto de vida, ou no dizer do Pregador, a vida deve ser vivida em abundância, o homem deve viver abundantemente, ou numa leitura mais consentânea, viver dignamente. “Eu vim para que tenham vida, e a tenham em abundância” (Jo: 10.10). Essa é a lição que encontramos talvez como uma das primeiras definições de dignidade humana, cujo conceito está traçado de forma simples, mas sintetiza a ideia de eminentes doutrinadores hodiernos, que ainda labutam para fechar o seu diagnóstico conceitual. O trabalhador deve viver abundantemente inclusive no seu local de trabalho, e é dever do empregador eliminar os ruídos, agentes poluidores físicos, mas também as tensões que causam os mesmos males. 67 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região 67 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região 67 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região 67 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 6 ASPECTO JURISPRUDENCIAL A jurisprudência, na ausência de parametrização legal, vem cumprindo seu papel para identificação do dano imaterial decorrentes de doenças psicossomáticas, tendo como premissa o nexo de causalidade, diante das suas múltiplas causalidades. Necessário, portanto, a caracterização da inércia do empregador quanto correto tratamento do seu ambiente de trabalho. Os pedidos de indenização por dano embasados nos prejuízos decorrentes do labor em condições labor-ambientais insalubres e desequilibradas, embora tenha registrado baixa frequência de ocorrência de julgados, representa a terceira categoria temática com maior frequência, de acordo com a pesquisa jurisprudencial realizada. Como exemplo, aponta-se o acórdão prolatado nos autos do recurso ordinário nº 0062200-64.2007.5.06.0015, tramitado perante o Tribunal Regional do Trabalho da 7ª Região (TRT&), em decisão prolatada por Sérgio Torres Teixeira, em 06 de Dezembro de 2013, cujo caso registra a procedência da pretensão indenizatória por dano existencial decorrente da contração de doença pelo trabalhador, em razão de condições labor- ambientais desequilibradas: DANO MORAL E MATERIAL. PROVA. RECONHECIMENTO. RECLAMANTE. DOENÇA. ACOMETIMENTO. NEXO CAUSAL. ATIVIDADE LABORAL. CONSTATAÇÃO. DANO EXISTENCIAL. OCORRÊNCIA. INDENIZAÇÕES. SENTENÇA. MANUTENÇÃO. Inúmeros são os documentos, no caderno processual, que demonstram a doença que acometeu a trabalhadora, por razões diretamente vinculadas ao seu ambiente de trabalho, e, sem sombra de dúvida, por culpa da conduta empresarial, que, por meio de superiores hierárquicos, exerciam intensa pressão psicológica, de forma desmedida sobre a parte hipossuficiente. Evidenciado, inclusive, autêntico dano existencial, este compreendido na própria “frustração do trabalhador em não realizar um projeto de vida e no prejuízo das relações sociais e familiares, em razão da privação do seu direito ao descanso”. Este cenário, inclusive, culminou com a emissão da Comunicação de Acidente de Trabalho - CAT, endereçada à Previdência Social, ensejando a percepção, do correspondente benefício previdenciário ao segurado, e, posteriormente, a aposentadoria por invalidez. Foi mais do que comprovada à atitude da entidade bancária, em colaborar, efetivamente, para os graves problemas de saúde que acometem a reclamante. Indenizações pelo dano moral e o material que se mostram pertinentes e 68 68 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região em valores condizentes com a razoabilidade e proporcionalidade. Sentença que se mantém. Recurso ordinário a que se nega provimento. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 6 ASPECTO JURISPRUDENCIAL (BRASIL, 2013) Não é outro o entendimento prolatado na decisão do relator Ricardo de Carvalho, da 3º turma do Tribunal Regional do Trabalho da 4º Região (TRT4), no Recurso Ordinário nº 0000221-16.2014.5.04.0661, em que reconhece a insalubridade emocional do meio ambiente de trabalho ao qual estava submetido o trabalhador, de modo a desencadear sintomas de depressão e afetar severamente seu convívio social: DANO EXISTENCIAL. INDENIZAÇÃO. Caso em que a condenação decorre das condições de trabalho a que foi submetida a demandante, levando-a a desenvolver depressão, ficando afastada do trabalho por três anos, bem como dos descontos injustos no salário, a ponto de nada receber no final do mês. (BRASIL, 2015a) O Tribunal Superior do Trabalho (TST) adota a teoria da responsabilidade subjetiva do empregador, todavia, não deixa de reconhecer, a exemplo dos Tribunais Regionais do Trabalho, a responsabilidade do empregador quando este contribui para a ocorrência da situação fática do adoecimento do trabalhador, como se verifica pela decisão que ora colamos a titulo de exemplificação, em decisão exarada no RR: 376008520075040030, Relator: José Roberto Freire Pimenta, Data de Julgamento: 15/04/2015, 2ª Turma, DEJT 24/04/2015: INDENIZAÇÃO POR DANOS MORAIS DECORRENTES DE DOENÇA EQUIPARADA A ACIDENTE DO TRABALHO - DEPRESSÃO. ASSÉDIO MORAL. EXISTÊNCIA DE CULPA. RESPONSABILIDADE SUBJETIVA DA RECLAMADA. Trata-se de pedido de indenização por danos morais, fundada em doença equiparada a acidente do trabalho, episódio depressivo em face de tratamento humilhante dispensado à autora e a outros empregados no local de trabalho durante o período em que esteve subordinada à supervisora (...). Conforme consta da fundamentação do acórdão recorrido, o juiz registrou que "a prova pericial comprova a existência de nexo causal entre o inapropriado tratamento no ambiente de trabalho e a moléstia desenvolvida durante o contrato, sendo evidentes os danos morais decorrentes da doença equiparada a acidente do trabalho". O Tribunal a quo, instância exauriente para análise de fatos e provas, com base em laudo pericial que diagnosticou quadro depressivo moderado e na prova oral colhida, assentou que "a doença ocupacional tem como concausa o ambiente laboral excessivamente humilhante, estressante, 69 69 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região prejudicial à saúde". E, além disso, concluiu que "o nexo de causalidade e a culpa da reclamada restam configurados, a última por meio da atitude da supervisora contratada". LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 6 ASPECTO JURISPRUDENCIAL Com efeito, considerando o contexto fático probatório consignado nos autos, acerca da doença adquirida pela reclamante, o dano moral dela emergente e o nexo causal entre o dano e o tratamento humilhante dispensado à reclamante, não há como afastar o direito à indenização. Ademais, ressalta-se que, para se chegar à conclusão diversa do Regional seria necessário o revolvimento do conjunto probatório, não permitido nesta instância recursal extraordinária, ante o óbice previsto na Súmula nº 126 do TST. (TRIGUEIROS, 2017) Merece destaque também a decisão proferida pelo TST no AI 806- 58.2011.5.15.0082.3ª Turma. Relator: Alexandre Agra Belmonte. De 25/03/2015. Merece destaque também a decisão proferida pelo TST no AI 806- 58.2011.5.15.0082.3ª Turma. Relator: Alexandre Agra Belmonte. De 25/03/2015. INDENIZAÇÃO POR DANO MORAL. PRIVAÇÃO DE FÉRIAS POR UM LONGO PERÍODO. DANO EXISTENCIAL. GOZO E PAGAMENTO. O e. TRT ao asseverar, que apesar do reconhecimento do direito ao pagamento das férias em dobro, a autora faz jus ao pagamento de indenização por dano moral, decorrente da longa privação do direito às férias (2005 a 2010), gozo e pagamento, mesmo porque restaram caracterizados os elementos ensejadores da respectiva indenização: o dano moral, a conduta do empregador e o nexo causal entre a atitude do empregador e o dano sofrido pela autora e ponderou: “... para, de um lado, coibir a reincidência do empregador em situações como a constatada na presente ação e, de outro, compensar o trabalhador pelo sofrimento causado sem, contudo, dar ensejo ao seu enriquecimento ilícito.”. Incólume o art. 5º, X, da Constituição Federal. Arestos oriundos de Turmas do TST, inservíveis nos termos do art. 896, alínea “a”, da CLT. Agravo de instrumento conhecido e desprovido. (BRASIL, 2015b) INDENIZAÇÃO POR DANO MORAL. PRIVAÇÃO DE FÉRIAS POR UM LONGO PERÍODO. DANO EXISTENCIAL. GOZO E PAGAMENTO. O e. TRT ao asseverar, que apesar do reconhecimento do direito ao pagamento das férias em dobro, a autora faz jus ao pagamento de indenização por dano moral, decorrente da longa privação do direito às férias (2005 a 2010), gozo e pagamento, mesmo porque restaram caracterizados os elementos ensejadores da respectiva indenização: o dano moral, a conduta do empregador e o nexo causal entre a atitude do empregador e o dano sofrido pela autora e ponderou: “... LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 6 ASPECTO JURISPRUDENCIAL para, de um lado, coibir a reincidência do empregador em situações como a constatada na presente ação e, de outro, compensar o trabalhador pelo sofrimento causado sem, contudo, dar ensejo ao seu enriquecimento ilícito.”. Incólume o art. 5º, X, da Constituição Federal. Arestos oriundos de Turmas do TST, inservíveis nos termos do art. 896, alínea “a”, da CLT. Agravo de instrumento conhecido e desprovido. (BRASIL, 2015b) Como se vê pelas decisões supra, os Tribunais do Trabalho parecem estar sensíveis a tais situações, todavia trabalham com os casos que lhes são apresentados, e não dão conta, evidentemente, da realidade social que acomete os trabalhadores no cenário produtivo Nacional. Desta forma não operam de forma preventiva, e não contribuem para tal mister, vez que ainda a eles, não raro, são arrogadas as criticas de serem muitos insensíveis ao arbitrarem os valores a serem compensados em casos de danos imateriais. Sem trazer a baila discussão não afeta a esta reflexão, e sem querer adentrar na discussão da inconstitucionalidade do tabelamento estabelecido pelo artigo 223-G da CLT - que entendemos que será referendado pelo STF, a exemplo de outros equivocados referendos já ocorridos -, os Tribunais trabalhistas parecem querer dizer que o tabelamento mencionado 70 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região parece razoável. Não concordamos, mas este é assunto para outra reflexão. 70 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região 70 parece razoável. Não concordamos, mas este é assunto para outra reflexão. parece razoável. Não concordamos, mas este é assunto para outra reflexão. CONSIDERAÇÕES FINAIS O Direito do Trabalho é uma área da ciência jurídica que dialoga com outras áreas da ciência, notadamente com a área da saúde e engenharia, para prevenir a saúde e segurança dos trabalhadores. Esse dialogo torna-se profícuo quando visa a efetivação dos direitos dos trabalhadores, com o fim de garantir a sua dignidade humana. Como visto os tribunais não atuam preventivamente, a fiscalização do trabalho vem enfrentando sério projeto de sucateamento há décadas, e agora já se fala em extinção do Ministério do Trabalho. Diante de tão graves situações que surgem no campo político, tendentes a causar um verdadeiro retrocesso social, o clima é de apreensão e espanto, pois a sociedade caminha a passos largos e de forma desenfreada para uma situação diametralmente oposta, caminha a passos largos para a evolução econômica e social impulsionada pelo avanço tecnológico e informacional. Em decorrência deste avanço social, situações surgem e precisam ser amparadas. Como sabemos o cenário político não esta sensível a tais situações. Por esta razão ganha relevo a discussão da proteção integral do trabalhador, se não for por politicas públicas, que haja uma conscientização sobre a necessidade de se cuidar da saúde social, individual ou coletiva; e com isso o empregador passe a cuidar mais da saúde física e mental de seus empregados, pois não é somente o absenteísmo que causa prejuízo a empresa, mas também o presenteísmo, na mesma proporção ou em proporção mais significativa. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 ABREU, F. M. Depressão como doença do trabalho e suas repercussões jurídicas. São Paulo: LTr, 2005. ALMEIDA, Victor Hugo de. Consumo e trabalho: impactos no meio ambiente do trabalho e na saúde do trabalhador. 2013. 241 f. Tese (Doutorado em Direito) – Faculdade de Direito, Universidade de São Paulo, São Paulo, 2013. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 71 71 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região COSTA, Aline Moreira da; GONÇALVES, Leandro Krebs. Meio ambiente do trabalho e proteção jurídica do trabalhador: (re)significando paradigmas sob a perspectiva constitucional. In: FELICIANO, Guilherme Guimarães; URIAS, João. (Org.). Direito ambiental do trabalho: apontamentos para uma teoria geral. São Paulo: LTr, 2013. v. 1. BRASIL. MINISTÉRIO DA SAÚDE. SECRETARIA DE POLÍTICAS DE SAÚDE. Protocolo de investigação, diagnóstico, tratamento e prevenção de Lesão por Esforços Repetitivos e Distúrbios Osteomusculares Relacionados ao Trabalho, Brasília, jul. 2000. Disponível em: <http://bvsms.saude.gov.br/bvs/publicacoes/protocolo_ler.pdf>. Acesso em: 19 ago. 2018. BRASIL. MINISTÉRIO DA SAÚDE. Doenças relacionadas ao trabalho - manual de 44 Rev. Trib. Reg. Trab. 3ª Reg., Belo Horizonte, v.46, n.76, p.27-44, jul./dez.2007 procedimento para os serviços de saúde. Brasília, 2001. BRASIL. MINISTÉRIO DA SAÚDE. Doenças relacionadas ao trabalho - manual de 44 Rev. Trib. Reg. Trab. 3ª Reg., Belo Horizonte, v.46, n.76, p.27-44, jul./dez.2007 procedimento para os serviços de saúde. Brasília, 2001. BRASIL. MINISTÉRIO DA PREVIDÊNCIA SOCIAL. Diretrizes de conduta médico-pericial em transtornos mentais. Brasília, 2007. BRASIL. TRT 7ª Região. RO 0062200-64.2007.5.06.0015. Relator: Sergio Torres Teixeira. 2ª Turma. Julgamento: 04/12/2013. Publicação: 06/12/2013. (CEARÁ, 2017). BRASIL. TRT 4º Região. RO 0000221-16.2014.5.04.0661. Relator Ricardo Carvalho Fraga. 3ª Turma. Julgamento: 15/09/2015. 2015a. BRASIL. TRIBUNAL SUPERIOR DO TRABALHO. AI 806-58.2011.5.15.0082. 3ª Turma. Relator: Alexandre Agra Belmonte. Julgamento 25/03/2015. 2015b. DENNY, Ercílio A. Ética & Sociedade. Capivari/SP: Opinião E., 2001. DENNY, Ercílio A. Ética & Sociedade. Capivari/SP: Opinião E., 2001. DIAS, José de Aguiar. Da responsabilidade civil. Rio de Janeiro: Lumen Juris, 2012. DELGADO, Mauricio Godinho. Curso de direito do trabalho. 16. ed. São Paulo: LTr, 2017. DELGADO, Mauricio Godinho. Curso de direito do trabalho. 16. ed. São Paulo: LTr, 2017. DIAS, José de Aguiar. Da responsabilidade civil. Rio de Janeiro: Lumen Juris, 2012. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 72 72 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região FLEINER, Thomas. O que são direitos humanos. Trad. Andressa Cunha Cury. São Paulo: Max Limonad, 2003. FLEINER, Thomas. O que são direitos humanos. Trad. Andressa Cunha Cury. São Paulo: Max Limonad, 2003. GEOVANA, Nicole. O que é síndrome de burnout e quais são os sintomas? In Médico GEOVANA, Nicole. O que é síndrome de burnout e quais são os sintomas? In Médico Responde, s.d. Disponível em: <https://medicoresponde.com.br/o-que-e-sindrome-de- burnout-e-quais-sao-os-sintomas/>. Acesso em: 19 ago. 2018. RIGOYEN, Marie-France. Assédio moral: a violência perversa no cotidiano. 7. ed. Rio de neiro: Bertrand Brasil, 2005. HIRIGOYEN, Marie-France. Assédio moral: a violência perversa no cotidiano. 7. ed. Rio de Janeiro: Bertrand Brasil, 2005. JACQUES, M. G. O nexo causal em saúde/doença mental no trabalho: uma demanda para a psicologia. Universidade Federal do Rio Grande do Sul. Porto Alegre: Brasil, 2006. MORETTI, Silvinha. Qualidade de vida no trabalho x Auto-realização humana. [Blumenau], 2008. Disponível em: Acesso em: 24 set. 2018. MORETTI, Silvinha. Qualidade de vida no trabalho x Auto-realização humana. [Blumenau], 2008. Disponível em: Acesso em: 24 set. 2018. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018 JACQUES, M. G. O nexo causal em saúde/doença mental no trabalho: uma demanda para a psicologia. Universidade Federal do Rio Grande do Sul. Porto Alegre: Brasil, 2006. LIMA, M. E. A. Escritos de Louis Le Guillant - da Ergoterapia à Psicopatologia do Trabalho. Petrópolis, Rio de Janeiro: Ed. Vozes, 2006. GI, Camila Martinelli. O dano existencial aplicado ao âmbito juslaboral. 2015. 72 f. Trabalho de Conclusão de Curso (Graduação em Direito) – Faculdade de Ciências Humanas e Sociais, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Franca, 2015. 73 Revista Jurídica Trabalho e Desenvolvimento Humano Procuradoria Regional do Trabalho da 15ª Região SABONGI, Camila Martinelli. O dano existencial na jurisprudência trabalhista brasileira e a necessidade de harmonização de políticas públicas labor-ambientais para o seu enfrentamento. 2018. 103 f. Dissertação de Mestrado – Faculdade de Ciências Humanas e Sociais, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Franca, 2018. ASAKI SFS. Trabalho bancário e fatores associados ao presenteísmo e ao absenteísmo. 2013. Dissertação de Mestrado - Faculdade de Saúde Pública da Universidade de São Paulo, São Paulo, 2013. SELIGMAN, Martin E. P. Indefensión: em la depresión, el desarrollo y la muerte. Trad. Luis Aguado Aguilar. Madrid: Debate, 2000. TEIXEIRA, Sueli. A depressão no meio ambiente do trabalho e sua caracterização como doença do trabalho. Rev. Trib. Reg. Trab. 3ª Reg., Belo Horizonte, v.46, n.76, p.27-44, jul./dez.2007. TEIXEIRA, Sueli. A depressão no meio ambiente do trabalho e sua caracterização como doença do trabalho. Rev. Trib. Reg. Trab. 3ª Reg., Belo Horizonte, v.46, n.76, p.27-44, jul./dez.2007. VALADARES, C. Trabalhador mais protegido. UnBnotícias, Ano 10, n. 78, 2007. Disponível em: <http://www.secom.unb/unbnoticias.un0507-p7.htm>. Acesso em 25 mai.2018. WEBER, Max. Economia e sociedade: fundamentos da sociologia compreensiva. Brasília: Universidade de Brasília, 1999. WEBER, Max. Economia e sociedade: fundamentos da sociologia compreensiva. Brasília: Universidade de Brasília, 1999. LIMA, Leonardo T.; GONÇALVES, Vitória M.; CARDOSO, Jair A. O meio ambiente do trabalho e a saúde do trabalhador: desafios à efetivação da dignidade humana. Revista Jurídica Trabalho e Desenvolvimento Humano, Campinas, v. 1, n. 1, p. 57-73, 2018
https://openalex.org/W4388681108	https://mutiara.al-makkipublisher.com/index.php/al/article/download/92/120	English	null	Analysis of the Jigsaw Learning Model on Pancasila Implementation Material From Time to Time Phase D in the Independent Curriculum	Mutiara	2,023	cc-by-sa	5,709	P-ISSN: 2988-7860 E-ISSN: 2988-7992 P-ISSN: 2988-7860 E-ISSN: 2988-7992 ABSTRACT KEYWORDS Jigsaw, Independent Curriculum, Phase D ABSTRACT This study aims to analyze the Jigsaw learning model in the form of a sub-chapter of the material Implementation of Pancasila from Time to Time Phase D in the Independent Curriculum. This research uses the method of literature study or literature review using content analysis to better understand and interpret a study. The results showed a model that is very suitable for use in learning outcomes in the form of subchapters, namely the material Implementation of Pancasila From Time to Time Phase D. The jigsaw model makes students work hand in hand in mastering the material and encourages student activity in order to achieve maximum results. Each member or individual is taught to take responsibility for the submaterial they obtain. Students have independent responsibilities and teach students to communicate well, therefore this model emphasizes the development of civic knowledge citizenship aspects. Then the benefit of this model is to prevent competition and increase collaboration as contained in the independent curriculum. Liana Anisa Putri1, Mukhamad Murdiono2 Universitas Negeri Yogyakarta1,2 Email:lianaanisa.2021@student.uny.ac.id, mukhamad_murdiono@uny.ac.id KEYWORDS Jigsaw, Independent Curriculum, Phase D ANALYSIS OF THE JIGSAW LEARNING MODEL ON PANCASILA IMPLEMENTATION MATERIAL FROM TIME TO TIME PHASE D IN THE INDEPENDENT CURRICULUM Liana Anisa Putri1, Mukhamad Murdiono2 Universitas Negeri Yogyakarta1,2 Email:lianaanisa.2021@student.uny.ac.id, mukhamad_murdiono@uny.ac.id INTRODUCTION INTRODUCTION Education is a top priority and a key factor to shape and improve the quality of human resources in Indonesia (Manasikana & Anggraeni, 2018). Education can also develop the potential that exists in students can be used as a way to solve problems in learning and life. Therefore, there must be a curriculum in order to encourage the creation of good education. In the curriculum, teachers play an important role and are responsible for both students and the wider community in the implementation of learning (Wahyudin, 2018). The urgency in education today that many teachers face is the low activeness of students and students are often bored in learning (Andayani et.al, 2019). The independent learning curriculum comes with the main concept of freedom in thinking. Teachers have the freedom to understand the curriculum before learning (Izza., et.al, 2020). Freedom of learning is also found in the objectives, materials, models and evaluations of learning used. The learning model must be student-centered (Indarta, et.al, 2022). In the implementation of 21st century generation learning, teachers must have varied and innovative strategies, models and methods by continuing to improve knowledge and skills in teaching that are interesting and interactive by utilizing technology (Sumantri, 2019). So dairi is 614 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum p needed a model that focuses on students in the learning process that will encourage student activity. Teachers must be able to find ways to encourage activeness, creativity and motivation of students (Susanto, 2014). Teachers must have skills in choosing models that are suitable for diverse forms of material. As in the subject of Pancasila education (civic education), in the analysis of the model must also consider which aspects of citizenship will be raised. The constructivistic learning model is one of the variations of the model that aims to improve the learning outcomes of students who are more active in learning. One of the models that has characteristics This is the Jigsaw Learning Model (Rahman, 2018). The jigsaw learning model is a model whose implementation of students is formed in small groups with different levels of ability (Tastra, 2013). RESEARCH METHODS This research is a research using the method of literature study or literature review using content analysis to better understand and interpret a study. This research uses data collection techniques through Google Scholar, with journal criteria used based on research objectives and has been accredited. The research examined various literature, both international and national journals and books, to analyze the innovation of Jigsaw model analysis on pancasila material from time to time in phase D in the independent curriculum (Surur, 2021). In this literature study, the data from the analysis will be discussed descriptively both to provide views (criticize) and summarize (summarize) for the selected article. Using the Meta-Analysis procedure by analyzing the contents of the journal, then reviewing using the results of journal data that has been collected then the researcher provides views and then summarized and discussed to draw conclusions (Kaid, Baehaqie, & Rizal, 2021). INTRODUCTION The jigsaw learning model can be done in order for students to express opinions, manage all information obtained so that students can improve communication skills and be able to convey the material that has been learned (Mariyaningsih & Hidayati, 2018). In addition, this model teaches material to other group members (origin) in a group way (Suprihatin, 2017). This type of learning model can be highly recommended for material in the form of subchapters, such as in the implementation of pancasila from time to time. This learning model gives rise to situations where individual success is influenced by groups, has positive dependence and cooperation in carrying out tasks to achieve learning objectives. In this model, opportunities are wide open for students to increase motivated activeness in learning, of course, it can affect the value of student learning outcomes that increase after the learning process. 1. Jigsaw Type Learning Model Concept Aroson et al developed the jigsaw as a model of cooperative learning. Lei explained that the jigsaw model is a cooperative model that considers the backgrounds, experiences and differences of learners. This is done so that learning gets something meaningful. Jigsaw is a model that has similarities to the model of exchange between groups (Rahman, 2021). In this model, students learn with a group of peers where there is one expert who is responsible for covering the subject matter being studied (Hertiavi, dkk, 2010). Jigsaw is a cooperative learning model that is characterized by the importance of working in small groups (Sholihah, Koeswardani, & Fitriana, 2018). The approach, or jigsaw style, directs and has learners conduct their own self-directed learning in small groups with a mix of up to ten people. Responsibility, cooperation and positive dependency are positive impacts of this model. Students get many opportunities to express their opinions in lessons. Everyone is responsible for achieving learning goals (Haliza, 2020). Jigsaw as one of the active learning models where the members are 4-6 people heterogeneously, each student has power over the material he can and is able to explain it to other members (Sholihah, dkk. 2018). The learning material is in the form of text, students get one to be learned. Each member understands the subchapters they have acquired and when put together will form a complete knowledge. Each member who came from the original group sat together and discussed into a group of experts with the same discussion. After that, return to the original group and explain to the group mates (Sulhan, 2020). Slavin (2009) suggests that the jigsaw-type model is very suitable for beginners and teachers who are new to implementing a cooperative approach". According to Adams (2013), jigsaw learning is easy to use because learning focuses on students and teachers only as supervisors. Students are trained to be responsible and exchange information among others. It can be concluded that the jigsaw learning model is part of cooperative learning whose implementation of groups is divided heterogeneously (Lahir, Ma’ruf, & Tho’in, 2017). Then given the task of discussing the material in the origin group and forming an expert group. The goal is the cognitive aspect, which is aligned with the civic knowledge citizenship aspect. In addition, there are aspects of cooperation, responsibility and positive dependence between others. A. Learning Model Cooperative A. Learning Model Cooperative Cooperative learning which is originally said to be "cooperative" which means maximizing the learning of learners in improving skills and academic achievement both individuals and groups (Trianto, 2009: 57). According to Johnson, the cooperative learning model is the only model that fully encompasses conceptual learning. Cooperative or group education model is a teaching method used by group students in order to achieve predetermined learning objectives (Sitomorang, 2017). Thus, based on the evidence presented, the cooperative learning model attributed to it is one that encourages group interaction and cooperation in any learning process. Cooperative learning as a learning model 615 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum by doing and emphasizing teamwork or collaboration when doing work that is often done in groups of more than two people (Amri dan Ahmadi, 2010:90). Susmadewi (2011) suggests that cooperative learning is a strategy where in a group there are diverse students both in terms of background and ability. Mutual cooperation and mutual understanding of the subject matter given in completing group tasks. Cooperative model analysis aims to make students build their own concepts and study concepts scientifically (Rahmat, Suwatno, & Rasto, 2018). Cooperative learning can be said to be a unique learning model. Where learners in small groups exchange information to achieve learning objectives, this results in positive dependence. In obtaining lessons not only from teachers but can be from anywhere, including the students themselves (Syarifuddin, 2011). Cooperative learning has its advantages. Where students have high self-confidence, increase the ability to think independently, find information from various sources. Able to express opinions loudly and directly and can compare with other opinions or ideas (Situmorang, 2017). A. Learning Model Cooperative Jigsaw Type Benefits of the Jigsaw Type Learning Model There are several benefits of the jigsaw learning model, among others. Can cause the ability of each individual to increase, reduced apathy, mutual acceptance of lack, not individualism, reduced interpersonal conflict, deeper understanding of material and increased motivation to learn (Muaddab, 2018). In addition, the jigsaw learning model can prevent the aggressiveness of learners without sacrificing cognitive aspects (Rosyidah, U, 2016). According to Adams (2013) the jigsaw learning model is easy to learn because not only teachers but also students can be a source of information. 1. Steps of the Jigsaw Type Learning Model 1. Steps of the Jigsaw Type Learning Model 1. Steps of the Jigsaw Type Learning Model There are stages of Jigsaw model analysis in (Sholihah, 2018), The analysis steps include: p g yp g There are stages of Jigsaw model analysis in (Sholihah, 2018), The analysis steps include: There are stages of Jigsaw model analysis in (Sholihah, 2018), The analysis steps include: a. Used for material that has many subchapters or sections. a. Used for material that has many subchapters or sections. b. The teacher makes heterogeneous groups of students into several small groups according to the material. The youngest group of 4-6 people is the origin group and the expert group. Th h b h i i l i b b b. The teacher makes heterogeneous groups of students into several small groups according to the material. The youngest group of 4-6 people is the origin group and the expert group. to the material. The youngest group of 4 6 people is the origin group and the expert group. c. The group that became the original group sent its group members to become an expert group. c. The group that became the original group sent its group members to become an expert group. d. All members of the initial group of saplings get material or subchapters that are not the same as each other. d. All members of the initial group of saplings get material or subchapters that are not the same as each other. e. After a joint discussion, the expert group was over. All members of the origin group met again to present the material obtained from the expert group. f. Then a draw is held where each group will explain the results of the group discussi g. 1. Jigsaw Type Learning Model Concept This model is very appropriate to be used in learning in an independent curriculum 616 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum p that requires students to be independent and free of opinion but responsible. In addition, it is appropriate for material in the form of subchapters on certain learning outcomes. Elements of the Jigsaw Type Learning Model In a book entitled "Cooperative Learning" written by Anita Lie (2004) it is written that the jigsaw learning model is not just group learning but there are distinguishing characteristics (Tias, 2021). Among others as follows. First, the success of a task is due to positive dependence between group members. In order to build positive dependence, learners must feel that they belong to a group. If the group gets an award when it successfully achieves a goal, all should feel it. Both are individual responsibilities. Each member of the group gets each piece of material and is responsible for understanding it. There are ways to foster a sense of responsibility, by encouraging learners, recording each individual's progress (Abas, 2017). Third, communication between members where group members must be able to greet and listen to each other. This process is very beneficial in the mental and emotional development of learners. Fourth, evaluation is carried out by teachers to improve future learning outcomes to be even better. Benefits of the Jigsaw Type Learning Model At the final stage, the teacher concludes with the students and instructs the students to answer questions in the form of individual tests. h. Teachers give awards to the best individuals and groups. h. Teachers give awards to the best individuals and groups. Broadly speaking, according to (Rahman, 2021) the syntax or stages of the jigsaw learning model are shown in table 1. Broadly speaking, according to (Rahman, 2021) the syntax or stages of the jigsaw learning model are shown in table 1. 617 alysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Tim Time Phase D In The Independent Curriculum Table 1 Syntax or stages of the jigsaw learning model Phase Activities Phase 1 Provide motivation and convey learning objectives to students The teacher provides a motivation before learning to students and explains the direction and objectives of learning to be achieved. Phase 2 Display information Teachers stimulate students by providing information in the form of facts, experiences and everything that will be done in learning. Phase 3 Form a group or origin group Students are divided into heterogeneous groups whose members can be 5 to 6 people in the initial group. Where all members will get material that is not the same as each other. Phase 4 Forming an expert group Teachers instruct students who have similar material to join as expert groups. Phase 5 The expert group goes back to the original or original group Students sit back to the initial group in order to express what they have gained to all members of the initial group. Phase 6 Evaluation The teacher instructs the students to conclude together and the teacher gives a test that covers all the material. Phase 7 Giving awards Teachers give appreciation in the form of awards to the best individuals or groups. B. Review of Relevant Research Results B. Review of Relevant Research Results To strengthen the implementation of the jigsaw learning model in the Pancasila implementation material from time to time in class IX, the author will provide relevant research to clarify the implementation of this model in learning. Research relevant to the discussion of the jigsaw learning model can be described as shrinking. The first research was conducted by Hj. Pratini (2015) about increasing the activity of class VIII Civics learning outcomes of SMPN 18 Mataram with a jigsaw learner model". Where the purpose of this study is to analyze the effectiveness of the jigsaw learning model in relation to improving Civics learning outcomes from learners. Where the results of research are obtained increasing the activities and learning value of students. Then further research was conducted by Mardhiah (2021) on increasing student achievement in Civics learning using the class VIII jigsaw learning model at MTSN 6 Aceh Besar. This research has a purpose with the jigsaw learning model, activity or activity and student learning achievement can increase, especially in the position and function of Pancasila. It was found that using the jigsaw model resulted in an increase in the activity and value of student learning outcomes on the position and function of Pancasila material in class VIII MTsN 6 Aceh. 618 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum p Advantages or Advantages and Disadvantages of the Jigsaw Learning Model Advantages or Advantages and Disadvantages of the Jigsaw Learning Model g g g g g In the analysis of the jigsaw learning model on material that has many subchapters, it certainly has its advantages and disadvantages. This advantage can be used as a foothold by teachers in order to produce students who have motivation and good results in learning, especially in Civics learning (Mirdanda, 2019). The advantages of the jigsaw model include. First can motivate students to be able to think critically in learning, students can develop their thinking power and communicate to others (Rahman, 2018). Second, students are required to be able to choose the right diction and be easy to understand in explaining the material to their friends. In this case, the ability of sosail students can develop. Third, in discussions all active members are not only dominated by one learner in learning. Fourth, jigsaws can be used alongside other learning models. B. Review of Relevant Research Results These five models can teach confidence, obtain information from various sources and exchange information between learners. Sixth, encourage students to be able to express their ideas directly and hear ideas from their friends. This is very useful when solving problems so that learners respect each other and respect the opinions of others. Seventh, it can improve time management and help increase motivation. In addition to the advantages and advantages, a learning model has disadvantages in its analysis, among others. First, there is fear and reluctance from students if they issue ideas that will be judged badly by their group members. Secondly, not all teachers and learners understand exactly this model. Third, this model takes a lot of time and must be staged and reported in detail. Fourth, it is not easy to increase the confidence of students because of different experiences and backgrounds. 1. Learning Objectives Flow Learning Objective Flow (ATP) is a learning objective step or activity that is made systematically in a learning phase (Hidayat, Dlis, & Hanief, 2021). ATP is used as a benchmark by teachers and students in order to achieve learning objectives at the end of a phase. Can be implemented using ATP designed in the independent curriculum. ATP can also be said to be a learning module developed from learning outcomes (CP) where the Pancasila Student Profile is the main object to be developed. Referring to ATP, in this discussion, the elements of the Pancasila student profile are critical reasoning and mutual assistance. The implementation of critical reasoning here students process information before being passed on to others (Edi, 2022). Students who reason critically can process information well and then evaluate and conclude it. Furthermore, the form of mutual assistance is that students together collect material from various expert groups in order to produce complex material in the original group. A model that is very suitable for use in CP in the form of subchapters is this jigsaw model which can make students work hand in hand in mastering the material and encourage student activity in order to achieve maximum results (Kumalasari, 2021). In the material divided into subchapters. To implement this model in the Pancasila implementation material from time to time, it is necessary to know the learning outcomes, objectives and learning activities and the main material. It can be seen in Table 2. 619 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum Table 2 Learning Objectives Flow Element Learning Outcomes (CP) Learning Objectives Subject matter Learning Activities Pancasila Student Profile Teaching Model Valuation Pancasila Students understand the implement ation of Pancasila in state life from time to time. Students are able to analyze the implementat ion of Pancasila in state life from time to time well. Implementa tion of Pancasila from time to time. Students analyze, present reports on the results of analysis, implementat ion of Pancasila from time to time. Critical reasoning, Mutual assistance Discussions, Presentations, Explorations and lectures Observati on (Individua l and group) s Of The Jigsaw Learning Model On Pancasila Implementation Material From Time e Phase D In The Independent Curriculum Table 2 1. Learning Steps The process of implementing the jigsaw learning model of Pancasila elements with the subject matter of implementing Pancasila from time to time in state life. In this case the jigsaw modelcan be carried out by going through 7 phases. The learning steps are set out in table 3. Table 3 Learning Steps PHASE TEACHER ACTIVITIES STUDENT ACTIVITIES INTRODUCTION Phase 1 Provide motivation and convey learning objectives to students The teacher guides students to pray, then provides motivation and conveys learning objectives to students Students pray and listen to the teacher's direction related to the objectives in learning and increase self-motivation for the spirit of learning. INTI Phase 2 Display information Teachers provide information to students about the implementation of Pancasila from time to time using PPT. Students receive information about the implementation of Pancasila from time to time through CFT. Table 3 Learning Steps PHASE TEACHER ACTIVITIES STUDENT ACTIVITIES INTRODUCTION Phase 1 Provide motivation and convey learning objectives to students The teacher guides students to pray, then provides motivation and conveys learning objectives to students Students pray and listen to the teacher's direction related to the objectives in learning and increase self-motivation for the spirit of learning. INTI Phase 2 Display information Teachers provide information to students about the implementation of Pancasila from time to time using PPT. Students receive information about the implementation of Pancasila from time to time through CFT. Table 3 Learning Steps Table 3 Learning Steps PHASE TEACHER ACTIVITIES STUDENT ACTIVITIES INTRODUCTION Students pray and listen to the teacher's direction related to the objectives in learning and increase self-motivation for the spirit of learning. Students pray and listen to the teacher's direction related to the objectives in learning and increase self-motivation for the spirit of learning. Students receive information about the implementation of Pancasila from time to time through CFT. 620 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum PHASE TEACHER ACTIVITIES STUDENT ACTIVITIES Phase 3 Form a group or origin group The teacher divides the group heterogeneously with 6 members of the original group using whell of names. Each member of the group is assigned a different sub- subject. (The implementation of Pancasila started from the early days of independence then the old order, the new order era to reform. Learners join members of the origin group. Learners hold one sub theme. 1. Learning Steps Phase 4 Forming an expert group The teacher instructs students who have the same sub-topic to be able to discuss in groups. (Expert group). Students join their own teams or groups of experts. Phase 5 The expert group goes back to the original or original group The teacher allows each student to re-discuss with the initial / original group to explain the material they get. Students return to the original group and then explain the material discussed or obtained from the expert team. COVER Phase 6 Evaluation The teacher allows students to give conclusions. Then give the test covering all the material. Students conclude and then take the test. Phase 7 Giving awards The teacher gives awards to the best group. The best learners receive awards. COVER Pratini (2015) and Mardhiah (2021) where from the results of the study there is a very high influence when learning using the jigsaw type learning model where after using the jigsaw model there is an increase in the quality of learning activities and the value of student learning outcomes. This is also in line with the benefits of the Jigsaw model put forward by (Rosyidah, U, 2016) The benefits obtained if teachers use the jigsaw model in the implementation of learning. Where it can have a positive impact on individuals such as increasing skills, high motivation, preventing conflict, reducing apathy and most importantly can prevent individual aggressiveness in learning. In this learning model related to the implementation of Pancasila from time to time, it is more dominant in developing aspects of civic knowledge and civic skills (Widiatmaka, 2016). Kusnadi, E (2010) suggests that the jigsaw type learning model is very influential in honing students' knowledge or if in PPKn learning it is known as civic knowledge. In addition, it can also increase social motivation so that you can complete the task as soon as possible. In civic education, this jigsaw model is designed to sharpen students' understanding of civic knowledge, having responsibility for learning both to themselves and others (Faujiah, Tafsir, & Sumadi, 2018). Students can work together cooperatively in order to achieve the goals of the material to be discussed, because they not only learn the material but must be able to pass it on to their group members. Every learner will understand each part and if put together will become a complete knowledge or civic knowledge. The jigsaw-type learning model can train students to be positive interdependence, cooperate with their group mates and have responsibility for the material or assignment given to them (Muthi'ah dkk, 2018). Jigsaw learning is also more effective in the cognitive realm where learners are directed to appreciate ideas from others (Koc, 2010). In addition to being related to civic knowledge, this jigsaw model is also capable of making improvements to aspects of civic skills. This is in line with what was stated by Subiyantari, A. R., et.al (2019) the average value of psychomotor domain assessment increases through the jigsaw model, this is because they are accustomed to working individually and in groups. COVER The teacher allows students to give conclusions. Then give the test covering all the material. The best learners receive awards. Jigsaw Type Learning Model on Pancasila Implementation Material from Time to Time aw Type Learning Model on Pancasila Implementation Material from Time to Tim Jigsaw Type Learning Model on Pancasila Implementation Material from Time to Time To innovate in the development of citizenship aspects. Based on the explanation of the theory above which explains that the jigsaw type learning model is a model that has a main point in working in small groups (Gunarta, 2018). This model consists of 5 to 6 learning members who are formed heterogeneously. The existence of independent responsibility and positive dependence between members are things that can be used as advantages of this model. In addition, the opportunity for students to be wide open in expressing opinions to their group members, this can also train students' communication skills. This model is almost the same as the group exchange method where group members will get one part and if combined can produce complete knowledge (Nur Kumala, 2016). The purpose of this model is to train students to be responsible for discussions and to help their friends to understand the material together. This model, which focuses on origin groups and expert groups, is very suitable when used on material that has many subchapters (Sholihah et al., 2018). As well as the material for the implementation of pancasila from time to time which has a subchapter on the implementation of pancasila in the early days of independence 621 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum (which is further divided into several rebellions), then in the old order era, the new order era and the reform era (Salam & Akmal, 2018). This jigsaw model is very helpful for students to better understand the material to be discussed and be responsible for the tasks and material they take. Up to this model will produce students who have a sense of responsibility, independence and can work together positively. This is in line with the results of research conducted by Hj. COVER If related to civic skills or psychomotor, what is observed is the ability of students to deliver learning material to members of their home group (Walad, A. M., et.al, 2019). This model can also support students in solving problems, this is because students if learning with small groups will be more specific. Until finally the jigsaw learning model can make positive and significant changes to the style and values and motivation of learning students (Basyah & Muslem, 2017). CONCLUSION 6 The jigsaw type learning model is a cooperative learning model whose implementation focuses on the origin group and group or expert team. Each member or individual is taught to take responsibility for the submaterial they obtain and can share it with their group mates. This teaches students to have independent responsibility and teaches students to communicate well, therefore this model emphasizes the development of aspects civic knowledge citizenship. 622 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum Then the benefit of this model is to prevent competition and increase collaboration as contained in the independent curriculum. Based on the presentation of theory and some of the topics discussed above, the conclusion that we can draw is that the jigsaw learning model is very suitable for material that has many subchapters, which is in line with the material of pancasila implementation from time to time. The advantages of the jigsaw model are that it provides positive dependence between students, trains responsibility and increases confidence in the task at hand and can increase motivation. In addition, the drawback is that the jigsaw-type cooperative model teachers have to be extra in applying this model, insufficient time and often dominance by one of the students when discussing. Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum To Time Phase D In The Independent Curriculum Kompetensi Abad 21. El-Hikmah: Jurnal Kajian Dan Penelitian Pendidikan Islam, 13(2), 146–167. p , ( ), Widiatmaka, Pipit. (2016). Kendala Pendidikan Kewarganegaraan dalam membangun karakter peserta didik di dalam proses pembelajaran. Jurnal Civics, 13(2), 188–198. Wahyudin, Wahyudin. (2018). Optimalisasi peran kepala sekolah dalam implementasi kurikulum 2013. Jurnal Kependidikan, 6(2), 249–265. Surur, Miftahus. (2021). A. Model Dick And Carey. Perencanaan Pembelajaran, 39. Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum Kompetensi Abad 21. El-Hikmah: Jurnal Kajian Dan Penelitian Pendidikan Islam, 13(2) 146 167 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum Kompetensi Abad 21. El-Hikmah: Jurnal Kajian Dan Penelitian Pendidikan Islam, 13(2), 146–167. Surur, Miftahus. (2021). A. Model Dick And Carey. Perencanaan Pembelajaran, 39. Tias, Tri. (2021). Variasi Permainan Pembelajaran, Metode, dan Ice Breaking. Guepedia. Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum Kompetensi Abad 21. El-Hikmah: Jurnal Kajian Dan Penelitian Pendidikan Islam, 13(2), 146–167. Surur, Miftahus. (2021). A. Model Dick And Carey. Perencanaan Pembelajaran, 39. Tias, Tri. (2021). Variasi Permainan Pembelajaran, Metode, dan Ice Breaking. Guepedia. Wahyudin, Wahyudin. (2018). Optimalisasi peran kepala sekolah dalam implementasi kurikulum 2013. Jurnal Kependidikan, 6(2), 249–265. Widiatmaka, Pipit. (2016). Kendala Pendidikan Kewarganegaraan dalam membangun karakter peserta didik di dalam proses pembelajaran. Jurnal Civics, 13(2), 188–198. BIBLIOGRAPHY Abas, H. Erjati. (2017). Magnet Kepemimpinan Kepala Madrasah Terhadap Kinerja Guru (Revisi) STO Mohon Banyak Disebar Di Lampung. Elex Media Komputindo. Edi, Syamsi. (2022). BAB 4 Modul Ajar. Perencanaan Pembelajaran (Kurikulum Merdeka Belajar), 37. Faujiah, Ajri, Tafsir, Ahmad, & Sumadi, Sumadi. (2018). Pengembangan Karakter Anak Di Indonesia Heritage Foundation (Ihf) Depok. Jurnal Penelitian Pendidikan Islam,[SL], 6(2), 163–186. Gunarta, I. Gd. (2018). Pengaruh Model Pembelajaran TGT Berbantuan Media Question Card Terhadap Hasil Belajar IPA. Jurnal Pedagogi Dan Pembelajaran, 1(2), 112–120. Hidayat, Abdul Salam, Dlis, Firmansyah, & Hanief, Sofyan. (2021). Pengembangan Model Pembelajaran Atletik Nomor Lari Berbasis Permainan pada Siswa Sekolah Dasar. Penerbit CV. Sarnu Untung. Kaid, Lynda Lee, Baehaqie, Imam, & Rizal, M. (2021). Iklan Politik: Handbook Penelitian Komunikasi Politik. Nusamedia. Kumalasari, Intan. (2021). Strategi Pembelajaran Pendidikan Agama Islam Dalam Pembinaan Mental Anak Tunagrahita di Sekolah Inklusif Kota Medan. Universitas Islam Negeri Sumatera Utara. Lahir, Sri, Ma’ruf, Muhammad Hasan, & Tho’in, Muhammad. (2017). Peningkatan prestasi belajar melalui model pembelajaran yang tepat pada sekolah dasar sampai perguruan tinggi. Jurnal Ilmiah Edunomika, 1(01). gg Manasikana, Arina, & Anggraeni, Candra Widhi. (2018). Pendidikan karakter dan mutu pendidikan indonesia. Seminar Nasional Pendidikan 2018. Mirdanda, Arsyi. (2019). Mengelola aktivitas pembelajaran di sekolah dasar. PGRI Kalbar dan Yudha English Gallery. Muaddab, Hafis. (2018). Memfollow Sang Presiden. Tebuireng Institute. Nur Kumala, Farida. (2016). Pembelajaran IPA SD. Ediide Indografika. Rahman, Taufiqur. (2018). Aplikasi model-model pembelajaran dalam penelitian tindakan kelas. CV. Pilar Nusantara. Rahmat, Fitriyane Laila Apriliani, Suwatno, Rasto, & Rasto, R. (2018). Meningkatkan Pemahaman Konsep Siswa Melalui Teams Games Tournament. Sosio Didaktika: Social Science Education Journal, 5(1), 15–23. Salam, Aprinus, & Akmal, Ramayda. (2018). Pahlawan dan pecundang militer dalam novel- novel Indonesia. UGM Press. Sholihah, Hayu Almar’atus, Koeswardani, Nurul Fiadhia, & Fitriana, Visca Kenia. (2018). Metode pembelajaran jigsaw dalam meningkatkan keterampilan komunikasi siswa SMP. KoPeN: Konferensi Pendidikan Nasional, 1(1), 160–167. f ( ) Sumantri, Budi Agus. (2019). Pengembangan Kurikulum di Indonesia Menghadapi Tuntutan 623 Analysis Of The Jigsaw Learning Model On Pancasila Implementation Material From Time To Time Phase D In The Independent Curriculum Kompetensi Abad 21. El-Hikmah: Jurnal Kajian Dan Penelitian Pendidikan Islam, 13(2), 146–167. ( ) Surur, Miftahus. (2021). A. Model Dick And Carey. Perencanaan Pembelajaran, 39. Wahyudin, Wahyudin. (2018). Optimalisasi peran kepala sekolah dalam implementasi kurikulum 2013. Jurnal Kependidikan, 6(2), 249–265. p ( ) Widiatmaka, Pipit. (2016). Kendala Pendidikan Kewarganegaraan dalam membangun karakter peserta didik di dalam proses pembelajaran. Jurnal Civics, 13(2), 188–198. 624 624
https://openalex.org/W2072630178	https://research-repository.st-andrews.ac.uk/bitstream/10023/12523/1/Chudzinska_2015_Oikos_Habitatselection_CC.pdf	English	null	Using habitat selection theories to predict the spatiotemporal distribution of migratory birds during stopover – a case study of pink‐footed geese <i>Anser brachyrhynchus</i>	Oikos	2,015	cc-by	9,780	Magda E. Chudzi n´ ska , Floris M. van Beest , Jesper Madsen and Jacob Nabe-Nielsen Birds must accumulate fat to fuel their migration and breeding activities as well as protein to gain muscle strength and produce eggs (McDonald et al. 1973, Robbins 1993). A common migration strategy is to build up and maintain suﬃ cient nutritional reserves (protein and fat) by foraging on stopover sites in order to arrive at the breeding areas in near-optimal breeding condition – a strategy termed capital breeding (Drent et al. 1978, Klaassen et al. 2006, Stephens et al. 2014). In most heterogeneous landscapes, however, the availability and quality of food resources varies both spatially and temporally. Besides the nutritional con- tent of the available food resources, other factors such as Arctic-nesting birds have a limited time to prepare for breeding as the time window when conditions are suitable for migration and for breeding is often narrow (Alerstam and Lindstr ö m 1990, Prop and Black 1998, Drent et al. 2003). Birds must accumulate fat to fuel their migration and breeding activities as well as protein to gain muscle strength and produce eggs (McDonald et al. 1973, Robbins 1993). A common migration strategy is to build up and maintain suﬃ cient nutritional reserves (protein and fat) by foraging on stopover sites in order to arrive at the breeding areas in near-optimal breeding condition – a strategy termed capital breeding (Drent et al. 1978, Klaassen et al. 2006, Stephens et al. 2014). In most heterogeneous landscapes, however, the availability and quality of food resources varies both spatially and temporally. Besides the nutritional con- tent of the available food resources, other factors such as the costs related to searching and movement, predation or disturbance risk, and inter- and intraspeciﬁ c competition can also greatly inﬂ uence the spatiotemporal distribution of species (Sih 1980, Amano et al. 2006a, Sunde and Redpath 2006, van Beest et al. 2014). Th is process, termed habitat selection, is thus the outcome of tradeoﬀ s between the costs and beneﬁ ts in selecting certain foraging sites over others (Mysterud and Ims 1998, Godvik et al. 2009). To explain how tradeoﬀ s in habitat selection of animals emerge, observed patterns are often tested against predic- tions derived from a speciﬁ c theoretical model. Th e most commonly employed theories within a habitat selection framework are: maximising nutrient intake (Lindstr ö m 1991, Hedenstr ö m and Alerstam 1997), density-dependent habitat selection (Rosenzweig 1981, Shochat et al. © 2015 Th e Authors. Th is an Online Open article Subject Editor: Rob Robinson. Editor-in-Chief: Dries Bonte. Accepted 1 December 2014 Oikos 124: 851–860, 2015 doi: 10.1111/oik.01881 © 2015 Th e Authors. Th is an Online Open article Subject Editor: Rob Robinson. Editor-in-Chief: Dries Bonte. Accepted 1 December 2014 Oikos 124: 851–860, 2015 doi: 10.1111/oik.01881 © 2015 Th e Authors. Th is an Online Open article Subject Editor: Rob Robinson. Editor-in-Chief: Dries Bonte. Accepted 1 December 2014 doi: 10.1111/oik.01881 Magda E. Chudzi n´ ska , Floris M. van Beest , Jesper Madsen and Jacob Nabe-Nielsen M. E. Chudzi ń ska (chudzinskam@gmail.com), F. M. van Beest and J. Nabe-Nielsen, Dept of Bioscience, Aarhus Univ., Frederiksborgvej 399, DK-4000 Roskilde, Denmark. – J. Madsen, Dept of Bioscience, Aarhus Univ., Gren å vej 14, DK-8410 R ø nde, Denmark. Understanding how animals select for habitat and foraging resources therein is a crucial component of basic and applied ecology. Th e selection process is typically inﬂ uenced by a variety of environmental conditions including the spatial and temporal variation in the quantity and quality of food resources, predation or disturbance risks, and inter- and intraspe- ciﬁ c competition. Indeed, some of the most commonly employed ecological theories used to describe how animals choose foraging sites are: nutrient intake maximisation, density-dependent habitat selection, central-place foraging, and predation risk eﬀ ects. Even though these theories are not mutually exclusive, rarely are multiple theoretical models considered con- comitantly to assess which theory, or combination thereof, best predicts observed changes in habitat selection over space and time. Here, we tested which of the above theories best-predicted habitat selection of Svalbard-breeding pink-footed geese at their main spring migration stopover site in mid-Norway by computing a series of resource selection functions (RSFs) and their predictive ability ( k -fold cross validation scores). At this stopover site geese fuel intensively as a preparation for breeding and further migration. We found that the predation risk model and a combination of the density-dependent and central-place foraging models best-predicted habitat selection during stopover as geese selected for larger ﬁ elds where predation risk is typically lower and selection for foraging sites changed as a function of both distance to the roost site (i.e. central-place) and changes in local density. In contrast to many other studies, the nutritional value of the available food resources did not appear to be a major limiting factor as geese used diﬀ erent food resources proportional to their availability. Our study shows that in an agricultural landscape where nutritional value of food resources is homogeneously high and resource availability changes rapidly; foraging behaviour of geese is largely a tradeoﬀ between fast refuelling and disturbance/predator avoidance. Arctic-nesting birds have a limited time to prepare for breeding as the time window when conditions are suitable for migration and for breeding is often narrow (Alerstam and Lindstr ö m 1990, Prop and Black 1998, Drent et al. 2003). Th is 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. Using habitat selection theories to predict the spatiotemporal distribution of migratory birds during stopover – a case study of pink-footed geese Anser brachyrhynchus Magda E. Chudzi n´ ska , Floris M. van Beest , Jesper Madsen and Jacob Nabe-Nielsen Magda E. Chudzi n´ ska , Floris M. van Beest , Jesper Madsen and Jacob Nabe-Nielsen 2002, McLoughlin et al. 2010), central-place foraging (Orians and Pearson 1979, Rosenberg and McKelvey 1999, van Gils and Tijsen 2007), and predation risk eﬀ ects (Lima and Dill 1990, Lindstr ö m 1990, Creel et al. 2005). Although not mutually exclusive, rarely are multiple theories considered simultaneously to evaluate which framework, or combinations Th is 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. 851 of frameworks, best predict the observed habitat selection patterns of the study species. However, such tests are impor- tant as accurately predicting habitat selection patterns is a prerequisite for eﬀ ective management and conservation of species (Boyce and McDonald 1999). distance from the central-place increases (Schoener 1979). When applying this theory to a setting where local density changes rapidly but predictably (as for roosting birds at migratory stopover sites) animals should alter selection based on both the distance from the focal point (roost site) as well as temporal changes in the number of conspeciﬁ cs present. For birds migrating in steps, such as pink-footed geese Anser brachyrhynchus , stopover sites along the migration route are used shortly (in comparison to e.g. wintering areas) but intensively (Madsen et al. 1999). Based on a simple maximising rule, geese would be expected to focus their habitat selection purely on food resources that provide the greatest energy/protein return. However, many individual geese typically use stopover sites at the same time. As such, density related processes, such as intraspeciﬁ c resource com- petition, could seriously aﬀ ect the habitat selection process. Following density-dependent habitat selection theory, selec- tion for high quality resources should be strongest during periods of low intraspeciﬁ c competition (e.g. when the ﬁ rst geese arrive at stopover sites) while the strength of selection for quality resources should weaken as intraspeciﬁ c competi- tion increases (e.g. as more geese arrive at the stopover site) (McLoughlin et al. 2010, van Beest et al. 2014). p g p p Besides the nutritional value of a site and the number of conspeciﬁ cs competing for the same food resources around a central-place, predation risk and disturbance pressures within the landscape can also be crucial drivers of habitat selection (Manly et al. 1993, Madsen 1994). Study population and site Th e Svalbard-breeding population of pink-footed geese over- winters in Belgium, the Netherlands and Denmark. During Table 1. An overview of the considered resource selection function (RSF) models with the corresponding predictor variables and predictions for habitat selection of pink-footed geese at the spring migration stopover site in mid-Norway. Table 1. An overview of the considered resource selection function (RSF) models with the corresponding predic for habitat selection of pink-footed geese at the spring migration stopover site in mid-Norway. Models and the used parameters Predictions Maximising nutrient intake Food resources Geese select strongest for food resources with the highest nutritional value Density-dependent habitat selection (DD) Time Selection of foraging sites varies with temporal changes in goose density during the day Time Food resources Selection for the highest nutritional resources weaken as goose density increases (morning and evening) Central-place foraging (CPF) Distance to roost Geese reduce selection of foraging sites with increasing distance from roosts Distance to roost Food resources Geese increase selection for the highest nutritional food resources as the distance from roost sites increases while showing no distinct selection for food resources close to roost sites Predation/disturbance risk (PD) Field area Geese select strongest for foraging sites in larger ﬁ elds Field area Food resources Geese select strongest for foraging sites in larger ﬁ elds that contain the highest nutritional value DD and CPF Time Distance to roost Geese reduce selection of foraging sites at increasing distance from roost sites when the density of geese around the roosts is low (middays) and increase selection of foraging sites at increasing distance from roost sites when the density around the roosts is high (morning and afternoon) DD and PD Time Field area Geese increase selection of foraging sites in larger ﬁ elds when the density of geese is low (middays) and decrease selection of foraging sites in larger ﬁ elds when the density is high (morning and afternoon) PD and CPF Field area Distance to roost Geese increase selection of foraging sites in larger ﬁ elds at increasing distance from roost sites Predictions Magda E. Chudzi n´ ska , Floris M. van Beest , Jesper Madsen and Jacob Nabe-Nielsen Indeed, mini- mising predation risk is considered an important determi- nant of behaviour of migratory birds (Hedenstr ö m and Alerstam 1997, Weber et al. 1998, Jonker et al. 2010, Chudzi ń ska et al. 2013). Increased disturbance or predation risk may force individuals to increase selection of marginal habitat with reduced forage but greater cover or alternatively to congregate at larger ﬁ elds where predators are more easily detected (Amano et al. 2006a). In this study we quantiﬁ ed population-level habitat selec- tion of pink-footed geese at their major spring-migration stopover site in mid-Norway. Our aim was to evaluate which of the aforementioned theories, or a combination thereof (Table 1), best predicted the spatial distribution of pink- footed geese at their stopover site in mid-Norway as quanti- ﬁ ed by a series of resource selection functions (RSFs). A special case of the general optimal foraging theory is the central-place foraging theory. Th is framework may be espe- cially valuable to explain habitat selection patterns of geese at stopover sites as they frequently congregate at roosting areas during night and some periods of the day to rest and digest. A typical feature of central-place foraging is a declining prob- ability of use of sites at increasing distance from the focal point (Rosenberg and McKelvey 1999). Moreover, classic central-place foragers are expected to show no distinct selec- tion for food resources close to the focal point (where compe- tition for food is greatest and resource depletion most likely) and increase selection of high-quality food resources as the Geese select strongest for food resources with the highest nutritional value Selection of foraging sites varies with temporal changes in goose density during the day Selection for the highest nutritional resources weaken as goose density increases (morning and evening) Selection of foraging sites varies with temporal changes in goose density during the day Selection for the highest nutritional resources weaken as goose density increases (morning and evening) Geese reduce selection of foraging sites with increasing distance from roosts Geese increase selection for the highest nutritional food resources as the distance from roost sites increases while showing no distinct selection for food resources close to roost sites Geese increase selection of foraging sites in larger ﬁ elds when the density of geese is low (middays) and decrease selection of foraging sites in larger ﬁ elds when the density is high (morning and afternoon) Geese increase selection of foraging sites in larger ﬁ elds at increasing distance from roost sites 852 time. Geese use roost sites mainly at night but also parts of the day, particularly around middays (Madsen et al. 1997). Th e geese start arriving in mid-Norway in early April, and numbers peak during late April – early May (Madsen et al. 1999). Individual geese stay in mid-Norway for an average of 20 days before migrating further north (Bauer et al. 2008) (Fig. 2A). h their migration to the breeding grounds, the geese stop in Tr ø ndelag in mid-Norway, and Vester å len in north-Norway (Madsen et al. 1999). Mid-Norway is semi-mountainous and characterized by a patchwork of agricultural ﬁ elds and for- ests. Th e area is rich in lakes and coastal areas, both of which serve as roost sites for the geese (Fig. 1). Geese are rarely seen resting on the ﬁ elds and therefore the above-mentioned roosting sites constitute their main resting places. Roosting sites are also the main sources of drinking water for geese and usually located in the remote places, further away from settlements and roads. Th e location of roosts sites is mapped every year by trained observers and appears constant over g Th ere are four main food resources available to geese in this area: grass, barley stubble from the preceding autumn, newly sown/germinating barley grains, and ploughed barley stubble. Th ese four food resources are henceforth referred to as grass, stubble, grain and ploughed. Geese select strongest for food resources with the highest nutritional value (A) Changes in number of pink-footed geese during spring stopover season in mid-Norway. Due to changes in food resource availability, the stopover season was divided into four periods (P1 – P4) in order to study within seasonal changes in habitat selection of geese. (B) Average diurnal changes in density of geese (km 2 ) observed foraging on ﬁ elds. Th e solid red line shows the ﬁ tted values from a generalized additive model (GAM: F 1,16 3.53, p 0.03) using a non-parametric smoother function and the dotted red lines represent the 95% conﬁ dence interval. dom design to determine the availability of food resources at diﬀ erent distances from roost sites and to determine how the use of the ﬁ elds changed during the stopover season. Th e season was divided into four periods of approximately 8 days based on the frequency of food resources map- ping surveys. Th e periods roughly corresponded to habitat changes due to agricultural practices. Period 1 lasted from 15 – 25 April, period 2 from 26 April – 3 May, period 3 from 4 – 11 May and period 4 from 12 – 19 May. We randomly selected 900 ﬁ elds out of 2900 available for each period, which were equally distributed between distances to the roost sites (0 – 1; 1 – 2; 2 – 5 km) and within the study site. In order to get an equal spatial representation of the study area, the survey area was sub-divided into 28 squares of 5 5 km which contained all the selected ﬁ elds (squares which only contained water were excluded). Each square was visited once per period. During each period the squares were visited in a random order, and all the randomly selected ﬁ elds and those directly bordering these were surveyed. Each visited ﬁ eld was classiﬁ ed as grass, stubble, grain or ploughed and it was noted if geese were present at the time. Flock size was counted to nearest 10 individuals. Th e mapping eﬀ ort was equally distributed between 05:00 – 21:00 hours. For further analysis we merged all neighbouring ﬁ elds with the same food resource that were not separated by any physical object (road, forest etc.). Merging of the ﬁ elds, calculation of distances to the closest roost sites and ﬁ eld area were per- formed in ArcGIS ver. 10.1 (ESRI 2010 <www.esri.com>). Geese select strongest for food resources with the highest nutritional value Th e geese occasionally 853 p of the study area (28 33 km) in nord-Tr ø ndelag, mid-Norway, showing all the recorded agricultural ﬁ elds (grey polygons) rom roost sites (black squares). Water is marked with light blue. Th e map in the upper left corner shows the spring migration -footed geese with the mid-Norway study area marked in red and north-Norway stopover site in black. Figure 1. Map of the study area (28 33 km) in nord-Tr ø ndelag, mid-Norway, showing all the recorded agricultural ﬁ elds (grey within 5 km from roost sites (black squares). Water is marked with light blue. Th e map in the upper left corner shows the spring route of pink-footed geese with the mid-Norway study area marked in red and north-Norway stopover site in black. Figure 1. Map of the study area (28 33 km) in nord-Tr ø ndelag, mid-Norway, showing all the recorded agricultural ﬁ elds (grey polygons) within 5 km from roost sites (black squares). Water is marked with light blue. Th e map in the upper left corner shows the spring migration route of pink-footed geese with the mid-Norway study area marked in red and north-Norway stopover site in black. Figure 1. Map of the study area (2833 km) in nord-Trøndelag, mid-Norway, showing all the recorded agricultural ﬁel Figure 1. Map of the study area (28 33 km) in nord-Tr ø ndelag, mid-Norway, showing all the recorded agricultural ﬁ elds (grey polygons) within 5 km from roost sites (black squares). Water is marked with light blue. Th e map in the upper left corner shows the spring migration route of pink-footed geese with the mid-Norway study area marked in red and north-Norway stopover site in black. 853 Figure 2. (A) Changes in number of pink-footed geese during spring stopover season in mid-Norway. Due to changes in food resource availability, the stopover season was divided into four periods (P1 – P4) in order to study within seasonal changes in habitat selection of geese. (B) Average diurnal changes in density of geese (km 2 ) observed foraging on ﬁ elds. Th e solid red line shows the ﬁ tted values from a generalized additive model (GAM: F 1,16 3.53, p 0.03) using a non-parametric smoother function and the dotted red lines represent the 95% conﬁ dence interval. Figure 2. Geese select strongest for food resources with the highest nutritional value Th e average ﬁ eld size after merging was 0.3 km 2 . forage on waste potato ﬁ elds but this food resource is very scarce in the study area. Time-activity budgets for pink- footed geese in mid-Norway revealed that ploughed ﬁ elds are mainly used as resting sites (M. Chudzinska and B. A. Nolet unpubl.). Grass starts growing at the end of April and is readily available during the entire stopover season (Bjerke et al. 2014). Stubble ﬁ elds are gradually ploughed and subse- quently sown with barley, which starts germinating towards the end of the stopover season in mid-May (Madsen et al. 1997). Stubble is the most energetic food resource of all widely available food resources and is 2.8 times more ener- getic than grain and grass, which are comparable in energet- ics (Chudzinska et al. unpubl.). Grass, however, is the most protein-rich food resource out of all available at the study site (Prop and Spaans 2004). In mid-Norway geese are occasionally hunted by white- tailed eagles Haliaeetus albicilla (Madsen et al. 1998), but human disturbance (which can also be regarded as preda- tion risk; Tombre et al. 2005, Klaassen et al. 2006) is more frequent at the study site and is believed to be the main risk factor perceived by geese (Madsen 1998). Sampling of goose presence and food resource availability Because the availability of food resources in our study site changes dynamically from year to year and within the season we could not rely on standard land cover maps of mid-Nor- way as they would likely be incorrect at the resolution neces- sary here. Map errors can seriously bias estimates of habitat selection (Johnson and Gillingham 2008) and we therefore mapped the availability of the diﬀ erent food resources manu- ally within a 28 33 km area between Steinkjer, Verdal and Inder ø y municipalities, which we believe is representative for the mid-Norway stopover site. Arable land (crops and pastures) constituted 12% of the study area and was the only habitat type where geese were observed foraging. Mountain areas/forests (55%) and water (mainly fj ords and lakes; 31%) were dominant habitat types at the study site. Settle- ments and roads made up 2% of the study area. In order to map the study area, we selected all agricultural ﬁ elds 5 km from the roost sites, which is the area where most geese forage (Jensen et al. 2008) and all parts were accessible to geese (Fig. 1). For these ﬁ elds we applied a stratiﬁ ed ran- Resource selection functions (RSFs) Our maximising nutrient intake RSF model included only food resources (four-class categorical variable: grain, grass, ploughed and stubble, with stubble being a reference category because this is the most energetic food resource) as the only independent variable. Th e number of food resources considered in the RSFs diﬀ ered between periods, as grain was not present during period 1. Our densi- ty-dependent (DD) habitat selection RSFs included time of day (both linear and squared) as the only explanatory vari- able because the majority of geese forage in the mornings and evenings and spent time on a roost site around midday (Madsen et al . 1997, Chudzi ń ska et al. 2013). Th is allowed us to use time of day as a proxy for goose density as the number of geese foraging on ﬁ elds will vary during a day with highest density in the mornings and evenings and low- est around midday (Fig. 2B). Using time of day as proxy for density instead of using goose density/abundance directly in the RSF allowed us to determine whether goose behaviour diﬀ ers during periods when density is decreasing (morning – when competition declines) or increasing (evening – when competition rises) (Fig. 2B). Using goose density/abundance directly might mask such behavioural diﬀ erences over time when density/abundance is equal (e.g. at 9:00 am and 17:00 pm: Fig. 2B). We let time of day interact with food resources as an additional density-dependent RSF model. Our central- place foraging (CPF) model used distance to roost site as y ( ) In order to successfully manage species and their envi- ronment, a prerequisite is to accurately predict their habitat selection patterns (Boyce and McDonald 1999). As such, and to determine which RSF model best predicted habitat selection patterns, we employed the k -fold cross-validation procedure as proposed by Fielding and Bell (1997) and Boyce et al. (2002). Th is procedure withholds a fraction of the data using a k -fold partitioning, where k represents the number of partitions (so called test-training sets) (Fielding and Bell 1997). To examine model performance, the pattern of predicted RSF scores for partitioned test-training sets is investigated against categories of RSF scores (bins) (Boyce et al. 2002). For the k -fold cross-validation procedure, we calculated Spearman rank correlations (r s ) between ten RSF- bin ranks and ﬁ ve test-training sets (k 5). Resource selection functions (RSFs) Moreover, by separating all used sites from the availability sample we removed any potential contamination of the data (i.e. when available sites are in fact used), which leads to robust RSFs and facilitates model inference (Johnson et al. 2006). Our RSF analyses corresponded most closely to that of the landscape-scale, equivalent to the second-order selec- tion of Johnson (1980), and were based on population-level use – availability sampling designs (Design I; Th omas and Taylor 2006). RSFs were computed using generalised lin- ear models (GLMs) for binomial data, performed in R 3.0.1 ( www.r-project.org/ ). Period 1 Period 2 Period 3 Period 4 Sum(unique) Use 36 60 80 69 189 Availability 912 1260 1079 1175 2184 Th e outcome of our RSFs (logistic regression using GLM) are a population level estimate of the log-odds ratio of using a site or given resource in the study area. Because we employed used vs available sampling design we could not derive absolute probabilities of use (as in RSPFs) and we calculated the odds ratio of selection (relative probability of use) instead. By taking the log of the odds ratio the outcome becomes more intuitive, as any value below 0 indicates that a given site or resource unit is used less than available, while any value above 0 reﬂ ects that a given resource unit is used more than available. Values overlapping with 0 indicate that use of a given resource unit is proportional to availability (i.e. no selection). Indeed, calculation of log-odds ratios of a resource unit (a continuous variable) or relative to a reference level (for a categorical variable) is informative and common in RSF analyses (Godvik et al. 2009, Blix et al. 2014). p j g Because availability of the diﬀ erent food resources changed over the stopover season, we constructed separate RSFs for each period to exclude potential bias of functional responses in habitat selection (Mysterud and Ims 1998). For each period, we considered ten diﬀ erent RSFs, which were constructed a priori and speciﬁ cally followed predictions of habitat selection theory (maximising nutrient intake, cen- tral-place foraging, predation and density frameworks and a combination of these frameworks as explained in the Intro- duction and Table 1). Resource selection functions (RSFs) We repeated this procedure 100 times to determine whether the r s were sig- niﬁ cantly diﬀ erent from random (t-test). To identify which models best predicted habitat selection, we considered for each period the two models with the highest r s value. We chose the k -fold cross-validation procedure over a model selection procedure based on AIC (Akaike 1987), as AIC values are a tradeoﬀ between the goodness of ﬁ t and the complexity of the model rather than a degree of prediction accuracy. Resource selection functions (RSFs) We estimated habitat selection of geese by modelling resource selection functions (RSFs), deﬁ ned as any function that is proportional to the probability of use by an organism (Manly et al. 1993). RSFs compare environmental condi- tions at animal locations (e.g. food resource, density, distance to roost) to the attributes at random (i.e. available) points (Boyce and McDonald 1999, Manly et al. 2002). In our RSFs the used points were all the ﬁ elds where goose presence was observed during the survey and available points were all the remaining surveyed ﬁ elds (randomly selected ﬁ elds and all the ﬁ elds directly bordering the random ﬁ elds) (Table 2). Although such a sampling design resembles that of used vs 854 explanatory variable, in addition to the interactions with food resources in a second CPF model. Our predation and disturbance (PD) RSF model was constructed using ﬁ eld area size as the major explanatory variable, as we assumed that larger ﬁ elds are less disturbed and make it easier for geese to detect potential predators. We also let ﬁ eld area interact with food resources as an additional predation/disturbance risk RSF model. Finally, we ﬁ tted three RSFs models using com- binations of the diﬀ erent habitat selection frameworks. As such, our DD and CPF RSF model included an interaction between time of day and distance to roost. Our DD and PD model included an interaction between time of day and ﬁ eld area and ﬁ nally our PD and CPF model included the inter- action between ﬁ eld area and distance to roost (Table 1). h Table 2. The number of ‘ used ’ and ‘ available ’ sites surveyed during the four deﬁ ned periods within the spring stopover season of pink- footed geese in mid-Norway. ‘ Sum(unique) ’ refers to a number of unique ﬁ elds visited over the four periods. Period 1 Period 2 Period 3 Period 4 Sum(unique) Use 36 60 80 69 189 Availability 912 1260 1079 1175 2184 unused data, which may be modelled using a resource selec- tion probability function (RSPFs), we could not be certain that the unused sites in our dataset remained so during other times of the day or season outside our ﬁ eld visits. As such, we considered these ﬁ elds ‘ available ’ rather than ‘ unused ’ and continued our analyses with RSFs rather than RSPFs. Results Grain, grass, stubble and ploughed were dominant food resources of geese within the study area (Fig. 3A). Fields with waste potatoes constituted only 0.2% of the area 855 Figure 3. Proportion of four dominant food resources available (A) and used (B) by pink-footed geese at the spring migration stopover site in mid-Norway. Figure 3. Proportion of four dominant food resources available (A) and used (B) by pink-footed geese at the spring migration stopover site in mid-Norway. ratio] close to roost log[odds ratio] away from roost , Fig. 5A). Further- more, the relative probability of observing geese declined as distance from roost sites increased, a pattern that was evident for all times of the day. During period 2 and 3 when densi- ties of geese at the stopover site were highest, geese used areas close to the roost sites proportional to availability regardless of time of day (log [odds ratio] is close to 0 at distance close to roost sites for all time of day: Fig. 5B – C). During the same periods, the relative probability of using areas further from the roost sites declined in the mornings (log[odds ratio] 0 at distances further away from roost sites) but increased in the evenings (log[odds ratio] 0 as distance from roost sites increased: Fig. 5 B – C). covered by the studied agricultural ﬁ elds. Th e availability of grass ﬁ elds did not change during the study period and con- sisted on average 36% of all available food resources. Th e proportion of stubble decreased over the season and was almost absent in period 3 and 4. Th e availability of ploughed ﬁ elds also decreased when the season progressed. Grain was absent in the ﬁ rst period; then its availability increased, to become dominant in period 3 and 4 (Fig. 3A). Our sur- vey data suggested that, for each period, use of the diﬀ er- ent main foraging food resources (grain, grass and stubble) by geese did not diﬀ er from availability and that ploughed ﬁ elds were the only food resource that was used less than expected based on its availability (log-likelihood χ 2 -statistics (Manly et al. Results 2002): period 1: χ L 2 0.01, p 0.99, period 2: χ L 2 1.09, p 0.58, period 3: χ L 2 0.64, p 0.73, period 4: χ L 2 4.32, p 0.12) (Fig. 3A – B). Th is indicates that food resources alone did not determine habitat selec- tion of geese. Th is is corroborated by the fairly low k -fold cross validation values for the maximizing nutrient intake RSF models (Table 3). Discussion For each period, the models that best and second best predicted habitat selection of pink-footed geese at their mid-Norway stopover site are shown in bold and italics respectively. k -fold cross-validation values were calculated using 10 RSF bins and 5 test-training sets. This procedure was repeated 100 times. n.par shows number of parameters in the RSF model. Note that the number of param- eters used in the same model differed between periods as the number of available food resources varied between periods. All r s -values were statistically different from random at p 0.05 unless indicated by a superscript. Table 3. Overview of the k -fold cross-validation procedure showing the Spearman-rank correlations ( r s ) for all RSF models. For each period, the models that best and second best predicted habitat selection of pink-footed geese at their mid-Norway stopover site are shown in bold and italics respectively. k -fold cross-validation values were calculated using 10 RSF bins and 5 test-training sets. This procedure was repeated 100 times. n.par shows number of parameters in the RSF model. Note that the number of param- eters used in the same model differed between periods as the number of available food resources varied between periods. All r s -values were statistically different from random at p 0.05 unless indicated by a superscript. Discussion Period 1 Period 2 Period 3 Period 4 n.par r s n.par r s n.par r s n.par r s Maximising nutrient intake Food resources 3 0.61 * 4 0.55 4 0.60 * ,1 4 0.41 Density-dependent habitat selection (DD) Time 3 0.47 3 0.42 3 0.67 3 0.21 Time Food resources 9 0.50 12 0.42 12 0.63 12 0.60 Central-place foraging (CPF) Distance to roost 2 0.52 2 0.56 2 0.61 2 0.55 Distance to roost Food resources 6 0.52 8 0.63 8 0.58 8 0.65 Predation/disturbance risk (PD) Field area 2 0.63 2 0.74 2 0.80 2 0.81 Field area Food resources 6 0.54 8 0.65 8 0.71 8 0.73 DD and CPF Time Distance to roost 6 0.68 6 0.73 6 0.77 6 0.59 DD and PD Time Field area 6 0.55 6 0.69 6 0.70 6 0.69 PD and CPF Field area Distance to roost 4 0.59 4 0.48 4 0.65 4 0.70 * k -fold value were calculated using 5 bins and 50 test-training sets as models did not converge using the settings employed in the other RSFs. 1 p 0.02 available in the landscape as stubble and grain are a good source of energy and grass is rich in protein. As such, choos- ing one forage resource over the other might not provide much beneﬁ t in terms of nutrient gain. Furthermore, the geese are foraging in a highly dynamic, agricultural land- scape where the food type on a given patch may change from one day to the next. In such a scenario, where indi- viduals are unlikely to have suﬃ cient knowledge about the environment, choosing patches at random may be more beneﬁ cial than returning to a previously visited patch as it reduces searching time and energy expenditure (Amano et al. 2006a, b). Similar random choice of foraging habitat has been found for other species living in highly dynamic landscapes (e.g. fallow deer Dama dama : Focardi et al. 1996) . Th e only ﬁ elds that geese used less than proportional were barren ploughed. Th is is, however, to be expected, as there is no energetic beneﬁ t from spending time on this food resource. h Th e predation/disturbance risk model including ﬁ eld size as the main explanatory variable best predicted habitat selection of geese for almost the complete stopover season. Discussion All theory-based habitat selection models considered here were able to predict the spatiotemporal distribution of migratory pink-footed geese at their spring stopover site in mid-Norway. However, predictive performance varied sub- stantially between models and temporal scales (both within stopover season as well as on a diurnal basis). We found most support for the predation/disturbance risk model and for the combined density-dependent and central-place foraging models. Th e k -fold cross validation score of all RSFs (r s and p-values of Spearman rank correlations) was signiﬁ cantly higher than expected from random, though we observed substantial variation in predictive performance among mod- els. Th e predation/disturbance model (PD) including only ﬁ eld area was the model with the highest predictive capac- ity for period 2 to 4 (mean r s 0.78, p 0.001, Table 3). Overall, larger ﬁ elds were used more than available and this tendency did not change between period 2 – 4, although geese selected larger ﬁ elds in period 4 than in the previ- ous periods (Fig. 4). Th e density-dependent RSF model (DD) and the central-place foraging model (CPF) did not predict habitat selection very well independently, but the combination of these models was the best predictor for period 1 and did rank among the two top models during period 2 – 3 (mean r s 0.73, p 0.001, Table 3). During the ﬁ rst period, the relative probability of observing geese was highest close to the roost site, as expected by CPF framework (log[odds Capital breeders, such as pink-footed geese, are expected to maximise nutrient (fat and protein) intake along the migra- tion route in preparation of the upcoming breeding period. Nevertheless, our habitat selection model based purely on maximising nutrient intake rules did not appear among the top-ranked models. Th is suggests that the nutritional value of the available food resources is not a major limiting factor of the habitat selection patterns of geese in this area. We found that geese used most food resources proportional to their availability, indicating that foraging sites were chosen independently of food quality. Th is ﬁ nding is likely inﬂ uenced by the overall high quality of the food resources 856 Table 3. Overview of the k -fold cross-validation procedure showing the Spearman-rank correlations ( r s ) for all RSF models. Discussion Th is ﬁ nding corroborates previous results that the behaviour of pink-footed geese along the complete migration route is strongly inﬂ uenced by disturbances (Madsen 1994, 1998, Chudzi ń ska et al. 2013, Simonsen 2014). Indeed, distur- bance and related predator avoidance behaviour is known to be an important factors shaping habitat selection of variety of species across diﬀ erent taxa (Blaustein et al. 2004, Pierce et al. 2004, Creel et al. 2005). Th ere are many sources of dis- turbances in our study area (e.g. passing cars, small planes, intentional scaring) and disturbance levels are typically lower on large ﬁ elds compared to smaller ﬁ elds (Amano et al. 2006a). Moreover, foraging on large open ﬁ elds is considered an eﬀ ective antipredator strategy in many species (e.g. white- fronted geese Anser albifrons , Amano et al. 2006a; mule deer Odocoileus hemionus , Altendorf et al. 2001). h Figure 4. Estimates of selection for ﬁ eld area during periods 2 – 4 (log odds ratio 95% conﬁ dence intervals) by pink-footed geese at the mid-Norway stopover site as computed by the predation/dis- turbance risk RSF model with ﬁ eld area as a the only predictor variable. Black line: period 2; red line: period 3; blue line: period 4. Estimates overlapping 0 (grey, horizontal line) indicate that the use is proportional to the availability ( ‘ no selection ’ ), estimates higher than 0 indicate use higher than availability whereas values below 0 indicate use below availability for a given site. Figure 4. Estimates of selection for ﬁ eld area during periods 2 – 4 (log odds ratio 95% conﬁ dence intervals) by pink-footed geese at the mid-Norway stopover site as computed by the predation/dis- turbance risk RSF model with ﬁ eld area as a the only predictor variable. Black line: period 2; red line: period 3; blue line: period 4. Estimates overlapping 0 (grey, horizontal line) indicate that the use is proportional to the availability ( ‘ no selection ’ ), estimates higher than 0 indicate use higher than availability whereas values below 0 indicate use below availability for a given site. Figure 4. Discussion Although the predictive performance of the central-place foraging and the density-dependent RSF models did not rank among the highest, the combination of these models revealed that variation in both local density and distance from the focal point (roost site) do inﬂ uence habitat selec- tion of geese both over the stopover season and during a day. In the mornings, when all geese leave roost sites to for- age, geese behaved like ‘ classical ’ central-place foragers, with a higher relative probability to stay close to the roost. At middays, when densities on ﬁ elds were lowest, geese used habitat independently of the distance to the nearest roost site. In the evenings, when densities increased again, geese selected areas further away from the roosts. If many birds are searching for suitable foraging patches around the same focal area, birds that depart from roost sites later in a day may be unable to ﬁ nd an unoccupied patch close to this area and are subsequently forced to select areas further away. We suggest that the cumulative eﬀ ect of increasing densities and intraspeciﬁ c competition over the day may force birds to search for available foraging sites further away from roost sites in the evening. Th e predictive capacity of the com- bined central-place foraging and density-dependent models was high only during the periods when the population was growing and therefore intraspeciﬁ c competition was rela- tively low (period 1 when not all geese have arrived at the stopover site, Madsen et al. 1999) or at its peak (period 2 and 3). At the end of the stopover season (period 4) when density is declining rapidly because geese depart the area, the combined model predicted habitat selection of geese substantially less well (Fig. 2A). the stopover site is used at the onset of the growing season leading to the increased availability and quick turnover of high-quality food resources. Indeed, a resource depletion model developed for our study site and based on the same ﬁ eld selection (all ﬁ elds 5 km from roost sites) showed that the amount of food resources available is suﬃ cient to accommodate the current number of geese (Baveco et al. pers. com.). What deﬁ nes the central-place foraging pattern is therefore not directly the quality or availability of food resources but the changes in goose density and intraspeciﬁ c competition for unoccupied patches. Discussion Estimates of selection for ﬁ eld area during periods 2 – 4 (log odds ratio 95% conﬁ dence intervals) by pink-footed geese at the mid-Norway stopover site as computed by the predation/dis- turbance risk RSF model with ﬁ eld area as a the only predictor variable. Black line: period 2; red line: period 3; blue line: period 4. Estimates overlapping 0 (grey, horizontal line) indicate that the use is proportional to the availability ( ‘ no selection ’ ), estimates higher than 0 indicate use higher than availability whereas values below 0 indicate use below availability for a given site. Th e Svalbard-breeding population of pink-footed geese has increased from approximately 15 000 individu- als in the 1960s to around 80 000 in 2012 (Madsen and Williams 2012), however no density dependent eﬀ ect has been observed so far during overwintering, migration 857 Figure 5. Estimates of selection for sites at increasing distance from roost sites at diﬀ erent times of the day (log odds ratio 95% conﬁ dence intervals) by pink-footed geese at the mid-Norway stopover site as computed by the density-dependent/central-place foraging RSF model with time of day (green line: morning (05:00 – 11:00); brown line: midday (11:00 – 16:00); grey line: evening (16:00 – 21:00)) and distance to the closest roost site as the predictor variables. Estimates overlapping 0 (grey, horizontal line) indicate that the use is proportional to the availability ( ‘ no selection ’ ), estimates higher than 0 indicate use higher than availability whereas values below 0 indicate use below avail- ability for a given site. Figure 5. Estimates of selection for sites at increasing distance from roost sites at diﬀ erent times of the day (log odds ratio 95% conﬁ dence intervals) by pink-footed geese at the mid-Norway stopover site as computed by the density-dependent/central-place foraging RSF model with time of day (green line: morning (05:00 – 11:00); brown line: midday (11:00 – 16:00); grey line: evening (16:00 – 21:00)) and distance to the closest roost site as the predictor variables. Estimates overlapping 0 (grey, horizontal line) indicate that the use is proportional to the availability ( ‘ no selection ’ ), estimates higher than 0 indicate use higher than availability whereas values below 0 indicate use below avail- ability for a given site. or breeding season (K é ry et al. 2006, Jensen et al. 2014). References Jensen, G. H. et al. 2014. Snow condition as an estimator of the breeding output in high-Arctic pink-footed geese Anser brach- yrhynchus . – Polar Biol. 37: 1 – 14. h Akaike, H. 1987. Factor analysis and AIC. – Psychometrika 52: 317 – 332. Å Johnson, D. H. 1980. Th e comparison of usage and availability measurements for evaluating resource preference. – Ecology 61: 65 – 71. Alerstam, T. and Lindstr ö m, Å . 1990. Optimal bird migration: the relative importance of time, energy, and safety. – In: Gwinner, E. (ed.), Bird migration. Springer, pp. 331 – 351. Johnson, C. J. and Gillingham, M. P. 2008. Sensitivity of species- distribution models to error, bias, and model design: an appli- cation to resource selection functions for woodland caribou. – Ecol. Modell. 213: 143 – 155. Altendorf, K. B. et al. 2001. Assessing eﬀ ects of predation risk on foraging behavior of mule deer. – J. Mammal. 82: 430 – 439. Amano, T. et al. 2006a. Foraging patch selection and departure by non-omniscient foragers: a ﬁ eld example in white-fronted geese. – Ethology 112: 544 – 553. Johnson, C. J. et al. 2006. Resource selection functions based on use-availability data: theoretical motivation and evaluation methods. – J. Wildl. Manage. 70: 347 – 357. Amano, T. et al. 2006b. Decision-making in group foragers with incomplete information: test of individual-based model in geese. – Ecol. Monogr. 76: 601 – 616. h Jonker, R. M. et al. 2010. Predation danger can explain changes in timing of migration: the case of the barnacle goose. – PLoS ONE 5: e11369. Bauer, S. et al. 2008. Th e consequences of climate-driven stop-over sites hanges on migration schedules and ﬁ tness of Arctic geese. – J. Anim. Ecol. 77: 654 – 660. K é ry, M. et al. 2006. Survival of Svalbard pink-footed geese Anser brachyrhynchus in relation to winter climate, density and land- use. – J. Anim. Ecol. 75: 1172 – 1181. Bjerke, J. W. et al. 2014. Reduced dairy grassland yields in central Norway after a single springtime grazing event by pink-footed geese. – Grass Forage Sci. 69: 129 – 139. Klaassen, M. et al. 2006. Modelling behavioural and ﬁ tness consequences of disturbance for geese along their spring ﬂ y- way. – J. Appl. Ecol. 49: 92 – 100. Blaustein, L. et al. 2004. References Oviposition habitat selection in response to risk of predation in temporary pools: mode of detection and consistency across experimental venue. – Oecologia 138: 300 – 305. pp Lima, S. L. and Dill, L. M. 1990. Behavioral decisions made under the risk of predation: a review and prospectus. – Can. J. Zool. 68: 619 – 640. Åh Blix, A. W. et al. 2014. Temporal scales of density-dependent habitat selection in a large grazing herbivore. – Oikos 123: 933 – 942. Lindstr ö m, Å . 1990. Th e role of predation risk in stopover habitat selection in migrating bramblings, Fringilla montifringilla . – Behav. Ecol. 1: 102 – 106. Å Boyce, M. S. and McDonald, L. L. 1999. Relating populations to habitats using resource selection functions. – Trends Ecol. Evol. 14: 268 – 272. Lindstr ö m, Å . 1991. Maximum fat deposition rates in migrating birds. – Ornis Scand. 22: 12 – 19. Madsen, J. 1994. Impacts on disturbance on migratory waterfowl. – Ibis 137: S67 – S74. Boyce, M. S. et al. 2002. Evaluating resource selection functions. – Ecol. Modell. 157: 281 – 300. Madsen, J. 1998. Changing tradeoﬀ s between predation risk and food intake: gaining access to feeding patches during spring- fattening in pink-footed geese Anser brachyrhynchus . – Norw. Polarinst. Skrifter 200: 305 – 312. Chudzi ń ska, M. et al. 2013. Diurnal variation in the behaviour of the pink-footed goose ( Anser brachyrhynchus ) during the spring stopover in Tr ø ndelag, Norway. – J. Ornithol. 154: 645 – 654. Madsen, J. and Williams, J. H. 2012. Inernational species manage- ment plan for Svalbard population of the pink-footed goose Anser brachyrhynchus . – AEWA Tech. ser. 48. Cibien, C. et al. 1989. Inﬂ uence of habitat characteristics on winter social organization in ﬁ eld roe deer. – Acta Th eriol. 34: 219 – 226. y y Madsen, J. et al. 1997. Spring migration strategies and stopover ecology of pink-footed geese. Results of ﬁ eld work in Norway, 1996 (Laursen, K, ed.). – NERI Tech. Rep. No. 204 Creel, S. et al. 2005. Elk alter habitat selection as an antipredator response to wolves. – Ecology 86: 3387 – 3397. Madsen, J. et al. 1998. Correlates of predator abundance with snow and ice conditions and their role in determining timing of nesting and breeding success in Svalbard light-bellied brent Drent, R. et al. 1978. Discussion Arable landscapes oﬀ er a readily accessible and highly nutritional food source which has led to an apparent increase in the use of pastures and agricultural crops by a variety of species (e.g. roe deer Capreolus capreolus : Cibien et al. 1989, various goose species: Madsen et al. 1999, Fox et al. 2005, buﬀ -breasted sandpipers Tryngites subruﬁ collis : McCarty et al. 2009). However, foraging in agricultural landscapes results in higher exposure to human presence compared to more natural and remote areas which can greatly inﬂ uence habitat selection of birds (such as geese as in this study), but also of mammals (e.g. roe deer: Hewison et al. 2001). In order to alleviate conﬂ icts between pink-footed geese foraging on arable ﬁ elds and farming interests, Norwegian authorities have tried to alleviate the conﬂ ict by oﬀ ering an annual ﬁ xed area rate payment to land-owners in exchange for providing accommodation areas where geese can forage undisturbed (Madsen et al. 2014). Th e criteria of such ref- uges is based on a range of environmental factors including distance to roost sites (Jensen et al. 2008, Madsen et al. 2014, Simonsen 2014). Based on the results from this study and previous observations, ﬁ eld size and goose density are impor- tant additional factors on which the location of accommoda- tion areas is already based (Madsen et al. 2014). Contrary to predictions of the central-place foraging theory, we did not ﬁ nd strong support for the model pre- dicting an increase in selection of high-quality food types with increasing distance from the focal point (distance to roost food resources model). Th is theoretical prediction is made under the assumptions that food quality is hetero- geneously distributed and high-quality food resources are rapidly depleted close to the central-place due to increased competition (Rosenberg and McKelvey 1999). However, forage depletion is unlikely to be a factor in our study as Comparing among a range of theory-driven habitat selec- tion models, we have shown that in landscapes where nutri- tional value of food resources is homogeneously high, the spatiotemporal distribution and foraging behaviour of pink- footed geese is largely a tradeoﬀ between fast refuelling and disturbance/predator avoidance. Th e analytical framework we adopted here can serve as a basis to try and understand 858 Drent, R. H. et al. 2003. Pay-oﬀ s and penalties of competing migratory schedules. Discussion – Oikos 103: 274 – 292. habitat selection patterns of other species as most factors con- sidered here are known to inﬂ uence the behaviour of most free-ranging animals (birds as well as mammals). Especially the foraging behaviour of species that regularly return to a central-place and forage in large groups in landscapes with anthropogenic disturbances may be inﬂ uenced by a combi- nation of environmental factors in a similar manner to those observed for pink-footed geese in our study. Overall, one single framework may be insuﬃ cient to explain the observed variation in foraging behaviour of animals as multiple condi- tions are expected to inﬂ uence habitat selection patterns. Fielding, A. H. and Bell, J. F. 1997. A review of methods for the assessment of prediction errors in conservation presence/ absence models. – Environ. Conserv. 24: 38 – 49. Focardi, S. et al. 1996. Do ungulates exhibit a food density thresh- old? A ﬁ eld study of optimal foraging and movement patterns. – J. Anim. Ecol. 65: 606 – 620. Fox, A. D. et al. 2005. Eﬀ ects of agricultural change on abundance, ﬁ tness components and distribution of two arctic-nesting goose populations. – Global Change Biol. 11: 881 – 893. Godvik, I. M. R. et al. 2009. Temporal scales, tradeoﬀ s and functional responses in red deer habitat selection. – Ecology 90: 699 – 710. Hedenstr ö m, A. and Alerstam, T. 1997. Optimum fuel loads in migratory birds: distinguishing between time and energy min- imization. – J. Th eor. Biol. 189: 227 – 234. h Acknowledgements – Th is study was part of MC ’ s PhD project funded by Aarhus University. Th e ﬁ eldwork was supported by the Norwegian Research Council project MIGRAPOP. We would like to thank Jannik Hansen and Robert Peel for help during data col- lection. h Hewison, A. J. et al. 2001. Th e eﬀ ects of woodland fragmentation and human activity on roe deer distribution in agricultural landscapes. – Can. J. Zool. 79: 679 – 689. Jensen, R. et al. 2008. Prioritizing refuge sites for migratory geese to alleviate conﬂ icts with agriculture. – Biol. Conserv. 141: 1806 – 1818. References Balancing the energy budget of arctic- breeding geese throughout the annual cycle; a progress report. – Verh. Ornithol. Gesellschaft Bayern 23: 239 – 264. 859 geese Branta bernicla hrota . – Norw. Polarinst. Skrifter 200: 221 – 234. Rosenberg, D. K. and McKelvey, K. S. 1999. Estimation of habitat selection for central-place foraging animals. – J. Wildl. Manage. 63: 1028 – 1038. Madsen, J. et al. 1999. Pink-footed goose Anser brachyrhynchus : Svalbard. – In: Madsen, J. et al. (eds), Goose population of the Western Palearctic. Wetlands Int. Publ. No. 48, Natl Environ. Res. Inst., Denmark, pp. 82. g Rosenzweig, M. L. 1981. A theory of habitat selection. – Ecology 62: 327 – 335. Schoener, T. W. 1979. Generality of the size – distance relation in models of optimal feeding. – Am. Nat. 114: 902 – 914. Madsen, J. et al. 2014. Regional management of farmland feeding geese using an ecological prioritization tool. – Ambio 43: 801 – 809. Shochat, E. et al. 2002. Density-dependent habitat selection in migratory passerines during stopover: what causes the devi- ation from IFD? – Evol. Ecol. 16: 469 – 488. Manly, B. F. J. et al. 1993. Resource selection by animals: statistical design and analysis for ﬁ eld studies. – Chapman and Hall. Sih, A. 1980. Can foragers balance two conﬂ icting demands? – Science 210: 1041 – 1043. Manly, B. J. F. et al. 2002. Resource selection by animals: statistical design and analysis of ﬁ eld studies. – Kluwer. Simonsen, C. E. 2014. Goose/agricultural conﬂ icts in Norway – building species distribution models: objective tools in local management when allocating subsidies and evaluations on scaring cost eﬀ ectiveness. – Aarhus Univ. www.gint.no . McCarty, J. P. et al. 2009. Behavior of buﬀ -breasted sandpipers ( Tryngites subruﬁ collis ) during migratory stopover in agricul- tural ﬁ elds. – PLoS ONE 4: e8000. McDonald, P. et al. 1973. Animal nutrition. – Oliver and Boyd. M L hli P D l 2010 C id i l i l d i Stephens, P. A. et al. 2014. Capital and income breeding: the role of food supply. – Ecology 95: 882 – 896. McLoughlin, P. D. et al. 2010. Considering ecological dynamics in resource selection functions. – J. Anim. Ecol. 79: 4 – 12. Sunde, P. and Redpath, M. 2006. References Combining information from range use and habitat selection: sex-speciﬁ c spatial responses to habitat fragmentation in tawny owls Strix aluco . – Ecography 29: 152 – 158. h Mysterud, A. and Ims, R. A. 1998. Functional responses in habitat use: availability inﬂ uences relative use in tradeoﬀ situations. – Ecology 79: 1435 – 1441. Orians, G. H. and Pearson, N. E. 1979. On the theory of central place foraging. – Ohio State Univ. Press, Columbus, OH. Th omas, D. L. and Taylor, E. J. 2006. Study designs and tests for comparing resource use and availability II. – J. Wildl. Manage. 70: 324 – 336. g g Pierce, B. M. et al. 2004. Habitat selection by mule deer: forage beneﬁ ts or risk of predation? – J. Wildl. Manage. 68: 533 – 541. Tombre, I. et al. 2005. Inﬂ uence of organised scaring on distribu- tion and habitat choice of geese on pastures in northern Nor- way. – Agric. Ecosyst. Environ. 111: 311 – 320. Prop, J. and Black, J. M. 1998. Food intake, body reserves and reproductive success of barnacle geese Branta leucopsis staging in diﬀ erent habitats. – Norw. Polarinst. Skrifter 200: 175 – 193. van Beest, F. M. et al. 2014. Increasing density leads to generalization in both coarse-grained habitat selection and ﬁ ne-grained resource selection in a large mammal. – J. Anim. Ecol. 83: 147 – 156. Prop, J. and Spaans, B. 2004. Body stores in pre-migratory brent geese: the consequence of habitat choice on protein deposition. – In: Food ﬁ nding. On the trail to successful reproduction in migratory geese. PhD thesis, pp. 71 – 85. van Gils, J. A. and Tijsen, W. 2007. Short-term foraging costs and long-term fueling rates in central-place foraging swans revealed by giving-up exploitation times. – Am. Nat. 169: 609 – 620. Weber, T. P. et al. 1998. Optimal avian migration: a dynamic model of fuel stores and site use. – Evol. Ecol. 12: 377 – 401. Robbins, C. T. 1993. Wildlife feeding and nutrition. – Academic Press. 860
https://openalex.org/W2022091129	https://espace.curtin.edu.au/bitstream/20.500.11937/52558/2/251964.pdf	English	null	The vertical distribution of buoyant plastics at sea: an observational study in the North Atlantic Gyre	Biogeosciences	2,015	cc-by	6,165	Correspondence to: J. Reisser (jureisser@gmail.com) Correspondence to: J. Reisser (jureisser@gmail.com) Received: 31 October 2014 – Published in Biogeosciences Discuss.: 26 November 2014 Revised: 15 February 2015 – Accepted: 16 February 2015 – Published: 26 February 2015 Biogeosciences, 12, 1249–1256, 2015 www.biogeosciences.net/12/1249/2015/ doi:10.5194/bg-12-1249-2015 © Author(s) 2015. CC Attribution 3.0 License. Biogeosciences, 12, 1249–1256, 2015 www.biogeosciences.net/12/1249/2015/ doi:10.5194/bg-12-1249-2015 © Author(s) 2015. CC Attribution 3.0 License. J. Reisser1,, B. Slat2,, K. Noble3, K. du Plessis4, M. Epp1, M. Proietti5, J. de Sonneville2, T. Becker6, and C. Pattiaratchi1 J. Reisser1,, B. Slat2,, K. Noble3, K. du Plessis4, M. Epp1, M. Proietti5, J. de Sonneville2, T. Becker6, and C. Pattiaratchi1 2The Ocean Cleanup Foundation, Delft, the Netherlands 5Instituto de Oceanograﬁa, Universidade Federal do Rio Grande, Rio Grande, Brazil 6Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Perth, Australia *These authors contributed equally to this work. 1 Introduction Abstract. Millimetre-sized plastics are numerically abun- dant and widespread across the world’s ocean surface. These buoyant macroscopic particles can be mixed within the up- per water column by turbulent transport. Models indicate that the largest decrease in their concentration occurs within the ﬁrst few metres of water, where in situ observations are very scarce. In order to investigate the depth proﬁle and physical properties of buoyant plastic debris, we used a new type of multi-level trawl at 12 sites within the North Atlantic sub- tropical gyre to sample from the air–seawater interface to a depth of 5 m, at 0.5 m intervals. Our results show that plastic concentrations drop exponentially with water depth, and de- cay rates decrease with increasing Beaufort number. Further- more, smaller pieces presented lower rise velocities and were more susceptible to vertical transport. This resulted in higher depth decays of plastic mass concentration (milligrams m−3) than numerical concentration (pieces m−3). Further multi- level sampling of plastics will improve our ability to predict at-sea plastic load, size distribution, drifting pattern, and im- pact on marine species and habitats. Plastics pose physical and chemical threats to the oceans’ ecosystem. Their widespread occurrence at the sea surface may be shifting the distribution and abundance of marine populations due to (1) enhanced ocean drift opportunities and (2) damaging effects on biota and habitats. Plastics harbour organisms – such as fouling microorganisms, invertebrates, and ﬁsh – that can widely disperse via this new type of habi- tat, potentially entering non-native waters (Winston et al., 1997; Barnes, 2002; Thiel and Gutow, 2005; Zettler et al., 2013; Reisser et al., 2014). Plastic objects can also entangle or be ingested/inhaled by marine animals, leading to impacts such as starvation, death, and hepatic stress (Derraik, 2002; Browne et al., 2008; Gregory, 2009; Rochman et al., 2013; Watts et al., 2014). Most of what is known about at-sea plastic characteris- tics and concentrations comes from surface net sampling, where the top few centimetres of the water column is ﬁltered to collect plastics larger than 0.2–0.4 mm (Hidalgo-Ruz et al., 2012). These sea surface samples have shown that the world’s sea surface contains many millimetre-sized plastic pieces known as “microplastics” when smaller than 5 mm in length (Arthur et al., 2009; Hidalgo-Ruz et al., 2012). This type of plastic pollution is widespread across oceans, with Published by Copernicus Publications on behalf of the European Geosciences Union. J. 1 Introduction Reisser et al.: The vertical distribution of buoyant plastics at sea 1250 1250 J. Reisser et al.: The vertical distribution of buoyant plastics at sea Figure 1. North Atlantic map indicating locations sampled during this study (orange dots) using the multi-level net displayed in the right panel. The four orange dots include all 12 multi-level net tows conducted in the study, which could not be shown separately due to scale. The map also shows the expedition departure and arrival location (Bermuda), as well as plastic accumulation zones predicted by ocean modelling (dotted lines) (Lebreton et al., 2012; Maximenko et al., 2012), and a surface net tow data set (solid gray line; grey dots show locations of net tows) (Law et al., 2010). Figure 1. North Atlantic map indicating locations sampled during this study (orange dots) using the multi-level net displayed in the right panel. The four orange dots include all 12 multi-level net tows conducted in the study, which could not be shown separately due to scale. The map also shows the expedition departure and arrival location (Bermuda), as well as plastic accumulation zones predicted by ocean modelling (dotted lines) (Lebreton et al., 2012; Maximenko et al., 2012), and a surface net tow data set (solid gray line; grey dots show locations of net tows) (Law et al., 2010). plastic mass and numerical concentration with depth and (2) demonstrate how these vary with sea state. We also provide the ﬁrst experimental measurements of the rise velocity of plastic pieces, evaluating its relationship to the type and size of pieces. higher contamination levels at convergence zones such as those within subtropical gyres (Carpenter and Smith, 1972; Maximenko et al., 2012; Lebreton et al., 2012; van Sebille et al., 2012; Cózar et al., 2014; Eriksen et al., 2014). Plas- tic debris collected by surface nets are mostly fragments of packaging and ﬁshing gear made of polyethylene and polypropylene (Barnes et al., 2009; Morét-Ferguson et al., 2010; Hidalgo-Ruz et al., 2012; Reisser et al., 2013). These two resins are less dense than seawater and account for ap- proximately 62 % of the plastic volume produced each year (Andrady, 2011). 2.1 At-sea sampling We conducted 12 multi-level net tows that sampled the up- per 5 m of the North Atlantic accumulation zone (Law et al., 2010; Maximenko et al., 2012; Lebreton et al., 2012) during daytime hours, from 19 to 22 May 2014, aboard the sailing vessel Sea Dragon (Fig. 1). We used a new collection device capable of sampling surface waters from the air–seawater in- terface to a depth of 5 m, at 0.5 m intervals. This equipment is composed of eleven frames with 0.5 m height × 0.3 m width ﬁtted with 2.1 m long 150 µm mesh polyester nets. These nets were stacked vertically and secured within an exter- nal frame that was dragged in the water from eight towing points, ensuring its stability and perpendicular position in re- lation to the sea surface, with the top net completely above mean water line (see Fig. 1). Tow durations ranged from 55 to 60 min and were all undertaken while the vessel was trav- elling at a speed of 1–1.9 kn. The captain, who has 20 years sailing experience, estimated wind speeds and sea state of each sampling period: Beaufort number 1 (N = 3 net tows), 3 (N = 4 net tows), and 4 (N = 5 net tows) (Reisser et al., 2015). After each tow, we transferred the collected contents Turbulence in the upper-ocean layer can vertically mix buoyant plastic particles. A model developed by Kukulka et al. (2012) predicted that the largest decrease in plastic con- centration occurs over the ﬁrst metres of the water column, where only a few low-resolution measurements exist (Lat- tin et al., 2004; Doyle et al., 2011; Kukulka et al., 2012; Isobe et al., 2014). As studying ocean turbulent transport is heavily dependent on observations (Ballent et al., 2012; D’Asaro, 2014), high-resolution multi-level plastic sampling is urgently needed to test this prediction. A better understand- ing of the vertical transport of buoyant plastics is fundamen- tal for improving estimates of concentration, size distribu- tion, and dispersal of plastics in the world’s ocean (Kukulka et al., 2012; Reisser et al., 2013; Law et al., 2014; Isobe et al., 2014). In this context, the present study aimed at obtaining depth proﬁles of plastic pollution in the top layer of the oceans (0– 5 m). 2.2 Estimating depth proﬁles of plastic contamination Secondly, we ﬁlled the tube with ﬁltered seawater, capped both ends with rubber stops, and locked it in place with a clamp. One of the tube ends was then opened, a plastic piece placed inside, the tube closed again (with no trapped air), and quickly turned upside down and locked in place with a clamp, using a spirit level to ad- just its vertical position. Finally, we recorded the time taken for the plastic piece to rise from one mark to the other (dis- tance = 75 cm) using a stopwatch. This was measured four times per plastic piece, and the average was used as the es- timation of its rise velocity (wb). Rise velocities of different plastic types were separately plotted against plastic lengths (l), and linear regressions of the form wb = al + b were ap- plied to assess the effect of plastics’ characteristics on its rise velocity. We also plotted the rise velocities of plastic pieces collected at different depths to visualise depth patterns. To quantify the variation of plastic concentration with depth and assess the effect of changing sea state on these ver- tical proﬁles, we ﬁrst divided the plastic concentration values of the samples by their corresponding surface concentration values. We then took the average of these normalised concen- trations between adjacent nets to estimate normalised plas- tic concentration values at depths of 0 m (top two nets), 1 m (third and fourth nets), 2 m (ﬁfth and sixth nets), 3 m (seventh and eighth nets), 4 m (9th and 10th), and 4.75 m (11th net). Finally, numerical and mass concentration values from tows collected under the same Beaufort number were grouped and ﬁtted to exponential decay models of the form N = e−λz, where N = normalised plastic concentration, z = depth, and λ = decay rate. Finally, we calculated the fractions of plastics of dif- ferent size classes (0.5–1, 1.5–2, 2.5–3, 3.5–4, 4.5–5, and > 5.5 mm) that were located at the sea surface (depth < 0.5 m) and in deeper layers (depth > 0.5 m) during sam- pling at Beaufort numbers of 1, 3, and 4. We calculated these fractions using all plastics collected, as well as separated by plastic type. We also predicted normalised plastic concentration depth proﬁles using the model described in Kukulka et al. 2.2 Estimating depth proﬁles of plastic contamination We measured the length of all plastic pieces using a transpar- ent ruler (0.5 mm resolution), and classiﬁed them into the fol- lowing types: hard plastic – fragments of rigid plastic; sheet We calculated plastic numerical and mass concentrations by dividing the number of plastic pieces and total plastic mass by the volume of ﬁltered seawater of each net sam- ple (pieces m−3 and milligrams m−3). Filtered volume was estimated using frame dimensions and readings from a me- chanical ﬂowmeter (32 cm per rotation). – fragments of thin plastic, with some degree of ﬂexibility; line – fragments of ﬁshing lines or nets; foam – expanded polystyrene fragments; and pellet – raw material used to pro- duce plastic items (Fotopoulou and Karapanagioti, 2012). We also identiﬁed the resin composition of 60 pieces using Ra- man spectroscopy (WITec alpha 300RA+), and measured the rise velocity of 0–3 plastics from each sample collected. – fragments of thin plastic, with some degree of ﬂexibility; line – fragments of ﬁshing lines or nets; foam – expanded polystyrene fragments; and pellet – raw material used to pro- duce plastic items (Fotopoulou and Karapanagioti, 2012). We also identiﬁed the resin composition of 60 pieces using Ra- man spectroscopy (WITec alpha 300RA+), and measured the rise velocity of 0–3 plastics from each sample collected. Samples were washed into a clear plastic container ﬁlled with ﬁltered seawater, and ﬂoating macroscopic plastics were organised into gridded petri dishes for counting and charac- terisation. Each search for plastic pieces was for at least 1 h per sample, with the aid of thumb forceps, dissecting nee- dles, magnifying glasses, and LED torches. The latter was particularly important for detecting thin transparent plastic fragments, which had low detection probability when not re- ﬂecting light. Two thin ﬁlaments resembling textile ﬁbres were discarded due to potential air contamination as noted in (Foekema et al., 2013). Once all plastics were counted and characterised, they were washed with deionised water, trans- ferred to aluminium dishes, dried at 60 ◦C, and weighed. y p p Our method of rise velocity measurement is an adaptation of an experiment to examine the fall velocity of various types of sediment particles in different ﬂuids (Allen, 1985). Firstly, we made two marks 12.5 cm from the ends of a 1 m long clear plastic tube (diameter = 40 mm). J. Reisser et al.: The vertical distribution of buoyant plastics at sea to a 150 µm sieve and stored them in aluminium bags that were kept frozen during transportation. 2.3 Characterising plastic length, type, resin, and rise velocity 2.3 Characterising plastic length, type, resin, and rise velocity We measured the length of all plastic pieces using a transpar- ent ruler (0.5 mm resolution), and classiﬁed them into the fol- lowing types: hard plastic – fragments of rigid plastic; sheet – fragments of thin plastic, with some degree of ﬂexibility; line – fragments of ﬁshing lines or nets; foam – expanded polystyrene fragments; and pellet – raw material used to pro- duce plastic items (Fotopoulou and Karapanagioti, 2012). We also identiﬁed the resin composition of 60 pieces using Ra- man spectroscopy (WITec alpha 300RA+), and measured the rise velocity of 0–3 plastics from each sample collected. 2.2 Estimating depth proﬁles of plastic contamination (2012): N = ezwbA 1 0 , where z = depth, wb = plastic rise velocity, and A0 = 1.5u∗wkHs, with u∗w = frictional velocity of wa- ter, k = 0.4 (von Karman constant), and Hs = signiﬁcant wave height. We considered wb = 0.0053 m s−1 (plastics’ median rise velocity, as estimated in this study), Hs = 0.1, 0.6, or 1 m (typical wave heights experienced at Beaufort numbers of 1, 3, and 4, respectively), and used the wind ranges of Beaufort of 1, 3, and 4 (1–3, 7–10, and 11– 16 kn, respectively) to estimate their respective u∗w val- ues through the approximation proposed by Pugh (1987): u∗w = 0.00012W10, where W10 = 10 m wind speed in m s−1. Thus, the considered numerical ranges of frictional veloc- ity of water (u∗w) were 0.0006–0.0019 m s−1 for Beaufort number 1, 0.0043–0.0062 m s−1 for Beaufort number 3, and 0.0068–0.0099 m s−1 for Beaufort number 4. 2.1 At-sea sampling We performed multi-level sampling with a new type of equipment to (1) quantify the exponential decay rates of Biogeosciences, 12, 1249–1256, 2015 www.biogeosciences.net/12/1249/2015/ 1251 3.1 Depth proﬁles of mass and numerical concentrations Black lines show model predictions (Kukulka et al., 2012) using median plastic rise velocity (0.0053 m s−1), and the typical range of frictional velocity of water (u∗w) at each of the sea states sampled. Figure 4. Glass jars with ﬁltered water and plastic samples collected under wind speeds of 1 kn (Beaufort number 1, top image) and 15 kn (Beaufort number 4, bottom image). From left to right: 0–0.5, 0.5–1, 1–1.5, 1.5–2, 2–2.5, 2.5–3, 3–3.5, 3.5–4, 4–4.5, and 4.5–5 m depth intervals. state (Fig. 3). Depth decay rate of numerical concentration went from 3.0 at Beaufort 1 (95 % conﬁdence interval – 95 %CI = 2.56–3.45), to 1.7 at Beaufort 3 (95 %CI = 1.51– 1.88), to 0.8 at Beaufort 4 (95 %CI = 0.62–0.98). Decay rate of mass concentration went from 3.8 at Beaufort 1 (95 %CI = 3.23–4.33), to 2.4 at Beaufort 3 (95 %CI = 1.63– 3.14), to 1.7 at Beaufort 4 (95 %CI = 1.50–1.94). These exponential ﬁts had relatively similar depth de- h di d b K k lk ’ d l f B Figure 4. Glass jars with ﬁltered water and plastic samples collected under wind speeds of 1 kn (Beaufort number 1, top image) and 15 kn (Beaufort number 4, bottom image). From left to right: 0–0.5, 0.5–1, 1–1.5, 1.5–2, 2–2.5, 2.5–3, 3–3.5, 3.5–4, 4–4.5, and 4.5–5 m depth intervals. Figure 4. Glass jars with ﬁltered water and plastic samples collected under wind speeds of 1 kn (Beaufort number 1, top image) and 15 kn (Beaufort number 4, bottom image). From left to right: 0–0.5, 0.5–1, 1–1.5, 1.5–2, 2–2.5, 2.5–3, 3–3.5, 3.5–4, 4–4.5, and 4.5–5 m depth intervals. state (Fig. 3). Depth decay rate of numerical concentration went from 3.0 at Beaufort 1 (95 % conﬁdence interval – 95 %CI = 2.56–3.45), to 1.7 at Beaufort 3 (95 %CI = 1.51– 1.88), to 0.8 at Beaufort 4 (95 %CI = 0.62–0.98). Decay rate of mass concentration went from 3.8 at Beaufort 1 (95 %CI = 3.23–4.33), to 2.4 at Beaufort 3 (95 %CI = 1.63– 3.14), to 1.7 at Beaufort 4 (95 %CI = 1.50–1.94). Figure 3. Depth proﬁles of normalised plastic numerical and mass concentrations under different Beaufort numbers: 1 (N = 3 net tows; 3283 plastic pieces), 3 (N = 4 net tows; 4049 plastic pieces), and 4 (N = 5 net tows; 5419 plastic pieces). 3.1 Depth proﬁles of mass and numerical concentrations Black lines show model predictions (Kukulka et al., 2012) using median plastic rise velocity (0.0053 m s−1), and the typical range of frictional velocity of water (u∗w) at each of the sea states sampled. These exponential ﬁts had relatively similar depth de- cay rates to those predicted by Kukulka’s model for Beau- fort 3 (λ = 2.36–3.37) and 4 (λ = 0.88–1.28). However, for Beaufort 1 the statistical ﬁt showed much smaller λ (2.56– 4.33) than those predicted by Kukulka’s model (λ = 141.73– 47.2492). Exponential models ﬁtted well with both numerical and mass concentrations (R2 = 0.99–0.84), with depth decay rates (λ) consistently higher for mass than numerical con- centration. Furthermore, both numerical and mass concen- tration decay rates were inversely proportional to Beaufort 3.1 Depth proﬁles of mass and numerical concentrations Plastic numerical and mass concentrations both decreased abruptly from their peak values at the sea surface, where me- dian values were equal to 1.69 pieces m−3 and 1.60 mg m−3 (Fig. 2). Concentration differences between the surface and deeper layers were higher in terms of mass than number of particles. For instance, median mass and numerical con- centrations at 0.5–1 m were, respectively 13.3 and 6.5 times lower than their median plastic peaks at 0–0.5 m. Biogeosciences, 12, 1249–1256, 2015 www.biogeosciences.net/12/1249/2015/ J. Reisser et al.: The vertical distribution of buoyant plastics at se J. Reisser et al.: The vertical distribution of buoyant plastics at sea 1252 1252 J. Reisser et al.: The vertical distribution of buoyant plastics at sea Figure 2. Boxplots of plastic numerical (left) and mass (right) concentrations at different depth intervals (N = 12 multi-level net tows). The central line is the median value, edges of the box are the 25th and 75th percentiles, whiskers extend to extreme data points not considered outliers, and outliers are plotted individually as crosses. Figure 2. Boxplots of plastic numerical (left) and mass (right) concentrations at different depth intervals (N = 12 multi-level net tows). The Figure 2. Boxplots of plastic numerical (left) and mass (right) concentrations at different depth intervals (N = 12 multi-level net tows). The central line is the median value, edges of the box are the 25th and 75th percentiles, whiskers extend to extreme data points not considered outliers, and outliers are plotted individually as crosses. Figure 3. Depth proﬁles of normalised plastic numerical and mass concentrations under different Beaufort numbers: 1 (N = 3 net tows; 3283 plastic pieces), 3 (N = 4 net tows; 4049 plastic pieces), and 4 (N = 5 net tows; 5419 plastic pieces). Black lines show model predictions (Kukulka et al., 2012) using median plastic rise velocity (0.0053 m s−1), and the typical range of frictional velocity of water (u∗w) at each of the sea states sampled. Fi 3 D th ﬁl f li d l ti i l d Figure 3. Depth proﬁles of normalised plastic numerical and mass concentrations under different Beaufort numbers: 1 (N = 3 net tows; 3283 plastic pieces), 3 (N = 4 net tows; 4049 plastic pieces), and 4 (N = 5 net tows; 5419 plastic pieces). J. Reisser et al.: The vertical distribution of buoyant plastics at sea 1253 Figure 5. Histogram of rise velocity of plastics (a), plots of plastic sizes versus rise velocities of different types of plastic (b), and boxplot of rise velocity for plastics collected at different depth intervals (c). In panel (c), the central dot is the median value, edges of the box are the 25th and 75th percentiles, whiskers extend to extreme data points not considered outliers, and outliers are plotted individually as crosses. Figure 5. Histogram of rise velocity of plastics (a), plots of plastic sizes versus rise velocities of different types of plastic (b), and box of rise velocity for plastics collected at different depth intervals (c). In panel (c), the central dot is the median value, edges of the box are 25th and 75th percentiles, whiskers extend to extreme data points not considered outliers, and outliers are plotted individually as crosses Figure 5. Histogram of rise velocity of plastics (a), plots of plastic sizes versus rise velocities of different types of plastic (b), and boxplot of rise velocity for plastics collected at different depth intervals (c). In panel (c), the central dot is the median value, edges of the box are the 25th and 75th percentiles, whiskers extend to extreme data points not considered outliers, and outliers are plotted individually as crosses. www.biogeosciences.net/12/1249/2015/ www.biogeosciences.net/12/1249/2015/ Biogeosciences, 12, 1249–1256, 2015 J. Reisser et al.: The vertical distribution of buoyant plastics at sea J. Reisser et al.: The vertical distribution of buoyant plastics at sea 3.2 Lengths, types, resins, and rise velocities of plastics locities differed among sampled depths, with particles at the surface (0–0.5 m) having a wider range of values and a higher median value than pieces at greater depths (Fig. 5c). We counted and classiﬁed 12 751 macroscopic plastic pieces with lengths varying from 0.5 to 207 mm (median = 1.5 mm; Fig. 4). They were mostly fragments of polyethylene (84.7 %), followed by polypropylene (15.3 %) items. Hard plastics (46.6 %) and sheets (45.4 %) were predominant, with lower presence of plastic lines (7.9 %), pellets (0.05 %), and foams (0.008 %). The vertical mixing process was size-selective and af- fected the size distribution of plastics located at the sea sur- face (Fig. 6), with the proportion of plastics at depths over 0.5 m generally increasing towards smaller plastic lengths (Fig. 7). For hard plastics and sheets, this trend was observed at all Beaufort numbers sampled. Plastic lines however, only displayed this trend at Beaufort 1, with different size classes showing similar and relatively high underwater proportions at Beaufort 3 and 4. Plastic rise velocity ranged from 0.001 to 0.0438 m/s (Fig. 5a). It was directly proportional to plastic length, with the slope of this linear relationship differing among types of plastic (Fig. 5b). While both hard plastics and sheets had a slope equal to 0.002 (95 % CI = 0.0017–0.0026 and 0.0012–0.0023, respectively), plastic lines had a ﬂatter slope of 0.00007 (95 % CI = 0.00002–0.00013), since their rise ve- locity increased only slightly towards longer pieces. Rise ve- Data sets produced and analysed in this study are available at ﬁgshare (Reisser et al., 2015). Biogeosciences, 12, 1249–1256, 2015 J. Reisser et al.: The vertical distribution of buoyant plastics at sea J. Reisser et al.: The vertical distribution of buoyant plastics at sea 1254 Figure 6. Size histograms of plastics collected at depths 0–0.5 and 0.5–5 m during Beaufort numbers 1 (top panel), 3 (middle panel), and 4 (bottom panel). Figure 7. Percentage of plastic pieces of different types and size classes located at depths greater than 0.5 m during sampling at Beaufort numbers 1, 3, and 4. Figure 7. Percentage of plastic pieces of different types and size classes located at depths greater than 0.5 m during sampling at Beaufort numbers 1, 3, and 4. Predictions of plastic vertical mixing are commonly used to correct numerical concentrations obtained by surface net sampling (Kukulka et al., 2012; Reisser et al., 2013; Cózar et al., 2014; Law et al., 2014). As determined in our study, the model described in Kukulka et al. (2012) performed rela- tively well in estimating the total number of plastic pieces at the wind-mixed surface layer. The major difference between this model and our observations occurred at the calmest sea state condition (Beaufort number 1): while the model pre- dicted that all plastics would be at the surface, we still ob- served some particles submerged at depths greater than 0.5 m below the water surface. This could have been a consequence of the presence of other types of vertical ﬂow at our sam- pled sites (e.g. downwelling) or the occurrence of plastics rising from deeper waters due to previous wind-driven mix- ing events. Figure 6. Size histograms of plastics collected at depths 0–0.5 and 0.5–5 m during Beaufort numbers 1 (top panel), 3 (middle panel), and 4 (bottom panel). www.biogeosciences.net/12/1249/2015/ www.biogeosciences.net/12/1249/2015/ www.biogeosciences.net/12/1249/2015/ J. Reisser et al.: The vertical distribution of buoyant plastics at sea tion sampling (Hidalgo-Ruz et al., 2012). Subsurface sam- ples are still scarce and the processes inﬂuencing distribution of plastics throughout the ocean’s water column are poorly understood. Further multi-level sampling across a broader range of sea states is necessary for better quantifying the vertical mixing of buoyant plastics. This will improve pre- dictions of ocean plastic concentration levels (Kukulka et al., 2012), size distributions (Cózar et al., 2014; Eriksen et al., 2014), drifting patterns (Isobe et al., 2014), and interactions with neustonic and pelagic species of the world’s oceans. trations. These authors estimated that the total plastic load in the world’s sea surface layer is between 7000 and 35 000 tons. On the other hand, Law et al. (2014) multiplied depth- integrated numerical concentrations by the average plastic particle mass (1.36 × 10−5 kg), and estimated that the mi- croplastic load at the North Paciﬁc accumulation zone alone is of at least 21 290 tons. Such differences evidence the im- portance of better predicting the vertical transport of ocean plastics in order to develop standard plastic load estimation methods. More sampling is required to better quantify both proﬁles of plastic mass and numerical concentration over a broader range of sea states, and translate these observations into prediction models. Such models may need to be three- dimensional, and account not only for wind mixing effects, but for also ocean plastic properties (e.g. particle size) and other types of vertical transport processes (e.g. Langmuir cir- culation). Acknowledgements. We thank The Ocean Cleanup and The University of Western Australia for funding, Pangaea Exploration for sea time, and the staff and crew of our expedition: Eric Loss, Shanley McEntee, Winston Ricardo, Bart Sturm, Beatrice Clyde- Smith, Kasey Erin, Mario Merkus, Max Muller, and Jennifer Gelin. The authors also acknowledge The Ocean Cleanup volunteers who helped design, build, and test the multi-level trawl (see http://www.theoceancleanup.com for details). J. Reisser received IPRS, UWA Completion, and CSIRO Postgraduate scholarships. As shown here, and in two modelling studies (Ballent et al., 2012; Isobe et al., 2014), vertical mixing affects the size distribution of plastics ﬂoating at the surface. We observed that the proportion of plastics mixed into deeper waters in- creases towards smaller sizes even under low wind speed (1 kn) conditions (see Fig. 7). This observation has implica- tions for studies assessing size distribution of plastics using surface sampling devices. Cózar et al. References Allen, J.: Sink or swim?, Physical Sedimentology, Springer, 1985. Andrady, A. L.: Microplastics in the marine environment, Mar. Pol- lut. Bull., 62, 1596–1605, 2011. Allen, J.: Sink or swim?, Physical Sedimentology, Springer, 1985. Andrady, A. L.: Microplastics in the marine environment, Mar. Pol- lut. Bull., 62, 1596–1605, 2011. Arthur, C., Baker, J., and Bamford, H.: Proceedings of the Interna- tional Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris, September 9–11, 2008, 2009. Ballent, A., Purser, A., de Jesus Mendes, P., Pando, S., and Thomsen, L.: Physical transport properties of marine mi- croplastic pollution, Biogeosciences Discuss., 9, 18755–18798, doi:10.5194/bgd-9-18755-2012, 2012. Barnes, D. K.: Biodiversity: invasions by marine life on plastic de- bris, Nature, 416, 808–809, 2002. Predicting the vertical mixing of buoyant plastics is also important as it affects the horizontal drifting patterns and ecological impacts of plastic pollution. For instance, larger pieces of plastic coming from land-based sources may stay trapped near the shore until further fragmentation due to a combination of their high buoyancy and the effect of Stokes drift produced by waves parallel to coastlines (Isobe et al., 2014). Furthermore, the vertical distribution of plastics will inﬂuence the likelihood of animals inhabiting different depths to encounter and potentially interact with plastic. For instance, sea birds, turtles, and mammals, which breathe air and use the sea surface for daily activities, present high rates of plastic ingestion and entanglement (Derraik, 2002; Tour- inho et al., 2010). These high interaction rates could be partly explained by the relatively high concentrations of plastic de- bris at the sea surface, as shown in this study. Barnes, D. K. A., Galgani, F., Thompson, R. C., and Barlaz, M.: Accumulation and fragmentation of plastic debris in global envi- ronments, Phil. Trans. Roy. Soc. B, 364, 1985–1998, 2009. Browne, M. A., Dissanayake, A., Galloway, T. S., Lowe, D. M., and Thompson, R. C.: Ingested microscopic plastic translocates to the circulatory system of the mussel, Mytilus edulis (L.), Environ. Sci. Technol., 42, 5026–5031, 2008. Carpenter, E. J. and Smith, K.: Plastics on the Sargasso Sea Surface, Science, 175, 1240–1241, 1972. Cózar, A., Echevarría, F., González-Gordillo, J. I., Irigoien, X., Úbeda, B., Hernández-León, S., Palma, Á. T., Navarro, S., García-de-Lomas, J., and Ruiz, A.: Plastic debris in the open ocean, Proc. Natl. Acad. Sci., 111, 10239–10244, 2014. D’Asaro, E. A.: Turbulence in the upper-ocean mixed layer, Ann. Rev. Mar. Sci., 6, 101–115, 2014. J. Reisser et al.: The vertical distribution of buoyant plastics at sea (2014) and Eriksen et al. (2014) quantiﬁed the size distribution of ocean plas- tics from worldwide sampling locations and concluded that there are major losses of small plastics from the sea surface. Here we show that at least a fraction of this “missing” plas- tic could be just under the sampled surface layer (0–0.5 m). For instance, 20 % of 0.5–1 mm, 13 % of 1.5–2 mm, and 8 % of 2.5–3 mm long plastics were between 0.5 and 5 m deep during our Beaufort number 1 net tows. More at-sea and ex- perimental work is required to further quantify this effect and estimate depth-integrated size distribution of buoyant plastics drifting at sea. Edited by: G. Herndl References Derraik, J. G.: The pollution of the marine environment by plastic debris: a review, Mar. Pollut. Bull., 44, 842–852, 2002. Our ﬁndings show that vertical mixing affects the number, mass, and size distribution of buoyant plastics captured by surface nets, a standard equipment for at-sea plastic pollu- Doyle, M. J., Watson, W., Bowlin, N. M., and Sheavly, S. B.: Plastic particles in coastal pelagic ecosystems of the Northeast Paciﬁc ocean, Mar. Environ. Res., 71, 41–52, 2011. Biogeosciences, 12, 1249–1256, 2015 4 Discussion This study describes high-resolution depth proﬁles of plastic concentrations, which were shown to decrease exponentially with depth, with decay rates decreasing towards stronger winds. It also provides the ﬁrst measurements of the rise ve- locity of ocean plastics, which varies with particle size and type. Furthermore, it shows that depth proﬁles of plastic mass are associated with higher decay rates than depth proﬁles of plastic numbers. This can be explained by our observation of smaller plastic pieces generally associated with lower rising velocities, being therefore more susceptible to vertical trans- port. Our results indicate that plastic numerical concentration decays at a lower rate than plastic mass concentration, as smaller plastics are more susceptible to vertical transport. The uncertainties related to how plastic numerical concen- tration translates into plastic mass concentration have al- ready led to differences between plastic load estimates aris- ing from different studies. For instance, Cózar et al. (2014) used a correlation based on simultaneous surface tow mea- surements of total mass and abundance of plastic to convert depth-integrated numerical concentrations into mass concen- Biogeosciences, 12, 1249–1256, 2015 www.biogeosciences.net/12/1249/2015/ 1255 J. Reisser et al.: The vertical distribution of buoyant plastics at sea J. Reisser et al.: The vertical distribution of buoyant plastics at sea 1256 Morét-Ferguson, S., Law, K. L., Proskurowski, G., Murphy, E. K., Peacock, E. E., and Reddy, C. M.: The size, mass, and composi- tion of plastic debris in the western North Atlantic Ocean, Mar. Pollut. Bull., 60, 1873–1878, 2010. Eriksen, M., Lebreton, L. C. M., Carson, H. S., Thiel, M., Moore, C. J., Borerro, J. C., Galgani, F., Ryan, P. G., and Reisser, J.: Plas- tic Pollution in the World’s Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Aﬂoat at Sea, PLOS ONE, 9, e111913, doi:10.1371/journal.pone.0111913, 2014. Pugh, D. T.: Tides, surges and mean sea-level: a handbook for engi- neers and scientists., John Wiley & Sonschichester, Chichester, 1987. Foekema, E. M., De Gruijter, C., Mergia, M. T., van Franeker, J. A., Murk, A. J., and Koelmans, A. A.: Plastic in North sea ﬁsh, Environ. Sci. Technol., 47, 8818–8824, 2013. Reisser, J., Shaw, J., Wilcox, C., Hardesty, B. D., Proi- etti, M., Thums, M., and Pattiaratchi, C.: Marine plas- tic pollution in waters around Australia: characteristics, concentrations, and pathways, PLOS ONE, 8, e80466, doi:10.1371/journal.pone.0080466, 2013. Fotopoulou, K. N. and Karapanagioti, H. K.: Surface properties of beached plastic pellets, Mar. Environ. Res., 81, 70–77, 2012. Gregory, M. R.: Environmental implications of plastic debris in ma- rine settings – entanglement, ingestion, smothering, hangers-on, hitch-hiking and alien invasions, Phil. Trans. Roy. Soc. B, 364, 2013–2025, 2009. Reisser, J., Shaw, J., Hallegraeff, G., Proietti, M., Barnes, D. K., Thums, M., Wilcox, C., Hardesty, B. D., and Pattiaratchi, C.: Millimeter-sized marine plastics: a new pelagic habitat for microorganisms and invertebrates, PLOS ONE, 9, e100289, doi:10.1371/journal.pone.0100289, 2014. Hidalgo-Ruz, V., Gutow, L., Thompson, R. C., and Thiel, M.: Mi- croplastics in the marine environment: a review of the methods used for identiﬁcation and quantiﬁcation, Environ. Sci. Technol., 46, 3060–3075, 2012. Reisser, J., Slat, B., Noble, K., du Plessis, K., Epp, M., Proietti, M., de Sonneville, J., Becker, T., and Pattiaratchi, C.: Data from “The vertical ditribution of buoyant plastics at sea: an observational study in the North Atlantic Gyre”, Figshare, 2015. Isobe, A., Kubo, K., Tamura, Y., Nakashima, E., and Fujii, N.: Se- lective transport of microplastics and mesoplastics by drifting in coastal waters, Mar. Pollut. Bull., 89, 324–330, 2014. Kukulka, T., Proskurowski, G., Morét-Ferguson, S., Meyer, D., and Law, K.: The effect of wind mixing on the vertical distribution of buoyant plastic debris, Geophys. Res. Lett., 39, 1–6, 2012. Rochman, C. www.biogeosciences.net/12/1249/2015/ Biogeosciences, 12, 1249–1256, 2015 J. Reisser et al.: The vertical distribution of buoyant plastics at sea M., Hoh, E., Kurobe, T., and Teh, S.: Ingested plastic transfers hazardous chemicals to ﬁsh and induces hepatic stress, Nature, 3, 3263, doi:10.1038/srep03263, 2013. Lattin, G. L., Moore, C. J., Zellers, A. F., Moore, S. L., and Weis- berg, S. B.: A comparison of neustonic plastic and zooplankton at different depths near the southern California shore, Mar. Pollut. Bull., 49, 291–294, 2004. Thiel, M. and Gutow, L.: The ecology of rafting in the marine en- vironment. II. The rafting organisms and community, Oceanogr. Mar. Biol., 43, 279–418, 2005. Tourinho, P. S., Ivar do Sul, J. A., and Fillmann, G.: Is marine debris ingestion still a problem for the coastal marine biota of southern Brazil?, Mar. Pollut. Bull., 60, 396–401, 2010. Law, K. L., Morét-Ferguson, S., Maximenko, N. A., Proskurowski, G., Peacock, E. E., Hafner, J., and Reddy, C. M.: Plastic accu- mulation in the North Atlantic subtropical gyre, Science, 329, 1185–1188, 2010. van Sebille, E., England, M. H., and Froyland, G.: Origin, dynam- ics and evolution of ocean garbage patches from observed sur- face drifters, Environ. Res. Lett., 7, 044040, doi:10.1088/1748- 9326/7/4/044040, 2012. Law, K. L., Moret-Ferguson, S., Goodwin, D. S., Zettler, E. R., De- Force, E., Kukulka, T., and Proskurowski, G.: Distribution of sur- face plastic debris in the eastern Paciﬁc Ocean from an 11-year dataset, Environ. Sci. Technol. 48, 4732–4738, 2014. Watts, A. J., Lewis, C., Goodhead, R. M., Beckett, S. J., Moger, J., Tyler, C. R., and Galloway, T. S.: Uptake and retention of microplastics by the shore crab Carcinus maenas, Environ. Sci. Technol., 48, 8823–8830, 2014. Lebreton, L.-M., Greer, S., and Borrero, J.: Numerical modelling of ﬂoating debris in the world’s oceans, Mar. Pollut. Bull., 64, 653–661, 2012. Winston, J. E., Gregory, M. R., and Stevens, L. M.: Encrusters, epibionts, and other biota associated with pelagic plastics: a re- view of biogeographical, environmental, and conservation issues, in: Marine Debris, Springer, 81–97, 1997. Maximenko, N., Hafner, J., and Niiler, P.: Pathways of marine de- bris derived from trajectories of Lagrangian drifters, Mar. Pollut. Bull., 65, 51–62, 2012. Zettler, E. R., Mincer, T. J., and Amaral-Zettler, L. A.: Life in the “Plastisphere”: Microbial communities on plastic marine debris, Environ. Sci. Technol., 47, 7137–7146, 2013. Biogeosciences, 12, 1249–1256, 2015 www.biogeosciences.net/12/1249/2015/ www.biogeosciences.net/12/1249/2015/
https://openalex.org/W1005729685	https://link.springer.com/content/pdf/10.1007%2Fs12062-015-9122-2.pdf	English	null	The Health of India’s Elderly Population: A Comparative Assessment Using Subjective and Objective Health Outcomes	Journal of population ageing	2,015	cc-by	7,623	Population Ageing (2015) 8:245–259 DOI 10.1007/s12062-015-9122-2 The Health of India’s Elderly Population: A Comparative Assessment Using Subjective and Objective Health Outcomes Jane Murray Cramm1 & Lisa Bornscheuer1 & Anna Selivanova1 & Jinkook Lee2 Received: 26 December 2014 /Accepted: 15 June 2015 /Published online: 24 July 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract This study examined relationships between and predictors of objective and subjective health measures among 766 individuals aged ≥45 years in India using the 2010 pilot wave of the Longitudinal Aging Study in India (LASI). Correlations between and gender differences in objective [grip strength, lung function] and subjective [self- rated health (SRH), dependence in activities of daily living (dADL)] health measures were examined. Multivariate logistic regression analyses, accounting for sample design, were conducted to identify predictors of poor health. Fewer individuals were classified as at risk according to subjective (SRH, 9 %; dADL, 12 %) than objective (lung function, 57 %; grip strength, 77 % women, 87 % men) indicators. Poor SRH was only weakly correlated with dADL (r=0.103, p≤0.05) and grip strength (r=−0.138, p≤0.001). From this study we conclude that older Indians tend to report more positive perception of health than the objective measures of health indicates, and that subjective and objective health indicators capture different aspects of health and only weakly correlated. Keywords Grip strength . Lung function . Self-rated health . Activities of daily living . Elderly. India 1 Department of Health Policy & Management (iBMG), Erasmus University Rotterdam, Oudlaan 50, 3062 PA Rotterdam, The Netherlands 2 Center for Economic & Social Research, University of Southern California, 635 Downey Way, Suite 312, Los Angeles, CA 90089-3332, USA * Jane Murray Cramm cramm@bmg.eur.nl 2 Center for Economic & Social Research, University of Southern California, 635 Downey Way, Suite 312, Los Angeles, CA 90089-3332, USA Introduction The global demographic transition that we are witnessing is caused by simultaneous trends of declining fertility rates coupled with longer life expectancies. Where * Jane Murray Cramm cramm@bmg.eur.nl 246 J.M. Cramm et al. economic development – which is closely linked to these trends via channels such as advancement in medical technologies – is particularly rapid, so is population ageing (Chan 2005; Singh et al. 2013). India falls under the United Nations’ definition of ‘ageing’ countries 1 and is a prime example of a context in which morbidity and mortality patterns are changing rapidly (Bhat and Dhruvarajan 2001; Prakash 1999). India’s elderly population has grown in relative and absolute terms (Bhat and Dhruvarajan 2001; Prakash 1999), and the United Nations Population Division projects that India’s population ages 50 and older will reach 34 % by 2050 (UN 2001). This demographic transition has important social, economic, and public health implications; one example is increased dependency ratios (Bhat and Dhruvarajan 2001; Chan 2005; Fikree and Pasha 2004). Some of these implications are direct consequences of the epidemiological transition that takes place concurrently. India’s changing health burden is characterised by the increased importance of non-communicable diseases (e.g., chronic illnesses) and social and behavioural disorders, relative to more traditional areas of health intervention that are focused on infectious diseases (Chan 2005; Johnson 2005). Efficient public health decisions require a thorough understanding of these developments, and the examination of health outcomes in ageing populations is one way of fostering this understanding. Against this background, this study aims to add to the literature examining the usefulness of different health indicators, with particular regard to self-rated and objec- tive health measures. 1 A country is defined as ‘ageing’ when the percentage of the population aged ≥60 years reaches 7 %. Literature Review SRH has repeatedly been proven to be a powerful and independent predictor of diverse health outcomes, and a stronger predictor of mortality than physician-assessed health as evident by a review of twenty-seven international community studies (Idler and Benyamini 1997) and empirical evidence from developed countries (Alexopoulos and Geitona 2009; Deeg and Bath 2003; Eriksson et al. 2001; McCullough and Laurenceau 2004). Even researchers who could not confirm the independent character of this predictive power, such as Bath (2003) in his study on the relationship between SRH and mortality in elderly men and women concede that differences in study design and cross-cultural variation may underlie the inconsistency of findings (Bath 2003). However, variations in the predictive ability of SRH have been found with regard to age, gender, and length of follow up (Bath 2003; Idler 2003; Jylhä 2009); for example, SRH seems to lose some predictive power in the assessment of older women’s health status (Benyamini et al. 2000; Cooper et al. 2010; McCullough and Laurenceau 2004). The origin of these variations is not clear (Smith and Goldman 2011; Spiers et al. 2003); several possible explanations have been proposed, such as the greater sensitivity of SRH to certain health conditions with differing prevalence across genders (Deeg and Bath 2003; Deeg and Kriegsman 2003). Reporting bias is another issue that compli- cates the use of SRH (Bago d'Uva et al. 2008). For example, Lindeboom and van Doorslaer (2004) confirmed the occurrence of index and cut-point shifting for age and gender when testing for differential reporting in subjective measures such as the McMaster Health Utility Index. If the variations observed in response patterns to health surveys are systematic and related to socio-demographic differences – as they likely are – they severely hinder the correct assessment of health (Bago d’Uva et al. 2008; Sen 2002). Therefore, the state of the art allows us only to state that SRH ‘clearly measure[s] something more – and something less – than objective medical ratings’ (Maddox and Douglass 1973; p. 92). A S (1993 2002) ll f f h i h i SRH i I di S Amartya Sen (1993, 2002) calls for further caution when using SRH in India. Sen argues that the individual’s perception of health might strongly be shaped by the respective socio-economic context. Literature Review A variety of measures have been used to examine individuals’ health status. The most frequently used measures are subjective ones, such as self-rated health (SRH) and self- perceived dependence in activities of daily living (ADL), and also recently few objective measures such as grip strength and lung function gained popularity (Ziebarth 2010). SRH and grip strength are static indicators, capturing deviations from a norm perceived as healthy (Murray and Chen 1992; Ziebarth 2010). By contrast, dependence in ADL is a functional indicator, capturing the inability to perform certain tasks. Dependence in ADL has been associated with various outcomes of interest, such as admission to retirement homes, health care utilisation, and mortality (Tsuji et al. 1994; Luppa et al. 2010; Scott et al. 1997). The measure is particularly useful for assessment of the health status of elderly populations, where prevalence rates are especially high (Wiener et al. 1990). Experience is lacking, however, in its use in the context of developing countries, such as India (Fillenbaum et al. 1999). Grip strength has been associated with a variety of health outcomes and can be used as a proxy for overall muscle strength. Low grip strength has been found to be a consistent predictor of mobility limitations and all-cause mortality (Bohannon 2008; Metter et al. 2002; Rantanen et al. 2000; Sallinen et al. 2010). Lung function has also been found to be a predictor of all-cause mortality in both sexes and different age groups, even when adjusting for factors such as body mass index or smoking (Hole The Health of India’s Elderly Population 247 et al. 1996; Knuiman et al. 1999; Mannino et al. 2003; Neas and Schwartz 1998; Schunemann et al. 2000). et al. 1996; Knuiman et al. 1999; Mannino et al. 2003; Neas and Schwartz 1998; Schunemann et al. 2000). Several studies have found that subjective health measures are independent predic- tors of a variety of health outcomes and can thus be said to predict different ‘parts’ of mortality than more conventional, objective measures (Idler and Benyamini 1997). SRH, among the most frequently used measures, is an ardently debated indicator (Idler and Benyamini 1997; Johnston et al. 2009; Smith and Goldman 2011). Literature Review Particularly, where there is an overall lack of (health) literacy - often the case in areas of poor socio-economic standing - measures of SRH might not be reliable because individuals simply fail to acknowledge certain morbidities, but perceive them to be normal. Sen’s hypotheses are supported by studies such as the one conducted by Sudha et al. (2007) in South India in an elderly population, finding that marital status may bias measures of SRH, and may do so differently across genders. Holding against these concerns is e.g., a study conducted by Subramanian et al. (2009), that maintains that those individuals with lower SES are in fact more likely to report morbidities, even when controlling for the objective level of health. Supporting the validity of self-rated health measures are also findings from a study amongst people of 50 years or older in Bangladesh, where SRH was found to be significantly associated 248 J.M. Cramm et al. with measured physical performance, even when controlling for other factors (Rahman and Barsky 2003). Furthermore, Hirve et al. (2012) found a significant association between SRH and death independent of SES for an elderly Indian population segment. However, this study again also points to potential differences in SRH validity across genders. The use of subjective health measures is the norm in surveys; objective measures and biomarkers are rarely used in this context. The introduction of such measures may provide an important complement to SRH and similar indicators (Ambrasat et al. 2011; Johnston et al. 2009; Kakwani et al. 1997). Thus, this study aimed to increase our understanding of health among elderly individuals in India by investigating relation- ships between subjective (SRH and dependence in ADL) and objective (grip strength and lung function) health indicators, and examining variation in predictor variables among health outcomes. Methods This study employed cross-sectional data from the 2010 pilot wave of the Longitudinal Aging Study in India (LASI), conducted in collaboration by the Harvard School of Public Health, the International Institute for Population Sciences, and the RAND Corporation. The primary objective of LASI is to provide Indian policymakers with the information needed to improve health status and health behaviour in the country’s ageing population. Social, economic, and health data (e.g., income, work/employment history, SRH, blood pressure, grip strength) were collected from a nationally represen- tative sample of the population aged ≥45 years. Sample The LASI pilot study took place in the northern Indian states of Rajasthan and Punjab, and the southern states of Kerala and Karnataka. The sampling plan was based on the 2001 Indian census. To adequately represent different socio-economic conditions, the eight districts that served as primary sampling units were stratified across urban and rural areas. Eligible households had at least one member aged ≥45 years, and eligible individuals were aged ≥45 years or married to an individual of that age. A total of 1546 households was randomly sampled, and 1683 interviews were conducted in 950 of these households. The response rates were 88.5 % for households and 90.9 % for individuals. Within the bounds of the individual survey, grip strength, lung function, and other biomarker data were collected from a smaller sample of 928 consenting eligible individuals (interviewees and their spouses aged ≥45 years) (Chien et al. 2013a, b, 2014). Grip Strength The LASI team measured grip strength using the sex-specific cut-off points reported by Sallinen and colleagues (2010): 21 kg for women (67 % sensitivity, 73 % specificity) and 37 kg for men (62 % sensitivity, 76 % specificity) (Chien et al. 2013a, b, 2014). This outcome measure was dichotomised as at risk (below cut-off value) or not at risk (cut-off value or above). Lung Function The LASI team used spirometry to measure lung function by examining the amount (volume) and speed (flow) of air that an individual was able to inhale or exhale. Measurement took place in the population setting. The flow electronic volume [forced expiratory volume in 1 s (FEV1) / forced vital capacity (FVC)] was used to identify lung problems. The criteria advanced by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) were used to classify the severity of chronic obstructive pulmonary disease (COPD, defined as FEV1 / FVC < 0.70). The severity of airflow limitation in COPD, was characterised as mild, moderate, severe, or very severe) (Chien et al. 2013a, b, 2014; Soriano et al. 2013). This health measure was dichotomised as normal or abnormal spirometry values. Self-Rated Health Self-Rated Health Self-Rated Health SRH was assessed by asking respondents to rate their current overall health on a five- point ordinal scale ranging from poor (1) to excellent (5) (full range: poor, fair, good, The Health of India’s Elderly Population 249 very good, excellent). This outcome variable was dichotomised as poor (1, 2) or good (3–5) (Chien et al. 2013a, b; Cramm and Lee 2014). very good, excellent). This outcome variable was dichotomised as poor (1, 2) or good (3–5) (Chien et al. 2013a, b; Cramm and Lee 2014). very good, excellent). This outcome variable was dichotomised as poor (1, 2) or good (3–5) (Chien et al. 2013a, b; Cramm and Lee 2014). Dependence in Activities of Daily Living Dependence in Activities of Daily Living Respondents’ ability to perform ADL was assessed using six variables: walking across the room, dressing oneself, bathing oneself, eating, getting out of bed, and using the toilet. Each item had four possible responses indicating that the respondent had difficulty (1) or no difficulty (2) with the task, or could not (3) or did not want to (4) perform the task. Responses 1 and 3 were considered to indicate some difficulty (coded as 1), 2 was considered to indicate no difficulty (coded as 0), and responses of 4 were recorded as missing values. The LASI team derived two summary ADL measures. The first measure used the ADL recommended by Wallace and Herzog (1995), including ‘some difficulty’ scores for the individual measures of bathing, dressing, and eating. The second summary measure additionally included getting in/out of bed and walking across the room. Summary measures were computed only for respondents with no missing value for any individual measure (Chien et al. 2013a, b). The summary variable was dichotomised as difficulty or no dependence in ADL. Statistical Analyses Descriptive analyses were used to examine objective and subjective health measures. We corrected health characteristics for age-gender variations, so we used age-gender standardized values. Since we use data for elderly population (age 45 and more), age variable was split into 3 categories: age 45–60, age 60–75 and age>75. In addition, we used concentration curves to investigate the association between socioeconomic status and health outcomes (objective and subjective). Pearson’s product–moment correlation analyses were then used to investigate relationships between these measures. Differ- ences in health outcomes between men and women were examined using chi-squared and independent-samples t-tests. Multivariate logistic regression analyses were used to identify and compare predictors of poor health across genders in India’s elderly population, while accounting for sample design effect. Socio-Demographic Measures The socio-demographic measures used in this research are age, marital status, educa- tional attainment, quintiles of income, place of residence, caste, and gender. Place of residence was categorised in terms of rural/urban environment. Educational status was classified using four categories (illiterate, primary, secondary and higher education). 250 J.M. Cramm et al. Castes were classified as scheduled castes, scheduled tribes, other backward group (OBG) and non-scheduled castes or tribes. The first two categories are historically disadvantaged social classes in India (Chien et al. 2013a, b). Furthermore, the variable smoking was included in the analysis. In addition, we constructed the variable ‘un- healthy environment’ which takes value of 1 if there is indoor smoking at home and/or the cooking is made under chimney, the variable takes value of 0 if there is no indoor smoking and cooking is not fulfilled under chimney. In such a way it is possible to observe if lung function is associated with smoking or unhealthy smoking environment at home. Castes were classified as scheduled castes, scheduled tribes, other backward group (OBG) and non-scheduled castes or tribes. The first two categories are historically disadvantaged social classes in India (Chien et al. 2013a, b). Furthermore, the variable smoking was included in the analysis. In addition, we constructed the variable ‘un- healthy environment’ which takes value of 1 if there is indoor smoking at home and/or the cooking is made under chimney, the variable takes value of 0 if there is no indoor smoking and cooking is not fulfilled under chimney. In such a way it is possible to observe if lung function is associated with smoking or unhealthy smoking environment at home. Results Descriptive analyses showed that the relative distributions of individual health out- comes differed between subjective and objective measures. Although 9 % of respon- dents reported poor health and 12 % reported difficulties in ADL, nearly 57 % of respondents had below-normal spirometry values and the majority of respondents of both sexes (77 % of women, 87 % of men) had below-threshold grip strength values. Tables 1 and 2 shows the descriptive statistics of health outcomes across socio- demographic characteristics among older men women in India (standardized). Concentration curves reveal a relationship between income and health outcomes (objective and subjective) (see Graph 1). Objective and subjective health measures showed no correlation or weak relation- ships. Poor health was significantly correlated with dependence in ADL (r=0.103, p≤ 0.05) and grip strength (r=−0.138, p≤0.001; Table 3). Tables 4 and 5 present the results of multivariate logistic regression analysis of subjective and objective health indicators among men and women, respectively. Among men, older age and being single increased the probability of being in poor health; age, educational level, income and tribe/caste/background class predicted lung function; older age and smoking increase the likelihood of dependence in ADL; educational level, income and indoor smoking predicted grip strength. Results Cramm et al. Results Among women, residence, age, marital status, income and indoor smoking predicted SRH; age, educational level, cooking under chimney and income predicted lung The Health of India’s Elderly Population 251 Table 1 Descriptive statistics of health outcomes across socio-demographic characteristics among older men in India(standardized) Over SRH (poor) n=712 Mean prevalence(SD) Lung function (abnormal) n=712 Mean prevalence(SD) ADL (difficulty) n=638 Mean prevalence(SD) Grip strength (low) n=712 Mean(SD) Age 45–60 7.4 % (4 % – 10.8 %) 56.8 % (49 % – 64.7 %) 7.1 % (4 % –10.2 %) 73.7 % (68.2 % – 79.3 %) 60–75 17.2 %(11 % – 23.4 %) 51.3 % (41.3 % –61.3 %) 16.6 % (10.3 % –23 %) 78.7 % (72 % –85.4 %) Older than 75 22.5 % (10.6 % – 34.5 %) 38.3 % (23.9 % – 52.8 %) 25.7 % (12.9 % – 38.5 %) 80.1 %(68.15 % –92.1 %) Education level Illiterate 11.1 % (6.8 % – 15.4 %) 49.7 % (42.4 % – 57 %) 11.6 % (7.7 % – 15.4 %) 78.1 % (73.5 % – 82.7 %) Primary school 16.7 % (10.9 % –22.4 %) 70 % (61.8 % – 78.2 %) 13.9 % (6.3 % – 21.4 %) 68.3 % (59.6 % – 77.1 %) Secondary school 13.1 % (6.6 % – 19.7 %) 69.1 % (60 % – 78.2 %) 10 % (6 % – 14 %) 73.1 % (65.3 % – 81 %) Higher education 6.7 % (1.6 % – 11.7 %) 66.7 % (54.7 % – 78.6 %) 13.4 % (6.7 % – 20.2 %) 73.3 %(64.5 % –82.2 %) Income quintiles 1 14.5 % (7.5 % – 21.4 %) 14.1 % (6.5 % – 21.7 %) 56 % (43.4 % – 68.5 %) 78.9 % (71 % – 86.9 %) 2 11.3 % (5.7 % – 17 %) 11 % (5.5 % – 16.5 %) 56.7 % (42.3 % – 71.2 %) 87.3 % (80.6 % – 93.9 %) 3 13 % (6.1 % – 20 %) 12 % (4.9 % – 19 %) 75.3 % (67.9 % – 82.7 %) 92.2 % (87.7 % – 96.7 %) 4 10.8 % (4.8 % – 16.7 %) 8.3 % (2.7 % – 14 %) 52.7 % (42.6 % – 62.8 %) 84.4 % (76.8 % – 91.9 %) 5 11.2 % (4.1 % – 18.3 %) 11.3 % (5 % – 17.5 %) 64.6 % (47.9 % – 81.3 %) 83.7 % (75.4 % – 92 %) Background Scheduled caste 13.6 % (5.2 %–21.9 %) 60.2 % (50.8 % – 69.6 %) 11.2 % (5.4 % – 17 %) 72.9 % (65.4 % – 80.4 %) Scheduled tribe 2.9 % (2.5 % – 8.2 %) 77.1 % (61.5 % – 92.8 %) 0 82.9 % (69.4 % – 96.3 %) Other background class 11.6 % (7.4 % – 15.8 %) 63.8 % (56.3 % – 71.2 %) 14.8 % (10.5 %–19.1 %) 77.5 % (72.4 % – 82.7 %) None 13.3 % (8.5 % – 18 %) 51.2 % (43.7 % – 58.7 %) 10.4 % (6.3 % – 14.4 %) 71.9 % (65.9 % – 77.8 %) 252 J.M. Results Table 2 Descriptive statistics of health outcomes across socio-demographic characteristics among older women in India(standardized) Over SRH (poor) n=723 Mean prevalence(SD) Lung function (abnormal) n=726 Mean prevalence(SD) ADL (difficulty) n=662 Mean prevalence(SD) Grip strength (low) n=725 Mean prevalence (SD) Age 45–60 10.1 % (6.2 % – 14 %) 57.7 % (51 % – 64.4 %) 10.9 % (6.5 % – 15.4 %) 73.8 % (68.8 % – 78.8 %) 60–75 19.8 % (13.3 % – 26.2 %) 44.4 % (37.8 % – 51.9 %) 18.3 % (12.3 % – 24.2 %) 69.9 % (63.4 % – 76.5 %) Older than 75 38 % (24.2 % – 52 %) 37.1 % (23.5 % –50.8 %) 36.4 % (22.2 % – 50.6 %) 70.1 % (56.5 % – 83.8 %) Education level Illiterate 16.7 % (12.4 % – 21 %) 48.2 % (42.6 % – 53.8 %) 15.6 % (11 % – 20 %) 73.2 % (68.9 % – 77.4 %) Primary school 19.2 % (8.9 % – 29.5 %) 64 % (52.9 % – 75.1 %) 23.9 % (13.9 % – 33.8 %) 52 % (42.2 % – 61.8 %) Secondary school 15.6 % (10.3 % – 21 %) 64.2 % (57.3 % – 71.1 %) 11.6 % (0.078 – 0.154) 70.9 % (61.8 % – 80.1 %) Higher education 6 % (2 % –14.2 %) 75.8 % (60.4 % – 91.1 %) 9.38 % (0.2 % – 19 %) 51.5 % (29.8 % – 73.2 %) Income quintiles 1 11.7 % (4.7 % – 18.8 %) 47.6 % (34 % – 61.1 %) 14 % (6.4 % – 21.7 %) 80.6 % (73.9 % – 87.3 %) 2 13 % (7.5 % – 18.6 %) 56.3 % (44 % – 68.5 %) 16.9 % (10.4 % – 23.4 %) 80.3 % (75.2 % – 85.4 %) 3 11.1 % (6 % – 16.3 %) 61.1 % (53 % – 69.2 %) 11.7 % (6 % – 17.4 %) 82.5 % (75.6 % – 89.5 %) 4 26.4 % (16.9 % – 36 %) 52.7 % (42.7 % – 62.7 %) 22 % (11.8 % – 32.2 %) 68.7 % (59.4 % – 78 %) 5 14.3 % (7.6 % – 20.9 %) 56 % (43 % – 69.2 %) 11.9 % (5.3 % – 18.6 %) 73.8 % (63.5 % – 84.2 %) Background Scheduled caste 11.5 % (5.9 %–17.1 %) 56.6 % (46.3 %–66.8 %) 15.3 % (8 %–22.6 %) 67.2 % (59.3 %–75.1 %) Scheduled tribe 19 % (8.5 % – 29.6 %) 50 % (26.9 % – 73.1 %) 17.5 % (5.3 % – 29.7 %) 81 % (72.6 % – 89.3 %) Other background class 15.8 % (10.9 % – 20.7 %) 57 % (50.8 % – 63.2 %) 11.5 % (7.9 % – 15.1 %) 66.2 % (61.5 % – 70.9 %) None 19.8 % (13.5 % – 26.1 %) 52.2 % (42.9 % – 61.5 %) 20.6 % (14.3 % – 26.9 %) 69.9 % (63.3 % – 76.5 %) The Health of India’s Elderly Population 253 function; age, income and smoking predicted ADL; educational level and smoking predicted grip strength. Results Graph 1 Cumulative population proportions Graph 1 Cumulative population proportions Graph 1 Cumulative population proportions function; age, income and smoking predicted ADL; educational level and smoking predicted grip strength. function; age, income and smoking predicted ADL; educational level and smoking predicted grip strength. Discussion This study added weight and detail to the findings of similar studies with the use of two objective health outcomes, as well as subjective measures, in the assessment of health in India’s elderly population. The most striking finding of this study is the weakness or lack of correlation between subjective and objective health indicators. These findings were also reported by Goverover and colleagues (2005) in their study investigating the relation between subjective and objective measures of everyday life activities in persons with multiple sclerosis. They concluded that all correlations between subjective and objective functional performance measures were low and non-significant, which is consistent with other studies in among patients with MS (Hoogervorst et al. 2003; Doble et al. 1994), Alzheimer patients (Bertrand and Willis 1999) and hospitalized older patients (Sager et al. 1992). The lack of a significant relation between objective and subjective health measures suggests that they each provide unique contributions to the evaluation of health status. Self-report can provide information about a person’s own perceptions regarding their health that cannot be measured using an objective assessment tool. Objective health measures are usually task oriented and are rated along a number of physical and/or cognitive dimensions enabling the observer to make a judgment as to a very specific aspect of health or health activity that can or cannot be performed independently (Gitlin 2001). As such, reliance solely on self-report health measures seem to provide information that may not reflect actual health performance able 3 Pearson correlations between subjective and objective health outcomes Table 3 Pearson correlations between subjective and objective health outcomes 1 2 3 1. Poor health (subjective) 2. Grip strength (objective) −0.138*** 3. Dependence in ADL (subjective) 0.103* −0.049 4. Abnormal spirometry value (objective) 0.052 0.052 −0.011 ADL activities of daily living. p≤0.05, p≤0.001 254 J.M. Cramm et al. Discussion Cramm et al. (e.g., grip strength or lung function) in everyday life (Goverover et al. 2005). Longi- tudinal research indeed showed that perceived or subjective health is a significant predictor of both mortality and morbidity even more so than objective health measures (Menec et al. 1999; Mossey 1995; Mossey and Shapiro 1982; Reuben et al. 1992). Furthermore, two literature reviews (Idler and Benyamini 1997) proved that subjective health remained a significant predictor of mortality, after controlling for objective health status as well as other covariates such as socioeconomic status, social support, and risk behaviours. This calls for the inclusion of both subjective as well as objective health indicators since they seem to catch ‘different parts’ of a person’s overall health. y p p The limitations of this study include the cross-sectional nature of the data, which prevented the examination of causality. Furthermore, it would have been beneficial to include a measure of economic dependency in the analysis, similar to the approach taken by Roy and Chaudhuri (2008). While this study included both objective and subjective health indicators and shed some light on their (lack of) relationship there is still a lot to learn. While in line with earlier studies (Goverover et al. 2005; Hoogervorst et al. 2003; Bertrand and Willis 1999; Doble et al. 1994; Sager et al. 1992) we found that objective and subjective health indicators were not or only very weakly related to each other use of other health measures, however, might lead to different findings. Still, we believe that a strength of this study is the inclusion of subjective as well as objective indicators of health. Biomarker data collection is a valuable contribution of the LASI study, as such information – crucial for the assessment of health status – is scarce given India’s poor health care infrastructure (Chien et al. 2013a, b, 2014). In conclusion, the main contribution of this research to the existing literature is the finding that no strong relationship between the health outcomes examined persists. The disparity between the small percentages of the population classified as ‘at risk’ accord- ing to subjective measures, and the majority of individuals determined to be at risk according to objective indicators, suggests that they reflect different ‘parts’ of mortality and morbidity. Discussion Table 4 Relationships between socio-demographic characteristics and health outcomes among older men in India SRH (poor) n=640 Adjusted OR Lung function (abnormal) n=640 Adjusted OR ADL (difficulty) n=637 Adjusted OR Grip strength (low) n=640 Adjusted OR Residence(rural) 1.341 (0.545 – 3.3) 0.666 (0.328 – 1.352) 0.819 (0.367 – 1.826) 0.683 (0.323 – 1.446) Age(45–60) 0.199(0.076 – 0.519)* 3.189 (1.419 – 7.168) * 0.112 (0.04 – 0.318)* 0.659 (0.24 – 1.809) Age(60–75) 0.518 (0.213 – 1.262) 1.698 (0.744 – 3.875) 0.332 (0.125 – 0.885) * 0.673 (0.219 – 2.066) Education(primary) 1.28 (0.675 – 2.426) 3.865 (2.02 – 7.397) * 1.045 (0.403 – 2.71) 0.368 (0.199 – 0.68) * Education(secondary) 1.146 (0.513 – 2.558) 3.02 (1.672 – 5.452) * 0.774 (0.356 – 1.684) 0.434 (0.248 – 0.758) * Education(higher) 0.503 (0.176 – 1.439) 2.308 (1.378 – 3.868)* 1.028 (0.403 – 2.62) 0.513 (0.25 – 1.05) Income quintile 2(upper middle) 0.75 (0.326 – 1.72) 0.968 (0.475 – 1.973) 0.607 (0.228 – 1.613) 2.14 (0.896 – 5.115) Income quintile 3(middle) 1.308 (0.573 – 2.986) 2.993 (1.487 – 6.026) * 1.18 (0.408 – 3.414) 2.859 (1.223 – 6.682) * Income quintile 4(poor) 0.946 (0.333 – 2.689) 0.748 (0.389 – 1.439) 0.603 (0.216 – 1.675) 1.504 (0.75 – 3.014) Income quintile 5(poorest) 0.95 (0.386 – 2.342) 1.354 (0.545 – 3.362) 0.996 (0.370 – 2.679) 1.74 (0.854 – 3.542) Marital status(single) 2.746 (1.102 – 6.845)* 1.193 (0.664 – 2.144) 0.873 (0.308 – 2.476) 1.343 (0.467 – 3.862) Scheduled tribe 1.28 (0.675 – 2.426) 2.228 (0.753 – 6.593) 1 0.9 (0.363 – 2.235) Other background class 1.146 (0.513 – 2.558) 0.791 (0.4 – 1.566) 1.305 (0.561 – 3.034) 1.48 (0.755 – 2.901) No tribe/caste/background class 0.503 (0.318 – 2.164) 0.521 (0.263 – 1.034) 0.982 (0.405 – 2.381) 0.98(0.519 – 1.848) Smoker(yes) 1.352 (0.782 – 2.337) 1.319 (0.734 – 2.371) 2.234 (0.966 – 5.168) 1.68 (0.866 – 3.529) Indoor smoking (yes) 1.165 (0.662 – 2.051) 0.932 (0.503 – 1.724) 0.735 (0.312 – 1.73) 0.585 (0.309 –1.108) Cooking under chimney(yes) 1.234 (0.644 – 2.365) 1.339 (0.848 – 2.113) 0.878 (0.467 – 1.653) 1.339 (0.764 – 2.347) Model F 1.76 3.64* 3.04* 2.39*** SRH self-rated health, OR odds ratio, ADL activities of daily living * p≤0.05; p≤0.1; * p≤0.001 The Health of India’s Elderly Population 255 Table 5 Relationships between socio-demographic characteristics and health outcomes among older women in India SRH (poor) n=667 Adjusted OR Lung function (abnormal) n=668 Adjusted OR ADL (difficulty) n=661 Adjusted OR Grip strength (low) n=668 Adjusted OR Residence(rural) 1.689 (0.849 – 3.362) 1.06 (0.604 – 1.86) 0.815 (0.396 – 1.678) 1.34 (0.888 – 2.046) Age(45–60) 0.2 (0.082 – 0.489)* 1.817 (0.907 – 3.64) 0.197 (0.099 – 0.392)* 0.609 (0.208 – 1.784) Age(60–75) 0.456 (0.187 – 1.113) 1.145 (0.556 – 2.359) 0.297 (0.123 – 0.392) * 0.634 (0.237 – 1.701) Education(primary) 1.179 (0.603 – 2.305) 2.592 (1.513 – 4.442) * 1.788 (0.881 – 3.626) 0.402 (0.222 – 0.727) * Education(secondary) 1.09 (0.538 – 2.205) 1.873 (1.196 – 2.933) * 0.857 (0.46 – 1.596) 1.164 (0.65 – 2.086) Education(higher) 0.433 (0.091 – 2.048) 3.571 (1.431 – 8.91) * 0.653 (0.159 – 2.689) 0.425 (0.153 – 1.178) ** Income quintile 2(upper middle) 0.956 (0.409 – 2.235) 1.366 (0.702 – 2.661) 1.086 (0.42 – 2.811) 1.001 (0.509 – 1.969) Income quintile 3(middle) 0.85 (0.34 – 2.126) 1.852 (1.003 – 3.419) * 0.703 (0.311 – 1.592) 1.079 (0.509 – 2.29) Income quintile 4(poor) 2.421 (1.072 – 5.466) * 1.254 (0.654 – 2.405) 1.461 (0.628 – 3.397) 0.471 (0.238 – 0.931)* Income quintile 5(poorest) 1.093 (0.445 – 2.687) 1.17 (0.506 – 2.708) 0.873 (0.345 – 2.213) 0.728 (0.32 – 1.659) Marital status(single) 2.971 (1.902 – 4.64) * 0.8 (0.546 – 1.173) 2.492 (1.407 – 4.412) * 1.369 (0.836 – 2.242) Scheduled tribe 1.666 (0.648 – 4.28) 1.011 (0.293 – 3.494) 1.272 (0.454 – 3.567) 1.777 (0.578 – 5.47) Other background class 1.735 (0.793 – 3.796) 1.13 (0.629 – 2.03) 0.881 (0.512 – 1.518) 0.872 (0.547 – 1.39) No caste/tribe/background class 2.075(0.889 – 0.844) 0.84 (0.427 – 1.65) 1.437 (0.737 – 2.8) 1.403 (0.856 – 2.3) Smoker(yes) 1.175 (0.502 – 2.754) 1.292 (0.645 – 2.587) 2.39 (1.05 – 5.444) 3.419 (0.807 – 14.486)*** Indoor smoking(yes) 1.846 (1.02 – 3.34)* 0.881 (0.495 – 1.567) 1.482 (0.787 – 2.789) 1.112 (0703–1.758) Cooking under chimney(yes) 0.634 (0.357 – 1.127) 1.72 (1.092 – 2.709)* 0.688 (0.409 – 1.157) 0.644 (0.383 – 1.082) Model F 3.27* 1.96* 3.35*** 2.22* SRH self-rated health, OR odds ratio, ADL activities of daily living * ≤0 05 ≤0 1 * ≤0 001 256 J.M. Conflict of interest No conflict of interest. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduc- tion 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. Discussion While ADL or SRH is expected to be low in our study sample the high proportion of older people in India with low grip strength and poor lung function is a concern. Further study is thus warranted to obtain a full understanding of the correct application and interpretation of different health indicators depending on the problem of interest, the context in which it is investigated, and the population involved. Alexopoulos, E. C., & Geitona, M. (2009). Self-rated health: inequalities and potential determinants. International Journal of Environmental Research and Public Health, 6(9), 2456–2469. References Alexopoulos, E. C., & Geitona, M. (2009). Self-rated health: inequalities and potential determinants. International Journal of Environmental Research and Public Health, 6(9), 2456–2469. The Health of India’s Elderly Population 257 Ambrasat, J., Schupp, J., & Wagner, G. G. (2011). Comparing the predictive power of subjective and objective health indicators: changes in handgrip strength and overall satisfaction with life as predictors of mortality. SOEPpapers on Multidisciplinary Panel Data Research, 398. Bago d’Uva, T., Van Doorslaer, E., Lindeboom, M., & O’Donnell, O. (2008). Does reporting heterogeneity bias the measurement of health disparities? Health Economics, 17(3), 351–375. Bath, P. A. (2003). Differences between older men and women in the self-rated health–mortality relationshi The Gerontologist, 43(3), 387–395. Benyamini, Y., Leventhal, E. A., & Leventhal, H. (2000). Gender differences in processing information f making self-assessments of health. Psychosomatic Medicine, 62(3), 354–364. Bertrand, R. M., & Willis, S. L. (1999). Everyday problem solving in Alzheimer’s patients (a comparison of subjective and objective assessments). Aging & Mental Health, 3, 281–293. Bhat, A. K., & Dhruvarajan, R. (2001). Ageing in India: drifting intergenerational relations, challenges and options. Ageing and Society, 21(5), 621–640. Bohannon, R. W. (2008). Hand-grip dynamometry predicts future outcomes in aging adults. Journal of Geriatric Physical Therapy, 31(1), 3–10. Chan, A. (2005). Aging in southeast and east Asia: issues and policy directions. Journal of Cross-Cultural Gerontology, 20(4), 269–284. Chien, S., Feeney, K., & Lee, J. (2013a). Longitudinal aging study in India: User guide for pilot data. RAND Labor and Population working paper series. Chien, S., Feeney, K., & Lee, J. (2013b). Harmonized LASI pilot data documentation: Version A. RAN Labor & Population. Chien, S., Feeney, K., & Lee, J. (2013b). Harmonized LASI pilot data documentation: Version A. RAND Labor & Population. Chi S & (2014) d l d d l b k d d Chien, S., Feeney, K., & Lee, J. (2014). Longitudinal aging study in India: Pilot biomarker data docume tation. RAND Cooper, R., Kuh, D., Hardy, R., FALCon Study Team, FALCon and HALCyon Study Teams, on behalf of the FALCon and HALCyon study teams, & HALCyon Study Team & Mortality Review Group. (2010). Objectively measured physical capability levels and mortality: systematic review and meta-analysis. BMJ Clinical Research, 341(7774), c4467. Cramm, J. M., & Lee, J. (2014). Smoking, physical activity and healthy aging in India. BMC Public Health, 14(1), 526. Deeg, D. J. H., & Bath, P. A. (2003). References Discussion: gender differences in self-rated health, in mortality, and in the relationship between the two. The Gerontologist, 43(3), 372–375. Idler, E. L., & Benyamini, Y. (1997). Self-rated health and mortality: a review of twenty-seven community studies. Journal of Health and Social Behavior, 38(1), 21–37. Johnson, M. L. (Ed.), in association with V. L. Bengtson, P. G. Coleman, and T. B .L. Kirkwood (2005). The Cambridge handbook of age and ageing. Cambridge: Cambridge University Press. Johnston, D. W., Propper, C., & Shields, M. A. (2009). Comparing subjective and objective measures of health: evidence from hypertension for the income/health gradient. Journal of Health Economics, 28(3), 540–552. Jylhä, M. (2009). What is self-rated health and why does it predict mortality? Towards a unified conceptu model. Social Science & Medicine, 69(3), 307–316. Kakwani, N., Wagstaff, A., & Van Doorslaer, E. (1997). Socioeconomic inequalities in health: measurement, computation, and statistical inference. Journal of Econometrics, 77(1), 87–103. Knuiman, M. W., James, A. L., Divitini, M. L., Ryan, G., Bartholomew, H. C., & Musk, A. (1999). Lung function, respiratory symptoms, and mortality: results from the Busselton Health Study. Annals of Epidemiology, 9(5), 297–306. Lindeboom, M., & Van Doorslaer, E. (2004). Cut-point shift and index shift in self-reported health. Journal Health Economics, 23(6), 1083–1099. Luppa, et al. (2010). Prediction of institutionalization in the elderly. A systematic review. Age and Ageing, 3 31–38. Maddox, G. L., & Douglass, E. B. (1973). Self-assessment of health: a longitudinal study of elderly subjects. Journal of Health and Social Behavior, 14(1), 87–93. Mannino, D. M., Buist, A. S., Petty, T. L., Enright, P. L., & Redd, S. C. (2003). Lung function and mortality in the United States: data from the first national health and nutrition examination survey follow up study. Thorax, 58(5), 388–393. McCullough, M. E., & Laurenceau, J. (2004). Gender and the natural history of self-rated health: a 59-ye longitudinal study. Health Psychology, 23(6), 651–655. Menec, V. H., Chipperfield, J. G., & Perry, R. P. (1999). Self-perceptions of health: a prospective analysis of mortality, control, and health. Journals of Gerontology: Biological Sciences & Medical Sciences, 54B, 85–93. Metter, E. J., Talbot, L. A., Schrager, M., & Conwit, R. (2002). Skeletal muscle strength as a predictor of all- cause mortality in healthy men. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 57(10), B359–B365. Mossey, J. M. (1995). Importance of self-perceptions for health status among older persons. In M. References Self-rated health, gender, and mortality in older persons: introduction to a special section. The Gerontologist, 43(3), 369–371. Deeg, D. J., & Kriegsman, D. M. (2003). Concepts of self-rated health: specifying the gender difference in mortality risk. The Gerontologist, 43(3), 376–386. Doble, S. E., Fisk, J. D., Fisher, A. G., Ritvo, P. G., & Murray, T. J. (1994). Functional competence of community-dwelling persons with multiple sclerosis using the assessment of motor and process skills. Archives of Physical Medicine and Rehabilitation, 83, 1070–1079. Eriksson, I., Undén, A. L., & Elofsson, S. (2001). Self-rated health. Comparisons between three different measures. Results from a population study. International Journal of Epidemiology, 30(2), 326–333. Fikree, F. F., & Pasha, O. (2004). Role of gender in health disparity: The South Asian context. England: BMJ Publishing Group. Fillenbaum, G. G., Chandra, V., Ganguli, M., Pandav, R., Gilby, J. E., Seaberg, E. C., Belle, S., Baker, C., Echement, D. A., & Nath, L. M. (1999). Development of an activities of daily living scale to screen for dementia in an illiterate rural older population in India. Age and Ageing, 28(2), 161–168. Goverover, Y., Kalmar, J., Gaudino-Goering, E., Shawaryn, M., Moore, N. B., Halper, J., & DeLuca, J. (2005). The relation between subjective and objective measures of everyday life activities in persons with multiple sclerosis. Archives of Physical Medicine and Rehabilitation, 86(12), 2303–2308. Griffith, K. A., Sherrill, D. L., Siegel, E. M., Manolio, T. A., Bonekat, H. W., & Enright, P. L. (2001). Predictors of loss of lung function in the elderly: the cardiovascular health study. American Journal of Respiratory and Critical Care Medicine, 163(1), 61–68. Hirve, et al. (2012). Does self-rated health predict death in adults aged 50 years and above in India? Evidence from a rural population under health and demographic surveillance. International Journal of Epidemiology, 41, 1719–1727. Hole, D. J., Watt, G. C., Davey-Smith, G., Hart, C. L., Gillis, C. R., & Hawthorne, V. M. D1996]. Impaired lung function and mortality risk in men and women: findings from the Renfrew and Paisley prospective population study. BMJ Clinical Research, 313D7059], 711–715 Ddiscussion 715–716]. D ] Hoogervorst, E. L., Eikelenboom, M. J., Uitdehaag, B. M., & Polman, C. H. (2003). One year changes in disability in multiple sclerosis (neurological examination compared with patient self-report). Journal of Neurology, Neurosurgery, and Psychiatry, 74, 439–442. 258 J.M. Cramm et al. Idler, E. L. (2003). References Gatz (Ed.), Emerging issues in mental health and aging (pp. 124–162). Washington, D.C: American Psychological Association. Mossey, J. M., & Shapiro, E. (1982). Self-rated health: a predictor of mortality among the elderly. America Journal of Public Health, 72, 800–808. Murray, C. J., & Chen, L. C. (1992). Understanding morbidity change. The Population and Development Review, 481–503. Neas, L. M., & Schwartz, J. (1998). Pulmonary function levels as predictors of mortality in a national sample of US adults. American Journal of Epidemiology, 147(11), 1011–1018. Prakash, I. J. (1999). Ageing in India. Prepared for the World Health Organization. http://www.popbook.com/ en/in/pdf/ageinginindia.pdf. Rahman, M. O., & Barsky, A. J. (2003). Self-reported health among older Bangladeshis: how good a health indicator is it? Gerontologist, 43(6), 856–863. Rantanen, T., Harris, T., Leveille, S. G., Visser, M., Foley, D., Masaki, K., et al. (2000). Muscle strength and body mass index as long-term predictors of mortality in initially healthy men. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 55(3), M168–M173. Reuben, D. B., Siu, A. L., & Kimpau, S. (1992). The predictive validity of self-report and performance-based measures of function and health. Journal of Gerontology: Medical Sciences, 47, M106–M110. Roy, K., & Chaudhuri, A. (2008). Influence of socioeconomic status, wealth and financial empowerment on gender differences in health and healthcare utilization in later life: evidence from India. Social Science & Medicine, 66(9), 1951–1962. Sager, M. A., Dunham, N. C., Schwantes, A., Mecum, L., Halverson, K., & Harlowe, D. (1992). Measurement of activities of daily living in hospitalized elderly (a comparison of self-report and performance-based methods). Journal of American Geriatrics Society, 40, 457–462. The Health of India’s Elderly Population 259 Sallinen, J., Stenholm, S., Rantanen, T., Heliövaara, M., Sainio, P., & Koskinen, S. (2010). Hand-grip strength cut points to screen older persons at risk for mobility limitation. Journal of the American Geriatrics Society, 58(9), 1721–1726. Schunemann, H. J., Dorn, J., Grant, B. J., Winkelstein, W., & Trevisan, M. (2000). Pulmonary function is a long-term predictor of mortality in the general population: 29-year follow-up of the Buffalo Health Study. CHEST Journal, 118(3), 656–664. ( ), Scott, W., Macera, C., Cornman, C., & Sharpe, P. (1997). Functional health status as predictor of mortality in men and women over 65. Journal of Clinical Epidemiology, 50(3), 291–296. Sen, A. (1993). Positional objectivity. Philosophy and Public Affairs, 22, 126–145. Sen, A. (2002). Health: perception versus observation. British Medical Journal, 324(7342), 860–861. References Singh, L., Arokiasamy, P., Singh, P. K., & Rai, R. K. (2013). Determinants of gend health among older population: evidence From India. SAGE Open, 3, 1–12. Singh, L., Arokiasamy, P., Singh, P. K., & Rai, R. K. (2013). Determinants of gender differences in self-rated health among older population: evidence From India. SAGE Open, 3, 1–12. S i h K V & G ld N (2011) M i h l h lf i i d h i i f Smith, K. V., & Goldman, N. (2011). Measuring health status: self-, interviewer, and physician reports overall health. Journal of Aging and Health, 23(2), 242–266. Soriano, J. B., Alfageme, I., Almagro, P., Casanova, C., Esteban, C., Soler-Cataluña, J. J., de Torres, J. P., Martinez-Camblor, P., Miravitlles, M., & Celli, B. R. (2013). Distribution and prognostic validity of the new global initiative for chronic obstructive lung disease grading classification new GOLD COPD grading. CHEST Journal, 143(3), 694–702. Spiers, N., Jagger, C., Clarke, M., & Arthur, A. (2003). Are gender differences in the relationship between self-rated health and mortality enduring? Results from three birth cohorts in Melton Mowbray, United Kingdom. The Gerontologist, 43(3), 406–411. Subramanian, S. V., Subramanyam, M., Selvaraj, S., & Kawachi, I. (2009). Are self-reports of health and morbidities in developing countries misleading? Evidence from India. Social Science & Medicine, 68(2), 260–265. Sudha et al. (2007). Marital Status, Family Ties, and Self-rated Health Among Elders in South India. Journal of Cross Cultural Gerontology, 21 (3–4). Tsuji, et al. (1994). The predictive power of self-rated health, activities of daily living and ambulatory activity for cause specific mortality among the elderly: a three-year follow-up in urban Japan. Journal of American Geriatrics Society, 42(2), 153–156. United Nations, Department of Economic and Social Affairs, Population Division (2001). World Population Ageing: 1950–2050, 271. Wallace, R. B., & Herzog, A. R. (1995). Overview of the health measures in the Health and Retirement Study. Journal of Human Resources, 84–107. iener, J. M., Hanley, R. J., Clark, R., & Van Nostrand, J. F. (1990). Measuring the activities of daily living: comparisons across national surveys. Journal of Gerontology, 45(6), S229–S237. Ziebarth, N. (2010). Measurement of health, health inequality, and reporting heterogeneity. Social Science & Medicine, 71(1), 116–124.
https://openalex.org/W2782475392	https://www.hal.inserm.fr/inserm-01843948/file/ijms-19-00148.pdf	English	null	Understanding the Progression of Bone Metastases to Identify Novel Therapeutic Targets	International journal of molecular sciences	2,018	cc-by	12,548	To cite this version: Annie Schmid-Alliana, Heidy Schmid-Antomarchi, Rasha Al-Sahlanee, Patricia Lagadec, Jean-Claude Scimeca, et al.. Understanding the Progression of Bone Metastases to Identify Novel Therapeu- tic Targets. International Journal of Molecular Sciences, 2018, 19 (1), Epub ahead of print. 10.3390/ijms19010148. inserm-01843948 Understanding the Progression of Bone Metastases to Identify Novel Therapeutic Targets Annie Schmid-Alliana 1,†, Heidy Schmid-Antomarchi 1,†, Rasha Al-Sahlanee 1,2, Patricia Lagadec 1, Jean-Claude Scimeca 1 and Elise Verron 3,4,5,* Annie Schmid-Alliana 1,†, Heidy Schmid-Antomarchi 1,†, Rasha Al-Sahlanee 1,2, Patricia Lagadec 1, Jean-Claude Scimeca 1 and Elise Verron 3,4,5,* 1 Centre National de la Recherche Scientiﬁque (CNRS), Université Côte d’Azur, Inserm, iBV, 06108 Nice, France; Annie.SCHMID-ALLIANA@unice.fr (A.S.-A.); Heidy.SCHMID-ANTOMARCHI@unice.fr (H.S.-A.); rasha.al-sahlanee@unice.fr (R.A.-S.); Patricia.Lagadec@unice.fr (P.L.); jean-claude.scimeca@unice.fr (J.-C.S.) 1 Centre National de la Recherche Scientiﬁque (CNRS), Université Côte d’Azur, Inserm, iBV, 06108 Nice, France; Annie.SCHMID-ALLIANA@unice.fr (A.S.-A.); Heidy.SCHMID-ANTOMARCHI@unice.fr (H.S.-A.); rasha.al-sahlanee@unice.fr (R.A.-S.); Patricia.Lagadec@unice.fr (P.L.); jean-claude.scimeca@unice.fr (J.-C.S.) 2 College of Sciences, Biotechnology Department, University of Bagdad, Bagdad, Iraq 3 Regenerative Medicine and Skeleton. RMeS-Lab, INSERM UMR 1229, University of Nantes, 44000 Nantes, France 4 rasha.al-sahlanee@unice.fr (R.A.-S.); Patricia.Lagadec@unice.fr (P.L.); jean-claude.scimeca@unice.fr (J.-C.S.) 2 College of Sciences, Biotechnology Department, University of Bagdad, Bagdad, Iraq 3 Regenerative Medicine and Skeleton. RMeS-Lab, INSERM UMR 1229, University of Nantes, College of Sciences, Biotechnology Department, University of Bagdad, Bagdad, Iraq 3 Regenerative Medicine and Skeleton. RMeS-Lab, INSERM UMR 1229, University of Nantes, 44000 Nantes, France 4 Chimie et Interdisciplinarité, Synthèse, Analyse, Modélisation (CEISAM), UMR CNRS 6230, University of Nantes 44300 Nantes France 5 Faculty of Pharmaceutical Sciences, University of Nantes, 44000 Nantes, France * Correspondence: elise.verron@univ-nantes.fr; Tel.: +33-2-53-48-43-05 † Contributed equally to this manuscript. † Contributed equally to this manuscript. Received: 21 November 2017; Accepted: 2 January 2018; Published: 4 January 2018 Abstract: Bone is one of the most preferential target site for cancer metastases, particularly for prostate, breast, kidney, lung and thyroid primary tumours. Indeed, numerous chemical signals and growth factors produced by the bone microenvironment constitute factors promoting cancer cell invasion and aggression. After reviewing the different theories proposed to provide mechanism for metastatic progression, we report on the gene expression proﬁle of bone-seeking cancer cells. We also discuss the cross-talk between the bone microenvironment and invading cells, which impacts on the tumour actions on surrounding bone tissue. Lastly, we detail therapies for bone metastases. Due to poor prognosis for patients, the strategies mainly aim at reducing the impact of skeletal-related events on patients’ quality of life. However, recent advances have led to a better understanding of molecular mechanisms underlying bone metastases progression, and therefore of novel therapeutic targets. Keywords: bone metastases; bone tropism; bone microenvironment; molecular mechanisms; therapeutic strategies HAL Id: inserm-01843948 https://inserm.hal.science/inserm-01843948v1 Submitted on 19 Jul 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. International Journal of Molecular Sciences International Journal of Molecular Sciences Int. J. Mol. Sci. 2018, 19, 148; doi:10.3390/ijms19010148 1. Introduction Metastasis is the process through which cancer cells leave the initial tumour and travel throughout the body to establish a secondary tumour in other organs and anatomical sites. Bone, is one of the most preferential metastatic target sites for cancers. Prostate, lung, breast, kidney, and thyroid primary cancers account for 80% of skeletal metastases [1]. Spine, pelvis, proximal femur, and skull are the most common sites of bone metastases. Bone metastases can be divided into two broad categories; (i) osteolytic metastases that are associated with bone destruction and (ii) osteosclerotic metastases that are characterized by new bone formation. Bone metastases can be predominantly osteolytic or osteosclerotic, or mixed with features of both types [2,3]. Although precise molecular mechanisms underlying preferential cancer metastasis to bone need to be elucidated, it seems likely that bone provides an attractive environment that allows circulating cancer cells to home, survive and proliferate. This notion is consistent with the “seed and soil” theory proposed by Paget [4] in which he postulated that cancer cells, somehow liberated from the primary Int. J. Mol. Sci. 2018, 19, 148; doi:10.3390/ijms19010148 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 148 2 of 20 tumour, would seek nurturing conditions that resemble their original environment to grow and create a new lesion. tumour, would seek nurturing conditions that resemble their original environment to grow and create a new lesion. 2. Seed and Soil Interactions Bone metastasis occurs either via a direct invasion of bone tissue or secondarily to bone marrow, the latter being the most common. Bone marrow is colonized by metastatic cancer cells which in turn spread to the ﬁrm bone matrix [5]. In light of this, interactions of metastatic cancer cells (seed) with bone/bone marrow cells (soil) are critical for establishment and development of bone metastases [6]. This need for reciprocal interactions between cancer cells and the microenvironment make difﬁcult to decipher mechanisms governing metastases formation, in particular the site speciﬁcity (i.e., preferentially in bones, lungs or liver for example) [6,7]. The binding of cancer cells at the metastatic site involves cell-to-cell interactions, or interactions between cells and the extracellular matrix, and these processes involves cell adhesion molecules like integrins [8]. It has been found that the endothelium represents the ﬁrst point of interaction between metastatic cancer cells released in the blood stream and a speciﬁc tissue [9]. The success of this interaction is dependent on chemo-attraction and adhesion events according to different cell-surface markers, to morphological phenotypes and to specialized functions of the endothelium. For example, the presence of a stromal-derived growth factor (SDF-1—CXCL12) and its receptor CXCR4 on bone marrow endothelial cells has been shown to be favourable to cancer cell extravasation from the circulation and their retention in bone marrow [10]. This is particularly true for breast and prostate cancers, and a similar “seed and soil” interaction mechanism operating in the lung and liver may account for preferential formation of metastases within these environments [6]. The role played by this “seed and soil” interaction mechanism is clearly demonstrated by the inconsistency of metastasis formation throughout the skeleton. Indeed, the axial skeleton represents a preferential target, in accordance with the haematopoietic bone marrow content, while almost no metastases develop within hands and feet where no haematopoietic bone marrow is present [4,11]. 3. Theories of Metastatic Progression Two main models of metastatic progression are currently accepted. The classical model is a linear progression model, and the more recent one is the parallel progression model that represents somewhat of a paradigm shift [12]. In the linear progression/late dissemination model, a normal epithelial cell acquires mutations that inactivate tumour suppressor genes and/or activate oncogenes, which promotes survival and uncontrolled proliferation of cancer cells. An increased proliferation rate may also lead to mutations in progeny cells, which result in a variety of subsequent mutations heterogeneously distributed throughout discrete tumour sub-populations [13–15]. Some of these mutations are associated with gene alterations that facilitate dissemination and invasion, and these mutated cells are capable of generating distal metastases. In this model, acquisition of malignant mutations is linked to tumours of large size and occurs at later stages of tumour growth [16–18]. The second model, is known as the parallel progression/early dissemination model [19] and has gained favour recently, although it dates from the 1950’s [20]. This model rejects the notion that tumours must reach a deﬁned size before acquiring the ability to disseminate, and it posits that dissemination occurs long before detection of the primary tumour [21–23]. Nevertheless, both models agree that cancer progression depends on clonal expansion of “ﬁttest” cells. The idea that tumour cells disseminate early is supported by the notion that in rare cases, patients with very small early-stage primary tumours (T1) present metastases at the time of diagnoses. This concept is further sustained by the fact that metastatic cancers of unknown primary origin represents 5–10% of all cancer diagnoses in the United States and Europe [24]. There is a third recent theory, known as the “pre-metastatic niche”, which is based on observations made during the last decade [25]. This theory has radically changed our view of metastatic colonization. Int. J. Mol. Sci. 2018, 19, 148 3 of 20 It holds that secreted factors (S100A8, S100A9, osteopontin OPN) are produced either by the primary tumour or by stromal cells in the metastatic site, which serve to “prime” tissues for later metastatic tumour engraftment [17,26,27]. These factors facilitate mobilization and recruitment of bone marrow-derived cells that can liberate pro-inﬂammatory cytokines such as TNF-α. These inﬂammatory cytokines “prime the soil” by activating endothelial cells and inducing matrix metallo proteins (MMPs) expression to promote subsequent cancer cell colonization and outgrowth [2]. 3. Theories of Metastatic Progression A key observation in the ﬁeld of cancer metastasis is that speciﬁc cancers tend to colonize particular sites. For example, breast cancer most commonly metastasizes to bone, followed by lung, brain, and liver [28], whereas colorectal cancers most frequently metastasize to liver, then lung, and rarely to bone and brain [29,30]. Stephen Paget ﬁrst offered in 1889 an explanation for this observation. His seminal “seed and soil” hypothesis suggested that to support the growth of metastases, the “soil” (i.e., metastatic environment) must be capable of nourishing the “seed” (i.e., a disseminated cancer cell) [4,30,31]. For instance, the bone microenvironment may provide sufﬁcient growth signals to facilitate breast cancer outgrowth while these same signals are less effective at promoting colon cancer outgrowth. This hypothesis was challenged nearly 40 years later when James Ewing claimed that differences in metastatic colonization by certain cancers could be explained by anatomy of the local circulatory system [32]. Thus, cancers will rather metastasize to secondary sites presenting the highest degree of accessibility via the vasculature and blood ﬂow patterns [31,33]. This is best exempliﬁed by the high propensity of colon cancers to metastasize to liver [34], which is the ﬁrst organ they encounter following intravasation into the portal circulation. It was later shown that while local recurrences correlated with increased perfusion rates, the formation of distant metastases could not be completely explained by anatomical vascular access patterns, and Paget’s “seed and soil” hypothesis prevailed and remained a guiding principle in the study of organ-speciﬁc metastatic process. 4. Gene Expression Proﬁling of Bone Metastases In recent years, gene expression proﬁling has become a standard technique used to identify genes that are deregulated in cancer. A better understanding of the various stages of metastatic progression has been gained by coupling gene-expression proﬁling with pre-clinical mouse models of breast cancer metastasis to bone. Some of these approaches, starting from heterogeneous cultures of breast cancer cells, involve derivation of sub-populations that preferentially spread to bones. These cell populations are isolated directly from established bone metastases that have been obtained following injection of the parental population into the left cardiac ventricle or the mammary fat pads of mice. Successive rounds of in vivo selection result in breast cancer cells that aggressively metastasize to bone tissue when compared to the original cell population. These approaches have facilitated the identiﬁcation of individual molecular mediators of metastatic process such as IL-8 [35], as well as sets of genes that work cooperatively including CXCR4, MMP-1 and the TGFβ-regulated genes CTGF and IL-11 [36–39]. On the other hand, the in vitro isolation of single cell progeny (SCP) yielded breast cancer cell-derived populations with vastly variable in vivo bone metastatic phenotypes [40]. Importantly, compared with aggressive bone metastatic cell populations that were derived by in vivo selection, aggressive bone metastatic SCPs present gene-expression proﬁles that are largely overlapping [16]. These studies, in addition to identifying novel mediators of metastasis, provide insights into the nature of the metastatic process. 5.1. Osteolytic Bone Metastases Osteolytic lesions arise when osteoclast-mediated bone resorption overcomes bone formation by osteoblasts, resulting in weakened structures that compromise bone integrity [5,6]. Various factors including cytokines and hormones control the bone remodelling equilibrium [5], and a vicious cycle involving osteoclasts and tumour cells progressively leads to the development of osteolytic lesions. Brieﬂy, the production of parathyroid hormone-related peptide (PTHrP) and the expression of receptor activator of nuclear factor—κB receptor ligand (RANKL) play important roles in osteolytic metastases, and in bone metastases arising from primary breast cancer. It has been observed that PTHrP expression was signiﬁcantly higher compared to metastases from other primary cancers [11]. PTHrP increases RANKL expression, which acts on osteoclasts to increase osteoclast maturation and resorptive activity. Subsequently, the resorptive activity releases TGFβ and other growth factors like EGFs or IGFs from the bone matrix [5,45,46], generating a feed-back loop that stimulates metastatic proliferation [45]. Once released, TGFβ in turn stimulates PTHrP production, which is further increased by a high local calcium concentration resulting from bone resorption [47,48]. Moreover, TGFβ regulates osteolytic and pro-metastatic agents, as well as other microenvironment factors like hypoxia [49], which promotes the growth of tumour cells. Indeed, TGFβ activates the epithelial to mesenchymal transition (EMT) [50], increases tumour cells invasiveness and angiogenesis, and displays also immunomodulation properties [51]. In addition, stromal cells present within the bone marrow microenvironment are involved in the establishment and the progression of bone metastases. These includes neurons, blood platelets and endothelial cells. Thus, sympathetic neurons activation by bone metastasis is responsible for severe pains [52], but also for increasing tumour proliferation and invasiveness [53]. On the other hand, tumour cells bind preferentially to endothelial cells and activate platelets aggregation, which induce angiogenesis and increases tumour survival and proliferation [54]. Indeed, platelet aggregation initiates a massive production of lysophosphatidic acid (LPA), which acts as a pro-metastatic lipid mediator that promotes survival, proliferation, motility and invasiveness of breast cancer cells for example [55]. Likewise, via the activation of G-protein-coupled receptors, LPA acts directly on tumour cells by stimulating the secretion of pro-osteoclastic interleukins such as interleukins (IL) IL-6 and IL-8 among others [56,57]. 5. Bone Metastasis Microenvironment Bone is a unique microenvironment made of proteins and calciﬁed hydroxyapatite crystals forming a dense matrix that is tightly interconnected to bone marrow, which contains osteoblast and osteoclast progenitors, as well as hematopoietic stem cells (HSCs). Bone marrow is a major site of metastatic diseases for breast and prostate carcinomas, and for multiple myeloma [25,41]. Although metastasis formation in lungs, liver and bones displays some similarities, the bone microenvironment undergoes constant remodelling events that deeply impacts metastasis onset and development [42]. 4 of 20 Int. J. Mol. Sci. 2018, 19, 148 This remodelling process results from an equilibrium between osteoblasts and osteoclasts activities, which are regulated by mechanical stress, cytokines and hormones. Should this balance be disturbed, osteolytic or osteosclerotic metastases appear, depending on the predominant activity occurring within lesions [5,43,44]. This remodelling process results from an equilibrium between osteoblasts and osteoclasts activities, which are regulated by mechanical stress, cytokines and hormones. Should this balance be disturbed, osteolytic or osteosclerotic metastases appear, depending on the predominant activity occurring within lesions [5,43,44]. 5.2. Osteosclerotic Bone Metastases Os eos e o i o e e as ases Osteosclerotic bone metastas Osteosclerotic bone metastases result from excessive bone formation, which produces often a disorganized and weak bone tissue. This imbalance is mostly encountered with metastases originating from prostate and breast primary tumours [5,6,29,66]. However, in contrast to osteolytic bone metastases, the mechanisms involved are less understood. The most inﬂuential factors so far identiﬁed are BMP and endothelin-1 (ET-1) [5,6,29]. The up-regulation of BMP expression by osteoblasts proportionally affects bone formation, and it is also involved in ectopic bone formation [6]. Compared to other primary cancers, BMP expression is high in cancerous prostate cells, correlating with the high occurrence of osteosclerotic metastases observed for primary prostate cancers [2]. Concerning ET-1, it is a potent vasoconstrictor, a direct stimulant of osteoblast progenitors mitogenesis, and it has been linked to the substantial pain resulting from osteosclerotic lesions [5,6,29]. , p disorganized and weak bone tissue. This imbalance is mostly encountered with metastases originating from prostate and breast primary tumours [5,6,29,66]. However, in contrast to osteolytic bone metastases, the mechanisms involved are less understood. The most influential factors so far identified are BMP and endothelin-1 (ET-1) [5,6,29]. The up-regulation of BMP expression by osteoblasts proportionally affects bone formation, and it is also involved in ectopic bone formation [6]. Compared to other primary cancers, BMP expression is high in cancerous prostate cells, correlating with the high occurrence of osteosclerotic metastases observed for primary prostate cancers [2]. Concerning ET-1, it is a potent vasoconstrictor, a direct stimulant of osteoblast progenitors mitogenesis, and it has been linked to the substantial pain resulting from osteosclerotic lesions [5,6,29]. Figure 1 summarizes the main steps of bone metastasis progression from the primary tumour to the development of osteolytic or osteosclerotic lesions. [ , , ] Figure 1 summarizes the main steps of bone metastasis progression from the primary tumour to the development of osteolytic or osteosclerotic lesions. Figure 1. Factors involved in hematogen dissemination and tissue invasion by cancer cells with a bone tropism. Factors involved in bone metastatic progression are mainly related to (i) migration of metastatic tumour cells within blood circulation, (ii) invasion of bone marrow niche, and (iii) development within bone tissue through two mechanisms leading to osteolytic or osteosclerotic lesions, both leading to severe skeletal-related events. Figure 1. Factors involved in hematogen dissemination and tissue invasion by cancer cells with a bone tropism. 5.1. Osteolytic Bone Metastases Concerning IL-6, it increases bone degradation via: (i) the production of RANKL, and the negative regulation of osteoprotegerin (OPG); (ii) the induction of proteins involved in bone resorption such as PTHrP, interleukin (IL) IL-8, IL-11 and Cox-2; (iii) an increase of oestradiol 17β-hydroxysteroid dehydrogenase activity (an inhibitor of the anti-osteoclast activity of oestrogens); (iv) the stimulation of DKK-1 expression by tumour cells and (v) the downregulation of collagen II and aggrecans by the osteoblasts [31]. Interestingly, these last two functions of IL-6 illustrate that the bone loss observed in osteolytic metastases is also partially due to the failure of osteoblasts to produce new osteoid and repair the bone matrix. In addition, other factors that play a role in osteolytic bone metastases are secreted or induced by metastatic tumour cells (IL-1, prostaglandin E2, granulocyte macrophage colony stimulating factor GM-CSF, tumour necrosis factor-alpha TNFα, matrix metallo protein MMPs, cathepsin K CTSK and osteopontin OPN) [54,58–63]. Finally, as already mentioned above, osteolytic lesions are not only the result of bone resorption mediated by osteoclasts. Indeed, they are also due to a decreased bone formation mediated by osteoblasts, and the antagonistic expression of Wnt and bone morphogenetic proteins (BMP) could play an important role [64,65]. Consequently, the bone metastatic niche favours a complex vicious cycle Int. J. Mol. Sci. 2018, 19, 148 I J M l S i 14 5 of 20 5 of 19 comprising interconnected processes, which feed on each other, that is bone metabolism regulations and tumour growth. A good understanding of these mechanisms and pathways is crucial to develop therapeutic strategies, and several pharmacological inhibitors are already being tested in clinical trials A good understanding of these mechanisms and pathways is crucial to develop therapeutic strategies, and several pharmacological inhibitors are already being tested in clinical trials [11,46,66,67]. g p g y g [11,46,66,67]. 6. Bone Metastasis Treatments Patients bearing bone metastases often suffer from many complications including pain, decreased mobility, neurologic compromise and pathologic fractures [68]. These complications, generically called skeletal-related events (SREs), impair the patient’s prognosis and increase the cost of treatments. Despite numerous studies attempting to better understand bone metastasis pathophysiology, there are as yet no established and efﬁcient clinical methods for their cure and/or their prevention. Currently, medical management of bone metastases is based on local approaches (i.e., surgery, radiation therapy) or systemic strategies such as molecular targeted therapies [66,69]. Depending on the case, local treatment of bone metastases can be done either by a consolidation procedure and/or tumour destruction by using surgery, irradiation (stereotactic) or interventional radiology techniques (i.e., vertebroplasty, radiofrequency, cryoablation). 6.1. Ablative Procedures Local treatments of bone metastases have a dual role: (i) mechanical stabilization to prevent pathological fractures; (ii) tumour destruction to block or slow tumour and, for example, to ﬁght against neurological complications in case of spinal metastases. It is noteworthy that in almost 90% of cases, ablative treatments lead to a relief from pain within 24 h [70]. 5.2. Osteosclerotic Bone Metastases Os eos e o i o e e as ases Osteosclerotic bone metastas Factors involved in bone metastatic progression are mainly related to (i) migration of metastatic tumour cells within blood circulation; (ii) invasion of bone marrow niche; and (iii) development within bone tissue through two mechanisms leading to osteolytic or osteosclerotic lesions, both leading to severe skeletal-related events. Figure 1. Factors involved in hematogen dissemination and tissue invasion by cancer cells with a bone tropism. Factors involved in bone metastatic progression are mainly related to (i) migration of metastatic tumour cells within blood circulation, (ii) invasion of bone marrow niche, and (iii) development within bone tissue through two mechanisms leading to osteolytic or osteosclerotic lesions, both leading to severe skeletal-related events. Figure 1. Factors involved in hematogen dissemination and tissue invasion by cancer cells with a bone tropism. Factors involved in bone metastatic progression are mainly related to (i) migration of metastatic tumour cells within blood circulation; (ii) invasion of bone marrow niche; and (iii) development within bone tissue through two mechanisms leading to osteolytic or osteosclerotic lesions, both leading to severe skeletal-related events. Int. J. Mol. Sci. 2018, 19, 148 6 of 20 6.1.2. Irradiation Techniques Ablative irradiation with tumouricidal doses are used for bone metastases limited in size and number (1 to 3 metastatic sites), and/or with a vertebral localisation close to the spinal cord. In 50 to 80% of cases, radiotherapy is used as an analgesic treatment, with the aim of getting an immediate relief, preventing complications, and improving the quality of life of patients. Most of the studies reported in the literature are related to the spine, while information concerning other metastatic sites (limbs, ribs) is limited [72,73]. 6.1.1. Surgical Techniques Surgical approaches are generally used for patients with impending or complete pathological fracture, or for patients exhibiting spinal cord instability or compression. Indeed, surgery may represent an emergency solution in the case of compression, a situation which requires the restoration of skeletal integrity to limit neurological impact. Concerning extra spinal bone metastases, about 92% of surgical procedures result from complete or imminent fractures [71]. 6.1.3. Thermal and Chemical Ablation In 50% of cases, a partial or complete response is observed, and this method is often very effective in treating initial metastases (especially from breast cancer) [72,74]. • Chemoembolization—Chemoembolization consists of delivering directly to tumours an antimitotic infusion or antimitotic-loaded microparticles (carboplatin and adriamycin). This technique is used to treat bone lesions that have been previously irradiated, or which are unresectable and resistant to other treatments. In 50% of cases, a partial or complete response is observed, and this method is often very effective in treating initial metastases (especially from breast cancer) [72,74]. Figure 2 recapitulates the main ablative procedures proposed to patients. Figure 2 recapitulates the main ablative procedures proposed to patien Figure 2 recapitulates the main ablative procedures proposed to patients. Figure 2 recapitulates the main ablative procedures proposed to patien gure 2 recapitulates the main ablative procedures proposed to patients. Figure 2 recapitulates the main ablative procedures proposed to patients. Figure 2. Tumours ablative therapies. They are selected according to the clinical status of patients, the tumour resistance, characteristics of metastases in terms of size/number/localization, and therapeutic objectives such as preventing development of skeletal-related events, achieving analgesic effect and getting a mechanical stabilization (see references [70–75]). Figure 2. Tumours ablative therapies. They are selected according to the clinical status of patients, the tumour resistance, characteristics of metastases in terms of size/number/localization, and therapeutic objectives such as preventing development of skeletal-related events, achieving analgesic effect and getting a mechanical stabilization (see references [70–75]). Figure 2. Tumours ablative therapies. They are selected according to the clinical status of patients, the tumour resistance, characteristics of metastases in terms of size/number/localization, and therapeutic objectives such as preventing development of skeletal-related events, achieving analgesic effect and getting a mechanical stabilization (see references [70–75]). Figure 2. Tumours ablative therapies. They are selected according to the clinical status of patients, the tumour resistance, characteristics of metastases in terms of size/number/localization, and therapeutic objectives such as preventing development of skeletal-related events, achieving analgesic effect and getting a mechanical stabilization (see references [70–75]). 6.1.3. Thermal and Chemical Ablation Interventional radiology uses several techniques (cementoplasty, radiofrequency ablation, cryotherapy, chemoembolization) to achieve bone consolidation with varying degrees of tumour cells destruction [74,75]. • Radiofrequency ablation - Radiofrequency ablation of bone metastases is a recent radiological percutaneous technique, using alternative current (450–600 kHz) applied through a needle entered in the metastasis under radioscopic control. Radiofrequency energy generates an overheating (60–70 ◦C), and cancer cells necrosis is obtained by heat destruction within a volume of 3 cm in diameter. This technique cannot be used in the close vicinity of nerve tissues, since they do not support a temperature above 45 ◦C. It is also difﬁcult to implement this technique for metastases affecting ﬂat bones (i.e., sternum, iliac wing). Radiofrequency can also be used in combination with cementoplasty, although this combination has not yet shown signiﬁcant improvement compared to cementoplasty alone [72,74]. • Cryotherapy—Cryotherapy is the more recent technique that enables tumoral destruction, over areas up to 5 cm, by low temperature using a needle-like applicator and liquid argon. This technique involves generally little pain and it can be realized under simple sedation, contrary Int. J. Mol. Sci. 2018, 19, 148 7 of 20 to radiotherapy that usually requires general anaesthesia. On the other hand, cryotherapy cannot be combined with cementoplasty since tissue cooling exerts an inhibitory effect on cement polymerization [72,74]. Int. J. Mol. Sci. 2018, 19, 148 7 of 19 • Chemoembolization—Chemoembolization consists of delivering directly to tumours an antimitotic infusion or antimitotic-loaded microparticles (carboplatin and adriamycin). This technique is used to treat bone lesions that have been previously irradiated, or which are unresectable and resistant to other treatments. In 50% of cases, a partial or complete response is observed, and this method is often very effective in treating initial metastases (especially from breast cancer) [72,74]. • Chemoembolization—Chemoembolization consists of delivering directly to tumours an antimitotic infusion or antimitotic-loaded microparticles (carboplatin and adriamycin). This technique is used to treat bone lesions that have been previously irradiated, or which are unresectable and resistant to other treatments. In 50% of cases, a partial or complete response is observed, and this method is often very effective in treating initial metastases (especially from breast cancer) [72,74]. • Chemoembolization—Chemoembolization consists of delivering directly to tumours an antimitotic infusion or antimitotic-loaded microparticles (carboplatin and adriamycin). This technique is used to treat bone lesions that have been previously irradiated, or which are unresectable and resistant to other treatments. 6.3.1. Bisphosphonates Due to their chemical and pharmacological properties, bisphosphonates (BPs), which have been initially developed for the treatment of osteoporosis, are also indicated for treatment of bone metastases [81]. Due to their chemical structure, BPs display a strong afﬁnity to hydroxyapatite crystals forming the mineral phase of the bone matrix. They are synthetic products derived from inorganic pyrophosphates (PPi), in which the P-O-P bond is replaced by a P-C-P bond to increase the chemical resistance to enzymatic cleavage. The phosphonates groups can chelate calcium ions present within biological hydroxyapatites, and this afﬁnity increases when R1 is a hydroxyl group [81]. Accordingly, two models of bonds have been presented: (i) a weak bond, corresponding to one single phosphonate group (etidronate); (ii) a stronger bond carrying two phosphonates and hydroxyl groups, (alendronate, zoledronate). Recently, it has been suggested that the R2 group could also impact BPs’ afﬁnity for hydroxyapatite crystals. Finally, it has been shown that, compared to compounds with a higher afﬁnity, low-afﬁnity molecules can penetrate more deeply within the mineral matrix, and so doing could reach the canalicular network [81]. Concerning the mechanisms of action, BPs’ afﬁnity decreases in acidic conditions (resorption lacuna) because of phosphonate-groups protonation. Once they are released from the bone matrix, BPs can be internalized into osteoclasts through endocytosis. Afterward, via the inhibition of the farnesyl diphosphate synthase, BP prevent the prenylation of several molecules (protein G trimeric subunits, phosphodiesterase subunits) involved in many cellular processes crucial for osteoclastic functions (cytoskeleton architecture, actin rings formation, wrinkling of the border, intracellular vesicles movements). The fusion of osteoclastic precursors is still possible and leads to multinucleated giant cells, which are inactive. At high concentration, BP can induce osteoclasts apoptosis. In all cases, by inhibiting bone resorption, they deprive tumour cells of growth factors released from the bone matrix upon osteoclastic action (TGFβ, IGFs) [81]. Much preclinical evidence suggests that BPs possess direct antitumor properties [82–84]. After internalization, BPs signiﬁcantly disrupt the function of small GTPases proteins by blocking their prenylation, which is essential for adhesion, migration, invasion and proliferation of tumour cells. Similarly, it has been suggested that BPs could inhibit αVβ3 integrin activation, which is involved in tumour cells adhesion on the bone surface [85]. Moreover, BPs could induce tumour cell apoptosis through cell structure alteration and DNA fragmentation [86]. BPs could have also an indirect antitumor activity by blocking angiogenesis. 6.2. Reconstructive Procedures 6.2. Reconstructive Procedures Cementoplasty, which is the simplest and most suitable technique to obtain vertebral body stabilization, represents the most common interventional radiology technique. It consists of injecting percutaneously, under radiologic control, a surgical cement in bone lesions (mostly PMMA, poly (methyl methacrylate)). In almost 90% of cases, mechanical stabilization and analgesic effects are obtained. In addition to these effects, the in situ recurrence rate is low (around 14%). This phenomenon can be explained by heat generation induced by the cement polymerization (60–70 °C), and/or by a direct toxic action of cement monomers on tumour cells [76]. In addition to osteolytic metastasis treatment, cementoplasty can also be efficient in the context of osteosclerotic or mixed metastases. However, in these circumstances, the technical implementation is more complex and therefore, a high rate of postoperative complications is observed. Cementoplasty can be applied in numerous sites including spine, humeral head or intertrochanteric region. However, some locations remain out of the scope of this technique (femoral neck parts of the hip bone) especially if there is Cementoplasty, which is the simplest and most suitable technique to obtain vertebral body stabilization, represents the most common interventional radiology technique. It consists of injecting percutaneously, under radiologic control, a surgical cement in bone lesions (mostly PMMA, poly (methyl methacrylate)). In almost 90% of cases, mechanical stabilization and analgesic effects are obtained. In addition to these effects, the in situ recurrence rate is low (around 14%). This phenomenon can be explained by heat generation induced by the cement polymerization (60–70 ◦C), and/or by a direct toxic action of cement monomers on tumour cells [76]. In addition to osteolytic metastasis treatment, cementoplasty can also be efﬁcient in the context of osteosclerotic or mixed metastases. However, in these circumstances, the technical implementation is more complex and therefore, a high rate of postoperative complications is observed. Cementoplasty can be applied in numerous sites including spine, humeral head or intertrochanteric region. However, some locations remain out of Int. J. Mol. Sci. 2018, 19, 148 8 of 20 the scope of this technique (femoral neck, parts of the hip bone), especially if there is an associated fracture that requires a surgery procedure [72,75]. These procedures may also involve an endoprosthetic reconstruction or consolidation techniques using intramedullary nails or metallic equipments such as plates and screws [77]. 6.3. Pharmacological Approaches One has to keep in mind that systemic administration of chemotherapeutic agents may alter the normal skeletal development and bone remodelling, leading to increased risks of fractures [78]. Considering the complex set of interactions between metastatic cells, the microenvironment, and bone-resident cells, therapies cannot be based on the genetic proﬁle of primary tumours. In this context, the concept of vicious cycle has changed the therapeutic approach, and bone resorption-blocking strategies have emerged as new potential treatments [79], together with several other pharmacological approaches including therapies targeting cancer cells and tumour vascularization [80]. 6.3.2. Denosumab Denosumab is a human monoclonal antibody developed for the treatment of osteoporosis and bone metastases [78,98,99]. Denosumab therapy reduces and delays the risk of SREs in patients with bone metastases from breast cancer, prostate cancer, non-small cell lung cancer and other types of solid tumours. Indeed, it binds and neutralizes RANKL, similarly to the decoy receptor OPG that blocks RANKL receptor activation on the surface of osteoclasts and their precursors, preventing osteoclastogenesis, as well as osteoclasts activity and survival [95]. As a result, bone resorption is decreased and the tumour-induced bone destruction is inhibited. Denosumab is administered by subcutaneous injections, which provides an alternative route of administration and eliminates the need for routine test of renal function prior to administration [100]. Hence, its use is widely recommended for patients with renal failure or receiving a nephrotoxic chemotherapy like cisplatin [101]. Regarding long-term impact, a study in postmenopausal women with low bone mass and treated with denosumab reported that stopping the treatment resulted in decreased bone density at lumbar spine and hip sites within 12 months following the ﬁnal dose [102]. Since denosumab use is recent, no data are available about patients’ compliance. However, observance should be better compared to treatments with BP. Indeed, good tolerance is reported, and the administration mode is more adaptable and less restrictive [81,99]. 6.3.1. Bisphosphonates Actually, BPs regulate the proliferation, the migration and the adhesion of endothelial cells, and they inhibit the development of capillaries. Finally, BPs could have an indirect antitumor action via the modulation of the immune system through the activation of γδτ T lymphocytes [87]. This activation is caused Int. J. Mol. Sci. 2018, 19, 148 9 of 20 by: (i) structural similarity between BPs and non-peptidic ligands that activates γδτ T cells; (ii) the recognition by the TCR (T cell receptor) of isopentyl pyrophosphate accumulated after the inhibition of farnesyl diphosphate synthase [81,82,88,89]. BPs (clodronate, pamidronate, ibandronate, zoledronate) are effective for the prevention and the retardation of SREs in patients with bone metastases from solid tumours or with multiple myeloma osteolytic lesions. Interestingly, several generation of compounds are available [90]. It has been shown that N-BPs of the second and the third generation (nitrogen-containing BP: zoledronate, palmidronate, alendronate, ibandronate) are more efﬁcient in reducing SREs related to bone metastases coming from solid tumours other than breast and prostate cancers [91] or from castration-resistant prostate cancers [92]. BP administration is proposed for all patients with bone metastases from solid tumours or with bone lesions from multiple myeloma [93]. Similar to the strategy used for the patients with breast cancers, BP therapy should be initiated as soon as the discovery of X-ray evidence of bone destruction, or abnormal bone scan images combined with localized pain, and it should be continued throughout the disease [94]. In other words, BPs should be started at the ﬁrst diagnosis of bone metastases [95]. Effectiveness of BP therapies requires a high level of patient compliance. However, this can be compromised by the extended duration of treatment, the use of another treatment, or private health insurance concerns in certain countries. Oral administration of BPs often triggers gastrointestinal side effects, and it requires strictly controlled administration conditions. In particular, patients must stay in a vertical position up to 2 h after drug intake, and they are advised to avoid food that could interact with BP metabolism. Concerning intravenous (iv) administration of BP, it is associated with development of symptoms similar to ﬂu, principally after the ﬁrst administration. In some cases, and following IV administration of high doses, severe side effects such as osteonecrosis of the jaw have been reported [81,96,97]. 6.3.3. Other Agents Targeting Bone Tissue Therapeutic strategies targeting osteoclastic bone resorption represents an area of intense clinical research [81]. This includes inhibitors of RANK/RANKL interaction (osteoclasts formation), as well as Ctsk and αVβ3 integrin inhibitors [89,103]. Interestingly, these therapies also impact on tumour cells and on stromal components involved in bone metastatic disease [57]. The RANK/RANKL/OPG triad appears as a key player in tumour expansion within bone tissue. Indeed, RANKL level is increased in osteolytic lesions related to malignant tumours, while OPG level Int. J. Mol. Sci. 2018, 19, 148 10 of 20 is increased in osteosclerotic lesions [104]. In animal models of bone metastases from breast cancer, OPG inhibits bone destruction and reduces bone tumour mass. This reduction of tumour development is probably related to the inhibition of osteoclastic resorption, since OPG does not affect tumour growth in soft tissue. In addition, RANK-related RANKL inhibitors (Fc-OPG) [105], neutralizing antibodies directed against RANKL, and soluble antagonists of RANK (Fc-RANK) are inhibitors of bone metastases, as observed in preclinical and clinical models [80,106]. The use of the semi-metallic element gallium (Ga) offers promising prospects. Indeed, through its action on osteoclastogenesis, Ga is an inhibitor of bone resorption [107]. Moreover, previous experiments have established that Ga would represent an attractive additive to calcium phosphate-based biomaterials for bone reconstructive surgery [108]. Ga’s direct effects on tumour cells have been recently studied in the context of bone metastases. The in vitro data obtained suggest that, through its action against the vicious cycle involving bone cells and tumour cells, Ga represents a relevant and promising candidate for a local delivery upon the resection of bone metastases from breast cancer [109]. As new potential diagnosis and therapeutic markers for bone metastases, microRNAs (miRNAs) are also currently investigated. During the last years, the list of miRNAs involved in the progression of breast and prostate cancers has grown, including those directly involved in cancer cells metastasis to the bone [110]. Analysis of osteoclasts-derived miRNAs that are induced in the presence of tumour cells also led to the discovery of speciﬁc miRNAs (miR-16, miR-378, miR-141) that might have an inﬂuence on osteolytic bone metastases [111–113]. As another example, MiR-34a is considered as an anti-osteoclastogenic factor negatively regulated by transforming growth factor-β-induced factor 2 (Tgif2) [114]. By consequence, Tgif2 deletion may reduce bone resorption by abolishing miR-34a regulation. Several strategies targeting TGFβ signalling pathways are under study in the context of cancer therapies. 6.3.4. Agents Targeting Tumour Cells As mentioned above, bone metastases result from selection and enrichment of pre-existing cellular subpopulations displaying gene expression signatures associated with metastatic bone development, (CXCR4, MMP-1, FGF5, CTGF, IL-11, OPN, follistatin, proteoglycan 1). Identiﬁcation of these genes, and the corresponding signalling pathways, could provide potential therapeutic targets. Indeed, anti-CXCR4 antibodies reduce signiﬁcantly the number of bone metastases in a model of prostate cancer, and the blockage of CXCR4 expression by siRNAs (small interfering RNAs) decreases in vitro the invasive character of breast cancer cells and inhibits the metastatic phenomenon in an animal model [120]. Growth factors and cytokines signalling pathways protect metastatic cells from anticancer drugs-induced apoptosis. Thus, targeting these survival factors in bone (TGFβ, IGF, IL-6, FGF, ET-1 and others) could increase cancer cells sensitivity to chemotherapeutic agents. Identiﬁcation of metastases suppressor genes (MSGs:) also provides new insights into the formation of bone metastases. These genes block the metastatic capacity without impacting growth of the primary tumour. For example, the recovery of PTEN expression (Phosphatase and Tensin homolog) inhibits the development of bone metastases from prostate cancer, while it has no effect on lung metastases [121]. In parallel to MSGs, miRNAs have been identiﬁed as another class of molecules which regulates negatively the formation of bone metastases. This is the case of miR-335 and miR-126, which have been identiﬁed as metastasis suppressors in breast cancer [80,104]. 6.3.3. Other Agents Targeting Bone Tissue The different classes of TGFβ inhibitors used in preclinical models and clinical trials include: (i) monoclonal antibodies targeting TGFβ and preventing TGFβ interactions with its receptor; (ii) small molecules that inhibit TGFβRI kinase activity and TGFβRII-mediated activation of TGFβR1-SMAD proteins; (iii) a naturally derived product, halofuginone (HFG), which inhibits TGFβ [115]. Because TGFβ signalling pathways are activated in numerous cell types, the blockade of the TGFβ axis could have adverse effects on wound healing and on the immune system. Nevertheless, clinical studies (patients with advanced metastatic melanoma, colon cancer, prostate and breast cancers) have shown that a small inhibitory molecule (LY2157299) was well-tolerated and displayed minimal toxicity [116]. Considering the variety of factors released from the bone matrix upon osteoclastic resorption (IGF1, PDGFs, BMP, calcium etc.), and which stimulate the growth of certain tumours, this ﬁeld of research represents also a potential source of pharmacological targets capable of interfering with metastatic processes [57]. Endothelin 1 (ET-1) is one of the promising osteoblastic targets studied in preclinical and clinical trials. This vasoconstriction agent stimulates osteoblastic proliferation and differentiation, and it is involved in the development of skeletal metastases of prostate cancer [117]. Therefore, atrasentan was the ﬁrst receptor antagonist to ET-1 sub-type A studied in patients with prostate cancer, while zibotentan, a more selective inhibitor, is currently used in clinical trials [118]. Bone stroma and the osteoblastic niche can interact with cancer cells and thus promote a resistance to cytotoxic chemotherapy. Cancer cells are often resistant because they are maintained on the G0 phase of the cell cycle by contact with stromal cells present within bone marrow. A new therapeutic approach exploits the fact that tumour cells use CXCR4 and VLA4 proteins for bone marrow homing [119]. In addition, the use of mobilizing agents for MSCs (AMD3100, VLA4 target agents), which drive leukaemia and melanoma cells out of bone marrow, leads to a better response to chemotherapy in animal models. This approach is under investigation in clinical trials for the treatment of acute myeloid leukaemia and multiple myeloma [57]. Int. J. Mol. Sci. 2018, 19, 148 11 of 20 11 of 20 6.3.5. Agents Targeting Angiogenesis Antiangiogenic treatments appear as attractive and promising anticancer therapies, but they have been less studied in the bone context. Indeed, to reduce the blood supply to a tumour is a challenge since bone marrow is a highly vascularized site, which can be strongly affected by antiangiogenic inhibitors [80]. In bone metastasis models, the antiangiogenic agent avastin inhibits tumour growth via indirect targeting to osteoclasts [122]. On the other hand, the exact target of antiangiogenic therapies is unknown, since actions of such agents on circulating endothelial progenitors have not been clearly established. However, the combination of an antiangiogenic agent with a cytotoxic chemotherapy has given promising results in an animal model of bone metastases from breast primary tumour [123]. Endothelial progenitor cells derived from bone marrow represent potential targets of antiangiogenic strategies. The difﬁculty here lies in the absence of consistent markers for endothelial progenitors that could be used to target them. MTA1 (metastasis-associated protein 1) is a proangiogenic factor involved in prostate cancer progression [124]. Accordingly, silencing MTA1 effectively limits the expression of proangiogenic factors like vascular endothelial growth factor (VEGF) by prostate cancer cells, and represents thereby a new potential target for patients with prostate tumour [57]. 6.3.6. Agents Targeting Pain Bone pain is very difﬁcult to treat and tends to be resistant to opioids [125]. Indeed, bone tissue is densely innervated by primary afferent neurons and sympathetic neurons located in the intramedullary bone and the periosteum. Bone resorption inhibitors decrease substantially the pain caused by expanding bone metastases. Honore et al. demonstrated that osteoclastic resorption triggers recruitment of nervous ﬁbbers related to pain and leads to bone pain-inducing modiﬁcations of spinal cord neurochemical properties. OPG, an antagonist of RANKL, prevents activation of nerve ﬁbbers associated with pain, and reduces pain behaviours in mice with bone metastases [126]. In addition, tumour cells residing within bone secreting factors (NGF nerve growth factor, ET-1) activate neurons-associated pain perception. Therefore, bone resorption inhibitors, receptor A of ET-1 and NGF represent therapeutic targets for bone pain treatment. Another approach for patients with 12 of 20 Int. J. Mol. Sci. 2018, 19, 148 bone metastases is the use of radiopharmaceutical molecules (i.e., strontium, samarium and radium) that have a high avidity for calciﬁed bone matrix. Finally, these agents have a slight antitumor effect via localized radiations, but they have substantial effects on bone pain and hence they are principally indicated because of their analgesic action [57,127]. 7. Conclusions: Immunotherapy Perspectives Identifying chemokines and their receptors involved in cancer biology is of growing interest due to their involvement in multiple steps of the metastatic process including proliferation, extravasation, survival of tumour cells and tumour angiogenesis [128–132]. Chemokines have been shown to control angiogenesis: (i) directly through interactions with their receptors expressed on endothelial cells, as described for CXCL5, CXCL8 and CXCL12; (ii) indirectly through the recruitment and the stimulation of leukocytes resulting in the production of angiogenic factors [133,134]. Lastly, tumour invasion is also controlled by chemokines and their receptors and among them, CXCR4 is the most frequently overexpressed receptor [135]. Interestingly, through their ability to recruit many leukocyte sub-populations, chemokines and their receptors regulate also the immune response toward the tumour [82,136–139]. Understanding these mechanisms provides numerous molecular targets for potential therapeutic applications. Several types of therapy can be explored including blocking interactions between chemokines and their receptors, interfering with receptor transduction pathways, or providing a local delivery of chemokines able to trigger an anti-cancer immune response [140–142]. In that respect, Bidwell et al. identiﬁed an innate immune pathway (Irf7 pathway) intrinsic to breast cancer cells involved in the development of metastasis. Based on this result, they suggested that the restoration of Irf7 may reduce bone metastases [143]. However, despite the development of these novel therapies, few studies address speciﬁcally the issue of bone metastases treatment, and this research area will certainly continue to expand. Indeed, unlike “passive” strategies involving the use of antibodies, such innovative and ambitious therapies can be considered as “active”, since they trigger the immune system response against cancer cells, including an immune memory resulting in long-term beneﬁts. Acknowledgments: The authors are grateful for the funding support from the University of Nantes (AAP Interdisciplinaires 2017—phase 1). Author Contributions: All authors have contributed to the manuscript. Heidy Schmid-Antomarchi, Annie Schmid-Alliana, Patricia Lagadec and Rasha Al-Sahlanee have extensively participated to the bibliographic research and have commented the studies. Jean-Claude Scimeca and Elise Verron have written and supervised the works. Conﬂicts of Interest: The authors declare no conﬂict of interest. Abbreviations BMP bone morphogenetic protein BP bisphosphonate CTGF connective tissue growth factor Ctsk cathepsin K Coll collagen CXCL C-X-C motif chemokine ligand CXCR4 C-X-C chemokine receptor type 4 EMT epithelial to mesenchymal transition ET-1 endothelin-1 FGF ﬁbroblast growth factor Ga gallium GM-CSF granulocyte macrophage colony stimulating factor Abbreviations 13 of 20 Int. J. Mol. Sci. 2018, 19, 148 HFG halofuginone HSCs hematopoietic stem cells IGF insulin-like growth factor IL interleukin LPA lysophosphatidic acid miRNAs microRNAs MMP matrix metallo protein MSC mesenchymal stem cells MSG metastases suppressor gene MTA1 metastasis- associated protein 1 NGF nerve growth factor OPG osteoprotegerin OPN osteopontin PDGF platelet -derived growth factor PgE2 prostaglandin E2 PMMA poly[methyl methacrylate] PPi pyrophosphate PTEN phosphatase and tensin homolog PTHrP parathyroid hormone-related peptide RANKL receptor activator of nuclear factor—κB receptor ligand SCP single cell progeny SDF-1 stromal-derived growth factor-1 (/CXCL12) siRNAs small interfering RNAs SRE skeletal-related event TCR T cell receptor TGFβ transforming growth factor-beta TNFα tumor necrosis factor-alpha VEGF vascular endothelial growth factor VLA4 very late antigen 4 References Iddon, J.; Byrne, G.; Bundred, N.J. Bone metastasis in breast cancer: The role of parathyroid hormone related protein. Surg. Oncol. 1999, 8, 13–25. [CrossRef] 12. Klein, C.A. Parallel progression of primary tumours and metastases. Nat. Rev. Cancer 2009, 9, 302–312. [CrossRef] [PubMed] 13. Klein, G. Foulds’ dangerous idea revisited: The multistep development of tumors 40 years later. Adv. Cancer Res. 1998, 72, 1–23. [PubMed] 4. Fearon, E.R.; Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 1990, 61, 759–767. [Cross 14. Fearon, E.R.; Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 1990, 61, 759–767. [CrossRef] 15. Cairns, J. Mutation selection and the natural history of cancer. Nature 1975, 255, 197–200. [CrossRef] [PubMed] 15. Cairns, J. Mutation selection and the natural history of cancer. Nature 1975, 255, 197–200. [CrossRef] [PubMed] 16. Olivier, M.; Langerod, A.; Carrieri, P.; Bergh, J.; Klaar, S.; Eyfjord, J.; Theillet, C.; Rodriguez, C.; Lidereau, R.; Bieche, I.; et al. The clinical value of somatic TP53 gene mutations in 1794 patients with breast cancer. Clin. Cancer Res. 2006, 12, 1157–1167. [CrossRef] [PubMed] 17. McAllister, S.S.; Gifford, A.M.; Greiner, A.L.; Kelleher, S.P.; Saelzler, M.P.; Ince, T.A.; Reinhardt, F.; Harris, L.N.; Hylander, B.L.; Repasky, E.A.; et al. Systemic endocrine instigation of indolent tumor growth requires osteopontin. Cell 2008, 133, 994–1005. [CrossRef] [PubMed] 18. Koscielny, S.; Tubiana, M.; Le, M.G.; Valleron, A.J.; Mouriesse, H.; Contesso, G.; Sarrazin, D. Breast cancer: Relationship between the size of the primary tumour and the probability of metastatic dissemination. Br. J. Cancer 1984, 49, 709–715. [CrossRef] [PubMed] 19. Koscielny, S.; Tubiana, M. Parallel progression of tumour and metastases. Nat. Rev. Cancer 2010, 10, 156. [CrossRef] [PubMed] 20. Collins, V.P.; Loefﬂer, R.K.; Tivey, H. Observations on growth rates of human tumors. Am. J. Roentgenol. Radium Ther. Nucl. Med. 1956, 76, 988–1000. [PubMed] 21. Husemann, Y.; Geigl, J.B.; Schubert, F.; Musiani, P.; Meyer, M.; Burghart, E.; Forni, G.; Eils, R.; Fehm, T.; Riethmuller, G.; et al. Systemic spread is an early step in breast cancer. Cancer Cell 2008, 13, 58–68. [CrossRef] [PubMed] 22. Podsypanina, K.; Du, Y.C.; Jechlinger, M.; Beverly, L.J.; Hambardzumyan, D.; Varmus, H. Seeding and propagation of untransformed mouse mammary cells in the lung. Science 2008, 321, 1841–1844. [CrossRef] [PubMed] 23. Schmidt-Kittler, O.; Ragg, T.; Daskalakis, A.; Granzow, M.; Ahr, A.; Blankenstein, T.J.; Kaufmann, M.; Diebold, J.; Arnholdt, H.; Muller, P.; et al. References 1. Davila, D.; Antoniou, A.; Chaudhry, M.A. Evaluation of osseous metastasis in bone scintigraphy. Semin. Nucl. Med. 2015, 45, 3–15. [CrossRef] [PubMed] 1. Davila, D.; Antoniou, A.; Chaudhry, M.A. Evaluation of osseous metastasis in bone scintigraphy. Semin. Nucl. Med. 2015, 45, 3–15. [CrossRef] [PubMed] 2. Camparo, P.; Vieillefond, A. Molecular aspects of prostate cancer: Recent data from the literature. Bull. Cancer 2007, 94, F77–F88. [PubMed] 2. Camparo, P.; Vieillefond, A. Molecular aspects of prostate cancer: Recent data from the literature. Bull. Cancer 2007, 94, F77–F88. [PubMed] 3. Hamaoka, T.; Madewell, J.E.; Podoloff, D.A.; Hortobagyi, G.N.; Ueno, N.T. Bone imaging in metastatic breast cancer. J. Clin. Oncol. 2004, 22, 2942–2953. [CrossRef] [PubMed] 3. Hamaoka, T.; Madewell, J.E.; Podoloff, D.A.; Hortobagyi, G.N.; Ueno, N.T. Bone imaging in metastatic breast cancer. J. Clin. Oncol. 2004, 22, 2942–2953. [CrossRef] [PubMed] 4. Paget, S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev. 1989, 8, 98–101. [PubMed] 4. Paget, S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev. 1989, 8, 98–101. [PubMed] 5. Cecchini, M.G.; Wetterwald, A.; van der Pluijm, G.; Thalmann, G.N. Molecular and Biological Mechanisms of Bone Metastasis. EAU Update Ser. 2005, 3, 214–226. [CrossRef] 5. Cecchini, M.G.; Wetterwald, A.; van der Pluijm, G.; Thalmann, G.N. Molecular and Biological Mechanisms of Bone Metastasis. EAU Update Ser. 2005, 3, 214–226. [CrossRef] 6. Buijs, J.T.; van der Pluijm, G. Osteotropic cancers: From primary tumor to bone. Cancer Lett. 2009, 273, 177–193. [CrossRef] [PubMed] 6. Buijs, J.T.; van der Pluijm, G. Osteotropic cancers: From primary tumor to bone. Cancer Lett. 2009, 273, 177–193. [CrossRef] [PubMed] 7. Yoneda, T.; Hiraga, T. Crosstalk between cancer cells and bone microenvironment in bone metastasis. Biochem. Biophys. Res. Commun. 2005, 328, 679–687. [CrossRef] [PubMed] 8. Ata, R.; Antonescu, C.N. Integrins and Cell Metabolism: An Intimate Relationship Impacting Cancer. Int. J. Mol. Sci. 2017, 18. [CrossRef] [PubMed] 9. Gasparics, A.; Rosivall, L.; Krizbai, I.A.; Sebe, A. When the endothelium scores an own goal: Endothelial cells actively augment metastatic extravasation through endothelial-mesenchymal transition. Am. J. Physiol. Heart Circ. Physiol. 2016, 310, H1055–H1063. [CrossRef] [PubMed] 10. Sipkins, D.A.; Wei, X.; Wu, J.W.; Runnels, J.M.; Cote, D.; Means, T.K.; Luster, A.D.; Scadden, D.T.; Lin, C.P. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 2005, 435, 969–973. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 148 14 of 20 11. References From latent disseminated cells to overt metastasis: Genetic analysis of systemic breast cancer progression. Proc. Natl. Acad. Sci. USA 2003, 100, 7737–7742. [CrossRef] [PubMed] 24. Tomuleasa, C.; Zaharie, F.; Muresan, M.S.; Pop, L.; Fekete, Z.; Dima, D.; Frinc, I.; Trifa, A.; Berce, C.; Jurj, A.; et al. How to Diagnose and Treat a Cancer of Unknown Primary Site. J. Gastrointestin. Liver Dis. 2017, 26, 69–79. [PubMed] 25. Kaplan, R.N.; Psaila, B.; Lyden, D. Bone marrow cells in the ‘pre-metastatic niche’: Within bone and beyond. Cancer Metastasis Rev. 2006, 25, 521–529. [CrossRef] [PubMed] 26. Hess, K.R.; Varadhachary, G.R.; Taylor, S.H.; Wei, W.; Raber, M.N.; Lenzi, R.; Abbruzzese, J.L. Metastatic patterns in adenocarcinoma. Cancer 2006, 106, 1624–1633. [CrossRef] [PubMed] 27. Psaila, B.; Lyden, D. The metastatic niche: Adapting the foreign soil. Nat. Rev. Cancer 2009, 9, 285–293. [CrossRef] [PubMed] 27. Psaila, B.; Lyden, D. The metastatic niche: Adapting the foreign soil. Nat. Rev. Cancer 2009, 9, 285–293. [CrossRef] [PubMed] 28. Nguyen, D.X.; Bos, P.D.; Massague, J. Metastasis: From dissemination to organ-speciﬁc colonization. 8. Nguyen, D.X.; Bos, P.D.; Massague, J. Metastasis: From dissemination to organ-speciﬁc coloniza Nat. Rev. Cancer 2009, 9, 274–284. [CrossRef] [PubMed] 29. Fidler, I.J. The pathogenesis of cancer metastasis: The ‘seed and soil’ hypothesis revisited. Nat. Rev. Cancer 2003, 3, 453–458. [CrossRef] [PubMed] 30. Gupta, G.P.; Minn, A.J.; Kang, Y.; Siegel, P.M.; Serganova, I.; Cordon-Cardo, C.; Olshen, A.B.; Gerald, W.L.; Massague, J. Identifying site-speciﬁc metastasis genes and functions. Cold Spring Harb. Symp. Quant. Biol. 2005, 70, 149–158. [CrossRef] [PubMed] 31. Ara, T.; Declerck, Y.A. Interleukin-6 in bone metastasis and cancer progression. Eur. J. Cancer 2010, 46, 1223–1231. [CrossRef] [PubMed] 2. Ewing, J. Neoplastic Diseases: A Treatise on Tumors, 3rd ed.; WB Saunders: Philadelphia, PA, USA, 1928. 33. Chiang, A.C.; Massague, J. Molecular basis of metastasis. N. Engl. J. Med. 2008, 359, 2814–2823. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 148 15 of 20 34. Zarour, L.R.; Anand, S.; Billingsley, K.G.; Bisson, W.H.; Cercek, A.; Clarke, M.F.; Coussens, L.M.; Gast, C.E.; Geltzeiler, C.B.; Hansen, L.; et al. Colorectal Cancer Liver Metastasis: Evolving Paradigms and Future Directions. Cell. Mol. Gastroenterol. Hepatol. 2017, 3, 163–173. [CrossRef] [PubMed] 5. Waugh, D.J.; Wilson, C. The interleukin-8 pathway in cancer. Clin. Cancer Res. 2008, 14, 6735–6741. [Cross [PubMed] 36. Weidle, U.H.; Birzele, F.; Kollmorgen, G.; Ruger, R. Molecular Mechanisms of Bone Metastasis. Cancer Genom. Proteom. 2016, 13, 1–12. 37. References Goerge, T.; Barg, A.; Schnaeker, E.M.; Poppelmann, B.; Shpacovitch, V.; Rattenholl, A.; Maaser, C.; Luger, T.A.; Steinhoff, M.; Schneider, S.W. Tumor-derived matrix metalloproteinase-1 targets endothelial proteinase-activated receptor 1 promoting endothelial cell activation. Cancer Res. 2006, 66, 7766–7774. [CrossRef] [PubMed] 38. Jacobson, A.; Cunningham, J.L. Connective tissue growth factor in tumor pathogenesis. Fibrogenesis Tissue Repair 2012, 5, S8. [CrossRef] [PubMed] 39. Sotiriou, C.; Lacroix, M.; Lespagnard, L.; Larsimont, D.; Paesmans, M.; Body, J.J. Interleukins-6 and -11 expression in primary breast cancer and subsequent development of bone metastases. Cancer Lett. 2001, 169, 87–95. [CrossRef] 0. Rose, A.A.; Pepin, F.; Russo, C.; Abou Khalil, J.E.; Hallett, M.; Siegel, P.M. Osteoactivin promotes br cancer metastasis to bone. Mol. Cancer Res. 2007, 5, 1001–1014. [CrossRef] [PubMed] 41. Psaila, B.; Lyden, D.; Roberts, I. Megakaryocytes, malignancy and bone marrow vascular niches. J. Thromb. Haemost. 2012, 10, 177–188. [CrossRef] [PubMed] 42. Guise, T.A.; Mundy, G.R. Cancer and bone. Endocr. Rev. 1998, 19, 18–54. [CrossRef] [PubMed] 43. Msaouel, P.; Pissimissis, N.; Halapas, A.; Koutsilieris, M. Mechanisms of bone metastasis in prostate cancer: Clinical implications. Best Pract. Res. Clin. Endocrinol. Metab. 2008, 22, 341–355. [CrossRef] [PubMed] 44. Quail, D.F.; Joyce, J.A. Microenvironmental regulation of tumor progression and metastasis. Nat. Med. 2013, 19, 1423–1437. [CrossRef] [PubMed] 45. Buijs, J.T.; Stayrook, K.R.; Guise, T.A. The role of TGF-beta in bone metastasis: Novel therapeutic perspectives. Bonekey Rep. 2012, 1, 96. [CrossRef] [PubMed] 6. Juarez, P.; Guise, T.A. TGF-beta in cancer and bone: Implications for treatment of bone metastases. Bone 2 48, 23–29. [CrossRef] [PubMed] 47. Kiriyama, T.; Gillespie, M.T.; Glatz, J.A.; Fukumoto, S.; Moseley, J.M.; Martin, T.J. Transforming growth factor beta stimulation of parathyroid hormone-related protein (PTHrP): A paracrine regulator? Mol. Cell. Endocrinol. 1993, 92, 55–62. [CrossRef] 48. Kingsley, L.A.; Fournier, P.G.; Chirgwin, J.M.; Guise, T.A. Molecular biology of bone metastasis. Mol. Cancer Ther. 2007, 6, 2609–2617. [CrossRef] [PubMed] 49. McMahon, S.; Charbonneau, M.; Grandmont, S.; Richard, D.E.; Dubois, C.M. Transforming growth factor beta1 induces hypoxia-inducible factor-1 stabilization through selective inhibition of PHD2 expression. J. Biol. Chem. 2006, 281, 24171–24181. [CrossRef] [PubMed] 50. Moustakas, A.; Heldin, C.H. Induction of epithelial-mesenchymal transition by transforming growth factor beta. Semin. Cancer Biol. 2012, 22, 446–454. [CrossRef] [PubMed] 51. Flavell, R.A.; Sanjabi, S.; Wrzesinski, S.H.; Licona-Limon, P. The polarization of immune cells in the tumour environment by TGFbeta. Nat. Rev. Immunol. 2010, 10, 554–567. [CrossRef] [PubMed] 52. References Yoneda, T.; Hata, K.; Nakanishi, M.; Nagae, M.; Nagayama, T.; Wakabayashi, H.; Nishisho, T.; Sakurai, T.; Hiraga, T. Involvement of acidic microenvironment in the pathophysiology of cancer-associated bone pain. Bone 2011, 48, 100–105. [CrossRef] [PubMed] 53. Xu, X.T.; Xu, B.; Song, Q.B.; Zeng, H. The role of neural-related factors in the metastasis of the gastrointestinal cancer. J. Cancer Res. Ther. 2013, 9, S123–S128. [PubMed] 54. Artacho-Cordon, F.; Rios-Arrabal, S.; Lara, P.C.; Artacho-Cordon, A.; Calvente, I.; Nunez, M.I. Matrix metalloproteinases: Potential therapy to prevent the development of second malignancies after breast radiotherapy. Surg. Oncol. 2012, 21, e143–e151. [CrossRef] [PubMed] 55. Leblanc, R.; Lee, S.C.; David, M.; Bordet, J.C.; Norman, D.D.; Patil, R.; Miller, D.; Sahay, D.; Ribeiro, J.; Clezardin, P.; et al. Interaction of platelet-derived autotaxin with tumor integrin alphaVbeta3 controls metastasis of breast cancer cells to bone. Blood 2014, 124, 3141–3150. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 148 16 of 20 16 of 20 56. Peyruchaud, O.; Leblanc, R.; David, M. Pleiotropic activity of lysophosphatidic acid in bone metastasis. Biochim. Biophys. Acta 2013, 1831, 99–104. [CrossRef] [PubMed] 57. Weilbaecher, K.N.; Guise, T.A.; McCauley, L.K. Cancer to bone: A fatal attraction. Nat. Rev. Cancer 2011, 11, 411–425. [CrossRef] [PubMed] 58. Voronov, E.; Shouval, D.S.; Krelin, Y.; Cagnano, E.; Benharroch, D.; Iwakura, Y.; Dinarello, C.A.; Apte, R.N. IL-1 is required for tumor invasiveness and angiogenesis. Proc. Natl. Acad. Sci. USA 2003, 100, 2645–2650. [CrossRef] [PubMed] 59. Di Francesco, S.; Castellan, P.; Manco, R.; Tenaglia, R.L. Reciprocal cross-talk between Prostaglandin E2 and bone in prostate cancer: A current review. Cent. Eur. J. Urol. 2011, 64, 201–204. [CrossRef] [PubMed] 60. Diament, M.J.; Stillitani, M.I.; Puricelli, L.; Bal de Kier-Joffe, E.; Klein, S. GM-CSF secreted by murine adenocarcinoma cells modulates tumor progression and immune activity. Oncol. Rep. 2003, 10, 1647–1652. [CrossRef] [PubMed] 61. Kato, H.; Wakabayashi, H.; Naito, Y.; Kato, S.; Nakagawa, T.; Matsumine, A.; Sudo, A. Anti-tumor necrosis factor therapy inhibits lung metastasis in an osteosarcoma cell line. Oncology 2015, 88, 139–146. [CrossRef] [PubMed] 62. Verbovsek, U.; Van Noorden, C.J.; Lah, T.T. Complexity of cancer protease biology: Cathepsin K expression and function in cancer progression. Semin. Cancer Biol. 2015, 35, 71–84. [CrossRef] [PubMed] 63. Kruger, T.E.; Miller, A.H.; Godwin, A.K.; Wang, J. Bone sialoprotein and osteopontin in bone metastasis of osteotropic cancers. Crit. Rev. Oncol. Hematol. 2014, 89, 330–341. [CrossRef] [PubMed] 64. Davis, H.; Raja, E.; Miyazono, K.; Tsubakihara, Y.; Moustakas, A. References Mechanisms of action of bone morphogenetic proteins in cancer. Cytokine Growth Factor Rev. 2016, 27, 81–92. [CrossRef] [PubMed] 65. Valkenburg, K.C.; Steensma, M.R.; Williams, B.O.; Zhong, Z. Skeletal metastasis: Treatments, mouse models, and the Wnt signaling. Chin. J. Cancer 2013, 32, 380–396. [CrossRef] [PubMed] 66. Buijs, J.T.; Kuijpers, C.C.; van der Pluijm, G. Targeted therapy options for treatment of bone metastases; beyond bisphosphonates. Curr. Pharm. Des. 2010, 16, 3015–3027. [CrossRef] [PubMed] 67. Garrison, K.; Hahn, T.; Lee, W.C.; Ling, L.E.; Weinberg, A.D.; Akporiaye, E.T. The small molecule TGF-beta signaling inhibitor SM16 synergizes with agonistic OX40 antibody to suppress established mammary tumors and reduce spontaneous metastasis. Cancer Immunol. Immunother. 2012, 61, 511–521. [CrossRef] [PubMed] 68 C l R E Sk l t l li ti f li C 1997 80 1588 1594 [C R f] 8. Coleman, R.E. Skeletal complications of malignancy. Cancer 1997, 80, 1588–1594. [CrossRef] 9. Liepe, K.; Kotzerke, J. Internal radiotherapy of painful bone metastases. Methods 2011, 55, 258–270. [Cross [PubMed] 70. Kurup, A.N.; Callstrom, M.R. Expanding role of percutaneous ablative and consolidative treatments for musculoskeletal tumours. Clin. Radiol. 2017, 72, 645–656. [CrossRef] [PubMed] 71. Szendroi, M.; Antal, I.; Szendroi, A.; Lazary, A.; Varga, P.P. Diagnostic algorithm, prognostic factors and surgical treatment of metastatic cancer diseases of the long bones and spine. EFORT Open Rev. 2017, 2, 372–381. [CrossRef] [PubMed] 72. Thariat, J.; Vignot, S.; Bensadoun, R.J.; Mornex, F. Improvements of ablative local treatments modify the management of the oligometastatic disease. Cancer Radiother. 2012, 16, 325–329. [CrossRef] [PubMed] 73. Kougioumtzopoulou, A.; Zygogianni, A.; Liakouli, Z.; Kypraiou, E.; Kouloulias, V. The role of radiotherapy in bone metastases: A critical review of current literature. Eur. J. Cancer Care 2017, 26. [CrossRef] [PubMed] 73. Kougioumtzopoulou, A.; Zygogianni, A.; Liakouli, Z.; Kypraiou, E.; Kouloulias, V. The role of radiotherapy in bone metastases: A critical review of current literature. Eur. J. Cancer Care 2017, 26. [CrossRef] [PubMed] 74. Chiras, J.; Shotar, E.; Cormier, E.; Clarencon, F. Interventional radiology in bone metastases. Eur. J. Cancer Care 2017, 26. [CrossRef] [PubMed] 74. Chiras, J.; Shotar, E.; Cormier, E.; Clarencon, F. Interventional radiology in bone metastases. Eur. J. Ca Care 2017, 26. [CrossRef] [PubMed] 75. Laredo, J.D.; Chiras, J.; Kemel, S.; Taihi, L.; Hamze, B. Vertebroplasty and interventional radiology procedures for bone metastases. Jt. Bone Spine 2017. [CrossRef] [PubMed] 76. Pikis, S.; Goldstein, J.; Spektor, S. Potential neurotoxic effects of polymethylmethacrylate during cranioplasty. J. Clin. Neurosci. References 2015, 22, 139–143. [CrossRef] [PubMed] 77. Ratasvuori, M.; Wedin, R.; Keller, J.; Nottrott, M.; Zaikova, O.; Bergh, P.; Kalen, A.; Nilsson, J.; Jonsson, H.; Laitinen, M. Insight opinion to surgically treated metastatic bone disease: Scandinavian Sarcoma Group Skeletal Metastasis Registry report of 1195 operated skeletal metastasis. Surg. Oncol. 2013, 22, 132–138. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 148 17 of 20 78. Van Driel, M.; van Leeuwen, J.P. Cancer and bone: A complex complex. Arch. Biochem. Biophys. 2014, 561, 159–166. [CrossRef] [PubMed] 78. Van Driel, M.; van Leeuwen, J.P. Cancer and bone: A complex complex. Arch. Biochem. Biophys. 2014, 561, 159–166. [CrossRef] [PubMed] 79. Mundy, G.R. Metastasis to bone: Causes, consequences and therapeutic opportunities. Nat. Rev. Cancer 2002, 2, 584–593. [CrossRef] [PubMed] 80. Zou, X.; Zou, L.; He, Y.; Bunger, C. Molecular treatment strategies and surgical reconstruction for metastatic bone diseases. Cancer Treat. Rev. 2008, 34, 527–538. [CrossRef] [PubMed] 81. Verron, E.; Bouler, J.M. Is bisphosphonate therapy compromised by the emergence of adverse bone disorders? Drug Discov. Today 2014, 19, 312–319. [CrossRef] [PubMed] 82. Bernardini, G.; Gismondi, A.; Santoni, A. Chemokines and NK cells: Regulators of development, trafﬁcking and functions. Immunol. Lett. 2012, 145, 39–46. [CrossRef] [PubMed] 83. Gronich, N.; Rennert, G. Beyond aspirin-cancer prevention with statins, metformin and bisphosphonates. Nat. Rev. Clin. Oncol. 2013, 10, 625–642. [CrossRef] [PubMed] 84. Odri, G.; Kim, P.P.; Lamoureux, F.; Charrier, C.; Battaglia, S.; Amiaud, J.; Heymann, D.; Gouin, F.; Redini, F. Zoledronic acid inhibits pulmonary metastasis dissemination in a preclinical model of Ewing’s sarcoma via inhibition of cell migration. BMC Cancer 2014, 14, 169. [CrossRef] [PubMed] 85. Clezardin, P.; Ebetino, F.H.; Fournier, P.G. Bisphosphonates and cancer-induced bone disease: Beyond their antiresorptive activity. Cancer Res. 2005, 65, 4971–4974. [CrossRef] [PubMed] 86. Guise, T.A. Antitumor effects of bisphosphonates: Promising preclinical evidence. Cancer Treat. Rev. 2008, 34 (Suppl. S1), S19–S24. [CrossRef] [PubMed] 87. Clezardin, P. Mechanisms of action of bisphosphonates in oncology: A scientiﬁc concept evolving from antiresorptive to anticancer activities. Bonekey Rep. 2013, 2, 267. [CrossRef] [PubMed] 88. Benzaid, I.; Monkkonen, H.; Bonnelye, E.; Monkkonen, J.; Clezardin, P. In vivo phosphoantigen levels in bisphosphonate-treated human breast tumors trigger Vgamma9Vdelta2 T-cell antitumor cytotoxicity through ICAM-1 engagement. Clin. Cancer Res. 2012, 18, 6249–6259. [CrossRef] [PubMed] 89. Benzaid, I.; Monkkonen, H.; Stresing, V.; Bonnelye, E.; Green, J.; Monkkonen, J.; Touraine, J.L.; Clezardin, P. References High phosphoantigen levels in bisphosphonate-treated human breast tumors promote Vgamma9Vdelta2 T-cell chemotaxis and cytotoxicity in vivo. Cancer Res. 2011, 71, 4562–4572. [CrossRef] [PubMed] 90. Dominguez, L.J.; Di Bella, G.; Belvedere, M.; Barbagallo, M. Physiology of the aging bone and mech of action of bisphosphonates. Biogerontology 2011, 12, 397–408. [CrossRef] [PubMed] 91. Hirsh, V.; Tchekmedyian, N.S.; Rosen, L.S.; Zheng, M.; Hei, Y.J. Clinical beneﬁt of zoledronic acid in patients with lung cancer and other solid tumors: Analysis based on history of skeletal complications. Clin. Lung Cancer 2004, 6, 170–174. [CrossRef] [PubMed] 92. Saad, F.; Gleason, D.M.; Murray, R.; Tchekmedyian, S.; Venner, P.; Lacombe, L.; Chin, J.L.; Vinholes, J.J.; Goas, J.A.; Zheng, M.; et al. Long-term efﬁcacy of zoledronic acid for the prevention of skeletal complications in patients with metastatic hormone-refractory prostate cancer. J. Natl. Cancer Inst. 2004, 96, 879–882. [CrossRef] [PubMed] 93. Terpos, E.; Confavreux, C.B.; Clezardin, P. Bone antiresorptive agents in the treatment of bone metastases associated with solid tumours or multiple myeloma. Bonekey Rep. 2015, 4, 744. [CrossRef] [PubMed] 93. Terpos, E.; Confavreux, C.B.; Clezardin, P. Bone antiresorptive agents in the treatment of bone metastases associated with solid tumours or multiple myeloma. Bonekey Rep. 2015, 4, 744. [CrossRef] [PubMed] 94. Van Poznak, C.H.; Von Roenn, J.H.; Temin, S. American society of clinical oncology clinical practice guideline update: Recommendations on the role of bone-modifying agents in metastatic breast cancer. J. Oncol. Pract. 2011, 7, 117–121. [CrossRef] [PubMed] 95. Coleman, R.; Gnant, M.; Morgan, G.; Clezardin, P. Effects of bone-targeted agents on cancer progression and mortality. J. Natl. Cancer Inst. 2012, 104, 1059–1067. [CrossRef] [PubMed] 96. Khosla, S.; Burr, D.; Cauley, J.; Dempster, D.W.; Ebeling, P.R.; Felsenberg, D.; Gagel, R.F.; Gilsanz, V.; Guise, T.; Koka, S.; et al. Bisphosphonate-associated osteonecrosis of the jaw: Report of a task force of the American Society for Bone and Mineral Research. J. Bone Miner. Res. 2007, 22, 1479–1491. [CrossRef] [PubMed] 97. Lezot, F.; Chesneau, J.; Battaglia, S.; Brion, R.; Castaneda, B.; Farges, J.C.; Heymann, D.; Redini, F. Preclinical evidence of potential craniofacial adverse effect of zoledronic acid in pediatric patients with bone malignancies. Bone 2014, 68, 146–152. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 148 18 of 20 18 of 20 98. Anderson, P.; Kopp, L.; Anderson, N.; Cornelius, K.; Herzog, C.; Hughes, D.; Huh, W. Novel bone cancer drugs: Investigational agents and control paradigms for primary bone sarcomas (Ewing’s sarcoma and osteosarcoma). References Expert Opin. Investig. Drugs 2008, 17, 1703–1715. [CrossRef] [PubMed] 99. D’Antonio, C.; Passaro, A.; Gori, B.; Del Signore, E.; Migliorino, M.R.; Ricciardi, S.; Fulvi, A.; de Marinis, F. Bone and brain metastasis in lung cancer: Recent advances in therapeutic strategies. Ther. Adv. Med. Oncol. 2014, 6, 101–114. [CrossRef] [PubMed] 100. Clemons, M.; Gelmon, K.A.; Pritchard, K.I.; Paterson, A.H. Bone-targeted agents and skeletal-related events in breast cancer patients with bone metastases: The state of the art. Curr. Oncol. 2012, 19, 259–268. [CrossRef] [PubMed] 101. Peddi, P.; Lopez-Olivo, M.A.; Pratt, G.F.; Suarez-Almazor, M.E. Denosumab in patients with cancer and skeletal metastases: A systematic review and meta-analysis. Cancer Treat. Rev. 2013, 39, 97–104. [CrossRef] [PubMed] 102. Miller, P.D.; Bolognese, M.A.; Lewiecki, E.M.; McClung, M.R.; Ding, B.; Austin, M.; Liu, Y.; San Martin, J.; Amg Bone Loss Study Group. Effect of denosumab on bone density and turnover in postmenopausal women with low bone mass after long-term continued, discontinued, and restarting of therapy: A randomized blinded phase 2 clinical trial. Bone 2008, 43, 222–229. [CrossRef] [PubMed] 103. Casimiro, S.; Guise, T.A.; Chirgwin, J. The critical role of the bone microenvironment in cancer met Mol. Cell. Endocrinol. 2009, 310, 71–81. [CrossRef] [PubMed] 104. Ando, K.; Heymann, M.F.; Stresing, V.; Mori, K.; Redini, F.; Heymann, D. Current therapeutic strategies and novel approaches in osteosarcoma. Cancers 2013, 5, 591–616. [CrossRef] [PubMed] 105. Fili, S.; Karalaki, M.; Schaller, B. Mechanism of bone metastasis: The role of osteoprotegerin and of the host-tissue microenvironment-related survival factors. Cancer Lett. 2009, 283, 10–19. [CrossRef] [PubMed] 106. Wittrant, Y.; Theoleyre, S.; Chipoy, C.; Padrines, M.; Blanchard, F.; Heymann, D.; Redini, F. RANKL/RANK/OPG: New therapeutic targets in bone tumours and associated osteolysis. Biochim. Biophys. Acta 2004, 1704, 49–57. [CrossRef] [PubMed] 107. Verron, E.; Loubat, A.; Carle, G.F.; Vignes-Colombeix, C.; Strazic, I.; Guicheux, J.; Rochet, N.; Bouler, J.M.; Scimeca, J.C. Molecular effects of gallium on osteoclastic differentiation of mouse and human monocytes. Biochem. Pharmacol. 2012, 83, 671–679. [CrossRef] [PubMed] 108. Strazic Geljic, I.; Melis, N.; Boukhechba, F.; Schaub, S.; Mellier, C.; Janvier, P.; Laugier, J.P.; Bouler, J.M.; Verron, E.; Scimeca, J.C. Gallium enhances reconstructive properties of a calcium phosphate bone biomaterial. J. Tissue Eng. Regen. Med. 2017. [CrossRef] [PubMed] 109. Strazic-Geljic, I.; Guberovic, I.; Didak, B.; Schmid-Antomarchi, H.; Schmid-Alliana, A.; Boukhechba, F.; Bouler, J.M.; Scimeca, J.C.; Verron, E. Gallium, a promising candidate to disrupt the vicious cycle driving osteolytic metastases. Biochem. Pharmacol. 2016, 116, 11–21. References [CrossRef] [PubMed] 110. Scimeca, J.C.; Verron, E. The multiple therapeutic applications of miRNAs for bone regenerative medicine. Drug Discov. Today 2017, 22, 1084–1091. [CrossRef] [PubMed] 111. Green, D.; Mohorianu, I.; McNamara, I.; Dalmay, T.; Fraser, W.D. miR-16 is highly expressed in Paget’s associated osteosarcoma. Endocr. Relat. Cancer 2017, 24, L27–L31. [CrossRef] [PubMed] 112. Dostalova Merkerova, M.; Krejcik, Z.; Votavova, H.; Belickova, M.; Vasikova, A.; Cermak, J. Distinctive microRNA expression proﬁles in CD34+ bone marrow cells from patients with myelodysplastic syndrome. Eur. J. Hum. Genet. 2011, 19, 313–319. [CrossRef] [PubMed] 113. Zhang, H.L.; Qin, X.J.; Cao, D.L.; Zhu, Y.; Yao, X.D.; Zhang, S.L.; Dai, B.; Ye, D.W. An elevated serum miR-141 level in patients with bone-metastatic prostate cancer is correlated with more bone lesions. Asian J. Androl. 2013, 15, 231–235. [CrossRef] [PubMed] 114. Krzeszinski, J.Y.; Wei, W.; Huynh, H.; Jin, Z.; Wang, X.; Chang, T.C.; Xie, X.J.; He, L.; Mangala, L.S.; Lopez-Berestein, G.; et al. miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2. Nature 2014, 512, 431–435. [CrossRef] [PubMed] 115. Colak, S.; Ten Dijke, P. Targeting TGF-beta Signaling in Cancer. Trends Cancer 2017, 3, 56–71. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 148 19 of 20 19 of 20 116. Herbertz, S.; Sawyer, J.S.; Stauber, A.J.; Gueorguieva, I.; Driscoll, K.E.; Estrem, S.T.; Cleverly, A.L.; Desaiah, D.; Guba, S.C.; Benhadji, K.A.; et al. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway. Drug Des. Dev. Ther. 2015, 9, 4479–4499. 117. Guise, T.A.; Mohammad, K.S. Endothelins in bone cancer metastases. Cancer Treat. Res. 2004, 118, 197–212. [PubMed] 118. Qiao, L.; Liang, Y.; Li, N.; Hu, X.; Luo, D.; Gu, J.; Lu, Y.; Zheng, Q. Endothelin-A receptor antagonists in prostate cancer treatment-a meta-analysis. Int. J. Clin. Exp. Med. 2015, 8, 3465–3473. [PubMed] 119. Bae, M.H.; Oh, S.H.; Park, C.J.; Lee, B.R.; Kim, Y.J.; Cho, Y.U.; Jang, S.; Lee, J.H.; Kim, N.; Park, S.H.; et al. VLA-4 and CXCR4 expression levels show contrasting prognostic impact (favorable and unfavorable, respectively) in acute myeloid leukemia. Ann. Hematol. 2015, 94, 1631–1638. [CrossRef] [PubMed] 120. Liang, Z.; Yoon, Y.; Votaw, J.; Goodman, M.M.; Williams, L.; Shim, H. Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res. 2005, 65, 967–971. [PubMed] 121. Wu, Z.; McRoberts, K.S.; Theodorescu, D. The role of PTEN in prostate cancer cell tropism to the bone micro-environment. Carcinogenesis 2007, 28, 1393–1400. [CrossRef] [PubMed] 122. References Peyruchaud, O.; Serre, C.M.; NicAmhlaoibh, R.; Fournier, P.; Clezardin, P. Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity. J. Biol. Chem. 2003, 278, 45826–45832. [CrossRef] [PubMed] 123. Krzeszinski, J.Y.; Wan, Y. New therapeutic targets for cancer bone metastasis. Trends Pharmacol. Sci. 2015, 36, 360–373. [CrossRef] [PubMed] 124. Malisetty, V.L.; Penugurti, V.; Panta, P.; Chitta, S.K.; Manavathi, B. MTA1 expression in human cancers—Clinical and pharmacological signiﬁcance. Biomed. Pharmacother. 2017, 95, 956–964. [CrossRef] [PubMed] 125. Lozano-Ondoua, A.N.; Symons-Liguori, A.M.; Vanderah, T.W. Cancer-induced bone pain: Mechanisms and models. Neurosci. Lett. 2013, 557 Pt A, 52–59. [CrossRef] [PubMed] 126. Honore, P.; Luger, N.M.; Sabino, M.A.; Schwei, M.J.; Rogers, S.D.; Mach, D.B.; O’Keefe, P.F.; Ramnaraine, M.L.; Clohisy, D.R.; Mantyh, P.W. Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord. Nat. Med. 2000, 6, 521–528. [PubMed] and pain-related neurochemical reorganization of the spinal cord. Nat. Med. 2000, 6, 521–528. [PubMed] 127. Chwistek, M. Recent advances in understanding and managing cancer pain. F1000Research 2017, 6, 945. [CrossRef] [PubMed] p g p 127. Chwistek, M. Recent advances in understanding and managing cancer pain. F1000Research 2017, 6, 945. [CrossRef] [PubMed] 128. Itatani, Y.; Kawada, K.; Inamoto, S.; Yamamoto, T.; Ogawa, R.; Taketo, M.M.; Sakai, Y. The Role of Chemokines in Promoting Colorectal Cancer Invasion/Metastasis. Int. J. Mol. Sci. 2016, 17. [CrossRef] [PubMed] 129. O’Hayre, M.; Salanga, C.L.; Handel, T.M.; Allen, S.J. Chemokines and cancer: Migration, intracellular signalling and intercellular communication in the microenvironment. Biochem. J. 2008, 409, 635–649. [CrossRef] [PubMed] 130. Rollins, B.J. Inﬂammatory chemokines in cancer growth and progression. Eur. J. Cancer 2006, 42, 760–767. [CrossRef] [PubMed] 131. Sarvaiya, P.J.; Guo, D.; Ulasov, I.; Gabikian, P.; Lesniak, M.S. Chemokines in tumor progression and metastasis. Oncotarget 2013, 4, 2171–2185. [CrossRef] [PubMed] 132. Vicari, A.P.; Caux, C. Chemokines in cancer. Cytokine Growth Factor Rev. 2002, 13, 143–154. [CrossRe 133. Santoni, M.; Bracarda, S.; Nabissi, M.; Massari, F.; Conti, A.; Bria, E.; Tortora, G.; Santoni, G.; Cascinu, S. CXC and CC chemokines as angiogenic modulators in nonhaematological tumors. Biomed. Res. Int. 2014, 2014, 768758. [CrossRef] [PubMed] 134. Stockmann, C.; Schadendorf, D.; Klose, R.; Helfrich, I. The impact of the immune system on tumor: Angiogenesis and vascular remodeling. Front. Oncol. 2014, 4, 69. [CrossRef] [PubMed] 135. Wang, Y.; Xie, Y.; Oupicky, D. Potential of CXCR4/CXCL12 Chemokine Axis in Cancer Drug Delivery. Curr. Pharmacol. Rep. 2016, 2, 1–10. [CrossRef] [PubMed] 136. © 2018 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/). References D’Ambrosio, D. Regulatory T cells: How do they ﬁnd their space in the immunological arena? Semin. Cancer Biol. 2006, 16, 91–97. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 148 20 of 20 20 of 20 137. Franciszkiewicz, K.; Boissonnas, A.; Boutet, M.; Combadiere, C.; Mami-Chouaib, F. Role of chemokines and chemokine receptors in shaping the effector phase of the antitumor immune response. Cancer Res. 2012, 72, 6325–6332. [CrossRef] [PubMed] 138. Hemdan, N.Y. Anti-cancer versus cancer-promoting effects of the interleukin-17-producing T help Immunol. Lett. 2013, 149, 123–133. [CrossRef] [PubMed] 139. Sallusto, F.; Lanzavecchia, A.; Mackay, C.R. Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. Today 1998, 19, 568–574. [CrossRef] 140. Viola, A.; Sarukhan, A.; Bronte, V.; Molon, B. The pros and cons of chemokines in tumor immunology. Trends Immunol. 2012, 33, 496–504. [CrossRef] [PubMed] 141. Dranoff, G. Cytokines in cancer pathogenesis and cancer therapy. Nat. Rev. Cancer 2004, 4, 11–22. [CrossRef] [PubMed] 142. Mantovani, A.; Savino, B.; Locati, M.; Zammataro, L.; Allavena, P.; Bonecchi, R. The chemokine system in cancer biology and therapy. Cytokine Growth Factor Rev. 2010, 21, 27–39. [CrossRef] [PubMed] 143. Bidwell, B.N.; Slaney, C.Y.; Withana, N.P.; Forster, S.; Cao, Y.; Loi, S.; Andrews, D.; Mikeska, T.; Mangan, N.E.; Samarajiwa, S.A.; et al. Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape. Nat. Med. 2012, 18, 1224–1231. [CrossRef] [PubMed] © 2018 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/).
https://openalex.org/W3198806248	https://www.frontiersin.org/articles/10.3389/fnmol.2021.726133/pdf	English	null	Sustained Baclofen-Induced Activation of GABAB Receptors After Cerebral Ischemia Restores Receptor Expression and Function and Limits Progressing Loss of Neurons	Frontiers in molecular neuroscience	2,021	cc-by	11,369	ORIGINAL RESEARCH published: 01 September 2021 doi: 10.3389/fnmol.2021.726133 Edited by: Bernhard Luscher, The Pennsylvania State University (PSU), United States Edited by: Bernhard Luscher, The Pennsylvania State University (PSU), United States Mohammad Hleihil1,2, Markus Vaas1†, Musadiq A. Bhat1, Karthik Balakrishnan1† and Dietmar Benke1,2* Mohammad Hleihil1,2, Markus Vaas1†, Musadiq A. Bhat1, Karthik Balakrishnan1† and Dietmar Benke1,2* 1 Institute of Pharmacology and Toxicology, University Zurich, Zurich, Switzerland, 2 Neuroscience Center Zurich, ETH Zurich, University of Zurich, Zurich, Switzerland Reviewed by: Imre Vida, Charité – Universitätsmedizin Berlin, Germany Paul Andrew Davies, Tufts University School of Medicine, United States Reviewed by: Imre Vida, Charité – Universitätsmedizin Berlin, Germany Paul Andrew Davies, Tufts University School of Medicine, United States One important function of GABAB receptors is the control of neuronal activity to prevent overexcitation and thereby excitotoxic death, which is a hallmark of cerebral ischemia. Consequently, sustained activation of GABAB receptors with the selective agonist baclofen provides neuroprotection in in vitro and in vivo models of cerebral ischemia. However, excitotoxic conditions severely downregulate the receptors, which would compromise the neuroprotective effectiveness of baclofen. On the other hand, recent work suggests that sustained activation of GABAB receptors stabilizes receptor expression. Therefore, we addressed the question whether sustained activation of GABAB receptors reduces downregulation of the receptor under excitotoxic conditions and thereby preserves GABAB receptor-mediated inhibition. In cultured neurons subjected to oxygen and glucose deprivation (OGD), to mimic cerebral ischemia, GABAB receptors were severely downregulated. Treatment of the cultures with baclofen after OGD restored GABAB receptor expression and reduced loss of neurons. Restoration of GABAB receptors was due to enhanced fast recycling of the receptors, which reduced OGD-induced sorting of the receptors to lysosomal degradation. Utilizing the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia, we veriﬁed the severe downregulation of GABAB receptors in the affected cortex and a partial restoration of the receptors after systemic injection of baclofen. Restored receptor expression recovered GABAB receptor-mediated currents, normalized the enhanced neuronal excitability observed after MCAO and limited progressive loss of neurons. These results suggest that baclofen-induced restoration of GABAB receptors provides the basis for the neuroprotective activity of baclofen after an ischemic insult. Since GABAB receptors regulate multiple beneﬁcial pathways, they are promising targets for a neuroprotective strategy in acute cerebral ischemia. Correspondence: Dietmar Benke benke@pharma.uzh.ch †Present address: Markus Vaas, Clinical Trial Center Zurich, University Hospital of Zurich, Zurich, Switzerland Karthik Balakrisnan, Department of Neurobiology, Harvard Medical School, Boston, MA, United States †Present address: Markus Vaas, Clinical Trial Center Zurich, University Hospital of Zurich, Zurich, Switzerland Karthik Balakrisnan, Department of Neurobiology, Harvard Medical School, Boston, MA, United States Specialty section: This article was submitted to Brain Disease Mechanisms, a section of the journal Frontiers in Molecular Neuroscience Received: 16 June 2021 Accepted: 12 August 2021 Published: 01 September 2021 Keywords: GABAB receptor, cerebral ischemia, MCAO, OGD, baclofen, neuroprotection INTRODUCTION the receptors stabilizes them at the cell surface and reduces their downregulation, making the baclofen treatment more eﬃcient. The main cause for progressing neuronal death in cerebral ischemia is excitotoxicity. Massive release of glutamate excessively stimulates glutamate receptors, resulting in neuronal overexcitation and apoptotic death (Lipton, 1999; Szydlowska and Tymianski, 2010; Kostandy, 2012; Bano and Ankarcrona, 2018; Choi, 2020). Under physiological conditions, the excitability of neurons is precisely controlled by G protein coupled GABAB receptors (Chalifoux and Carter, 2011a). GABAB receptors are assembled from two subunits, GABAB1 and GABAB2, and are abundantly expressed at pre- and postsynaptic areas of most neurons. Activation of GABAB receptors by the neurotransmitter GABA provides long lasting tonic inhibition mainly by suppression of voltage gated Ca2+ channel activity and activation of inwardly rectifying K+ channels. This inhibits neurotransmitter release via GABAB receptors located at presynaptic sites and reduces neuronal excitability via postsynaptic receptors (Gassmann and Bettler, 2012). One function of GABAB receptors is to act as an “emergency brake” that prevents neurons from shifting into overexcitation and apoptotic cell death (Chalifoux and Carter, 2011a). However, under the strong excitotoxic conditions caused by cerebral ischemia, GABAB receptors are downregulated (Kim et al., 2011; Zhu et al., 2015; Huang et al., 2017) and are apparently no longer able to protect neurons from overexcitation. Studies on cultured neurons revealed that downregulation of GABAB receptors is primarily due to aberrant sorting of the receptors to lysosomal degradation on the expense of recycling them to the cell surface (Guetg et al., 2010; Maier et al., 2010; Terunuma et al., 2010; Kantamneni et al., 2014). In this study, we tested whether sustained activation of GABAB receptors reduces their ischemia-induced downregulation using cultured neurons subjected to oxygen and glucose deprivation (OGD) and the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia. We found that sustained activation of GABAB receptors restored receptor expression by inhibiting the ischemia-induced aberrant sorting of the receptors to the lysosomal degradation pathway. This was due to enhanced fast recycling of the receptors. The re-established GABAB receptor-mediated inhibition reduced neuronal excitability and limited progressing loss of neurons. Citation: Hleihil M, Vaas M, Bhat MA, Balakrishnan K and Benke D (2021) Sustained Baclofen-Induced Activation of GABAB Receptors After Cerebral Ischemia Restores Receptor Expression and Function and Limits Progressing Loss of Neurons. Front. Mol. Neurosci. 14:726133. doi: 10.3389/fnmol.2021.726133 September 2021 \| Volume 14 \| Article 726133 1 Frontiers in Molecular Neuroscience \| www.frontiersin.org Baclofen Recovers GABAB Receptor Expression Hleihil et al. the receptors stabilizes them at the cell surface and reduces their downregulation, making the baclofen treatment more eﬃcient. In this study, we tested whether sustained activation of GABAB receptors reduces their ischemia-induced downregulation using cultured neurons subjected to oxygen and glucose deprivation (OGD) and the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia. We found that sustained activation of GABAB receptors restored receptor expression by inhibiting the ischemia-induced aberrant sorting of the receptors to the lysosomal degradation pathway. This was due to enhanced fast recycling of the receptors. The re-established GABAB receptor-mediated inhibition reduced neuronal excitability and limited progressing loss of neurons. Antibodies and Drugs g The following primary antibodies were used in this study: Rabbit anti NeuN (1:400 for microscopy, 1:1000 for Odyssey Scanner, Millipore Cat# ABN78), mouse anti GABAB2 (1:400 for microscopy, abcam Cat# ab181736), rabbit anti GABAB2 (1:2000 for Odyssey Scanner, abcam Cat# ab75838), mouse anti EEA1 (1:500, BD Biosciences Cat# 610456), mouse anti Rab11 (1:100, Millipore Cat# 05-853), and goat anti Rab7 (1:50, Santa Cruz Cat# sc-11303). Secondary antibodies: donkey anti rabbit Alexa Fluor Plus 488 (1:2000, Thermo Fisher Scientiﬁc Cat# A32790), donkey anti mouse Alexa Fluor Plus 555 (1:2000, Thermo Fisher Scientiﬁc Cat# A32773), and goat anti rabbit Alexa Fluor Plus 800 (1:2000, Thermo Fisher Scientiﬁc Cat# A32735). All chemicals used for electrophysiology solutions were purchased from Sigma- Aldrich and baclofen was purchased from Tocris Bioscience. Most G protein coupled receptors (GPCRs) undergo arrestin- mediated internalization upon agonist stimulation, which involves phosphorylation via G protein receptor kinases and uncoupling the receptors from G proteins. This process stops GPCR signaling and provides protection against excessive receptor activity (Gurevich and Gurevich, 2019). In contrast, activation of GABAB receptors appears to stabilize them at the cell surface. Although activation of GABAB receptors accelerates their internalization (Wilkins et al., 2008; Zhang et al., 2015), this appears to be counterbalanced by their enhanced recycling to the cell surface (Grampp et al., 2008; Zhang et al., 2015). Agonist stimulated recycling of GABAB receptors involves the recruitment of the small GTPase Rap1, which physically interacts with GABAB receptors and promotes receptor recycling by an unknown mechanism (Zhang et al., 2015). Animals Animal experiments were performed according to the national guidelines of the Swiss Federal act on animal protection. Experimental conditions were approved by the Cantonal Veterinary Oﬃce Zurich, Zurich, Switzerland (license ZH152/16 and ZH011/19). MCAO experiments were performed using 9– 12 weeks old C57BL/6J male mice purchased from ENVIGO (Netherlands). Mice were housed up to ﬁve per cage with a standard 12/12-h light/dark cycle. Food and water were available ad libitum. Primary neuron cultures were prepared from E18 embryos of time pregnant Wistar rats obtained from ENVIGO. Since sustained activation of GABAB receptors appears to stabilize receptor numbers on the cell surface, it might well be that this eﬀect contributes to the neuroprotective activity of the selective GABAB receptor agonist baclofen. Baclofen exhibit neuroprotection in in vitro and in vivo models of cerebral ischemia (Lal et al., 1995; Jackson-Friedman et al., 1997; Kulinskii and Mikhel’son, 2000; Babcock et al., 2002; Dave et al., 2005; Zhang et al., 2007; Xu et al., 2008; Cimarosti et al., 2009; Liu et al., 2015). This suggests that either sustained activation of remaining GABAB receptors after the ischemic insult is suﬃcient to promote neuronal survival or, alternatively, that persistent activation of Frontiers in Molecular Neuroscience \| www.frontiersin.org Middle Cerebral Artery Occlusion Middle Cerebral Artery Occlusion Transient MCAO was conducted using C57Bl/6J naïve male mice as previously described (Vaas et al., 2017). Before the surgery, mice were injected with buprenorphine (0.1 mg/kg, s.c.) and with lidocaine (5 mg/kg, s.c.) at the site of incision. The mice were anesthetized with 5% isoﬂurane and maintained at 1.5–2% isoﬂurane in a mixture of O2 and air during the surgery. The left common carotid artery was exposed through a midline incision in the neck and unilateral MCAO was conducted by inserting a 7-0 silicone rubber-coated monoﬁlament to occlude the middle cerebral artery (Catalog No.: 701956PK5Re, Doccol Corp., Sharon, MA, United States). The middle cerebral artery was occluded for 60 min and then the ﬁlament was withdrawn to allow reperfusion. Following the surgery, mice were transferred to a 37◦C chamber for recovery before moved back to the home cage. For sham operated mice, the surgery was identical to the animals undergoing MCAO except that the ﬁlament was inserted to occlude left MCA and immediately withdrawn to allow instant reperfusion. Buprenorphine was given every 6 h on the day of surgery and then supplied via the drinking water (1 mg/kg). To encourage eating, the mice received softened chow in a Petri-dish placed on the ﬂoor of their cages. Baclofen (25 µg/kg, i.p.) was dissolved in sterile PBS and injected immediately after starting reperfusion. Sham operated mice were kept for 1 h in a recovery box at 37◦C and were injected at the same time point as MCAO operated mice. Except for the experiment shown in Figure 3, sham operated mice were sacriﬁced time matched with MCAO operated mice. Successful MCAO surgery was assessed by testing for impaired neurological/motor function using the Bederson score (Schaar et al., 2010). Current-clamp experiments were performed using a series of current steps (in 50 pA increments from −350 to 600 pA, 250 ms duration). Neurons were maintained at their original resting membrane potential throughout the experiment. The input resistance was determined using Ohm’s law after calculating the slope of the current–voltage (I–V) curves obtained in current clamp mode. The voltage responses were measured following a series of 50 pA hyperpolarize current pulses, the resting membrane potential (Vm) was determined immediately after acquiring the whole cell patch mode and the ﬁring threshold (Vth) was determined by injection of 5 pA depolarization current steps until the ﬁrst action potential was generated. Middle Cerebral Artery Occlusion For agonist-induced currents, neurons were hold at −50 mV and changes in holding currents in response to bath application of 100 µM baclofen were measured for 15 min. GABAB receptor- mediated GIRK currents were conﬁrmed by application of the GABAB receptor antagonist CGP36742. For the analysis of GIRK currents in cultured neurons, the cultures were subjected to OGD and treated for 16 h with baclofen. After washing to remove baclofen, baclofen-induced currents were measured for a duration of 10 min. Series resistance (Rs) was monitored throughout the experiment and recordings were excluded from the analysis if the Rs varied by 25%. Currents were ﬁltered at 5 kHz and digitized at 20 kHz. All recordings were performed using a Multiclamp 700B ampliﬁer, acquired with Digidata 1550A, and analyzed oﬄine using Clampﬁt 10.5 (Molecular Devices). Electrophysiology and Acute Brain Slice Preparation For ex vivo electrophysiological recordings, sham or MCAO operated mice were sacriﬁced 1 h after baclofen or saline injection for slice preparation and recorded ex vivo within 3– 6 h. Isoﬂurane anesthetized mice were decapitated and their brains quickly transferred into ice-cold solution containing 65 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 7 mM MgCl2, 0.5 mM CaCl2, 25 mM glucose, and 105 mM sucrose saturated with 95% O2 and 5% CO2. Coronal sections (300 µm) containing the cortical areas aﬀected by MCAO were sliced using a vibratome (HM 650, Microm). Slices were transferred to a recovery chamber containing oxygenated Primary Neuron Culture Primary co-cultures of neurons and glia cells were prepared from E18 Wistar rat embryos as described in Buerli et al. (2007). Cells dissociated from minced cerebral cortex tissue were plated at a density of 60,000 cells onto poly-D-lysine coated 15 mm diameter coverslips and incubated overnight in complete Dulbecco’s modiﬁed Eagle Medium (DMEM; Thermo Fisher) at 37◦C and 5% CO2. Subsequently, medium was replaced by the MEM/Nu-Serum based growing medium and kept at 37◦C and 5% CO2 for 12–16 days in vitro (DIV). September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 2 Baclofen Recovers GABAB Receptor Expression Hleihil et al. artiﬁcial cerebrospinal ﬂuid (ACSF, 315 mOsm) saturated with 95% O2 and 5% CO2 and containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, and 25 mM glucose at 34◦C for 25 min and then at room temperature. Following an additional 30 min recovery period, the slices were transferred to a recording chamber perfused with oxygenated ACSF at a ﬂow rate of 1– 2 ml/min. All electrophysiology experiments were performed at 32–34◦C. Oxygen–Glucose Deprivation Oxygen–glucose deprivation was carried out by replacing the Nu-based culture medium by OGD medium (DMEM free of glucose, glutamine, and phenol red) depleted from oxygen by bubbling nitrogen for 15 min through the medium in a water bath at 37◦C. Coverslips containing the cells were incubated in OGD media in a hypoxic incubator (1% O2, 5% CO2, 37◦C) for 1 h. After the incubation, cells were transferred back to the original conditioned medium and incubated at 37◦C and 5% CO2 until further use. Neurons in the somatosensory cortex to be recorded were visualized by infrared illumination using an Olympus ﬂuorescent microscope (BX51WI). Whole-cell patch-clamp recordings were performed from excitatory neurons, which were identiﬁed based on their morphology and ﬁring patterns. The intracellular solution used to measure GABAB receptor- mediated GIRK currents and neuronal excitability consisted of 135 mM potassium gluconate, 2 mM NaCl, 4 mM KCl, 4 mM EGTA, 10 mM HEPES pH 7.3, 4 mM Mg-ATP, and 0.3 mM Na3GTP and was ﬁlled in borosilicate glass patch pipettes (3.5– 4.5 m). Confocal microscopy and quantitative image analysis Images were recorded with a LSM 510 Meta confocal microscope (Carl Zeiss) equipped with a Plan-Neoﬂuar 40×/1.3 DIC and Plan-Fluar 100×/1.45 DIC oil-immersion objective or with a LSM 800 confocal microscope (Carl Zeiss) using a Plan- Apochromat 40×/1.4 or 63×/1.4 DIC oil-immersion objective. Images containing 4–5 optical sections with 0.4–1.0 µm spacing were recorded at a resolution of 1024 × 1024 pixels and used for the analysis of ﬂuorescence intensities, counting of neurons or the number of ﬂuorescent spots in case of in situ PLA. Laser intensities and detector gain were adjusted so that all signals were below saturation. Images were analyzed using the ImageJ software1. Cell survival assay Neurons were subjected to OGD with a recovery period of 24 h. Thereafter, cells were washed with incubation buﬀer and incubated in 7.5 µM propidium iodide solution (Sigma Aldrich) for 25 min at 37◦C, 5% CO2. Subsequently, cultures were ﬁxated, permeabilized, and stained for neurons using NeuN antibodies. Images were taken with a LSM 510 Meta confocal microscope equipped with a Plan-Neoﬂuar 40×/1.3 DIC oil-immersion objective (Carl Zeiss) and the number of NeuN and propidium iodide positive neurons was determined. Staining of Brain Sections Immunostaining g Following either sham or MCAO surgery, mice were anaesthetized with pentobarbital (100 mg/kg, i.p.) and transcardiacally perfused with ice-cold ACSF. Brains were instantly extracted and embedded in NEG50 (Richard-Allan Scientiﬁc, Fisher Scientiﬁc, Switzerland) and stored at −80◦C. For immunostaining, 30 µm sections were cut using a cryostat (Hyrax 60, Carl Zeiss) and mounted onto glass slides (SuperFrost Plus, Thermo Scientiﬁc). The sections were ﬁxated using 4% PFA for 30 min at room temperature, followed by antigen retrieval in a PickCell 2100-Retriever in 10 mM citrate buﬀer pH 6.0, 0.05% Tween 20 for 20 min. Sections were then permeabilized (0.2% Triton X-100 in 10 mM Tris pH 7.4, 150 mM NaCl, 10% NGS) and incubated with primary antibody diluted in TBST (10 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween-20) containing 10% NGS overnight at 4◦C. After washing ﬁve times for 5 min with TBST, the sections were incubated with secondary antibodies (Alexa Fluor Plus 800) for 1 h at room temperature. After washing with TBST, sections were air dried in the dark and scanned with an Odyssey CLx infrared scanner (Licor) at a resolution of 21 µm (Figures 4A, 6A) and 42 mm (Figure 3C). In situ proximity ligation assay Proximity ligation assay was used to monitor the colocalization of GABAB receptors with endosomal marker proteins as previously described (Maier et al., 2014; Zemoura et al., 2019). Cells were washed with PBS, ﬁxated with 4% PFA for 20 min at room temperature followed by permeabilization with 0.2% Triton X- 100 in PBS for 10 min. The cells were then incubated overnight at 4◦C with the appropriate pair of antibodies for GABAB receptors and the endosomal marker protein diluted in PBS containing 5% BSA. Subsequently, in situ PLA was performed using the Duolink kit (Sigma Aldrich) exactly according to the manufacturer’s instructions. Cell surface staining For quantiﬁcation of in situ PLA signals, the cell soma was marked carefully to allow particles quantiﬁcation within the cell using the ImageJ plug-in Analyze Particles. Before counting signal dots, a Gaussian blur ﬁlter was applied (sigma = 1), followed by background subtraction (Rolling Ball radius = 30). The build- in Moments algorithm was applied to determine the signal threshold for all images. The number of dots representing the PLA signal was then determined. Cultured neurons were washed with incubation buﬀer (25 mM HEPES pH 7.4, 119 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose) and incubated with anti-GABAB receptor antibodies diluted in incubation buﬀer containing 10% NDS for 2 h on ice. After washing with ice-cold incubation buﬀer, the cultures were incubated with secondary antibodies for 1 h on ice, washed again and ﬁxated with 4% PFA containing 4% sucrose for 15 min at room temperature. The coverslips were then mounted onto glass slides with DAKO ﬂuorescence mounting medium for analysis by confocal microscopy. 1https://imagej.nih.gov/ij Total cell staining Neurons were washed with PBS, ﬁxated with 4% paraformaldehyde (PFA) containing 4% sucrose for 20 min and then permeabilized for 12 min with 0.2% Triton X-100 in PBS. Subsequently, neurons were incubated overnight at 4◦C with primary antibody diluted in PBS containing 10% September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 3 Baclofen Recovers GABAB Receptor Expression Hleihil et al. normal goat serum (NGS) or normal donkey serum (NDS) based on the antibody used. After extensive washing with PBS, secondary antibodies were added for 1 h at room temperature. Subsequently, the coverslips were washed with PBS and mounted onto glass slides with DAKO ﬂuorescence mounting medium for analysis by confocal microscopy. For quantiﬁcation of the cell surface staining, the optical sections of each z-stack were merged into one image and the outer and the inner perimeter of the cell surface containing the ﬂuorescence signals were exactly outlined. Then the mean ﬂuorescence intensity value obtained from the inner border was subtracted from the one of the outer border. Nissl staining Paraformaldehyde ﬁxated and permeabilized sections were incubated for 15 min in 0.5% cresyl violet solution, washed for 30 s in H2O and destained in ethanol for 20 s up to 2 min. The sections were then dehydrated by a series of isopropanol, isopropanol/xylene and xylene incubations and ﬁnally coversliped in Eukitt (Sigma Aldrich). RESULTS conditions considerably reduced cell surface expression of GABAB receptors (Figure 1A). To tested whether sustained stimulation of GABAB receptors aﬀects receptor surface expression following OGD, we administered baclofen during OGD or thereafter and determined cell surface expression of the receptors after a recovery period of 16 h. Baclofen treatment only during OGD did not aﬀect cell surface GABAB receptor expression, whereas application of baclofen after OGD restored receptor expression close to control levels (Figure 1A). Under these conditions, baclofen treatment only during OGD did not aﬀect loss of neurons tested 24 h after OGD, whereas treatment after OGD signiﬁcantly reduced loss of neurons (Figure 1B). Statistical Analysis Data represent means ± SD except for electrophysiological results which are indicated as mean ± SEM for better display. Statistical comparisons were made with either one-way or two- way analysis of variance (ANOVA) followed by Tukey’s post hoc tests. Statistical tests were performed using GraphPad Prism 8. Diﬀerences were considered statistically signiﬁcant when p < 0.05. September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 4 Baclofen Recovers GABAB Receptor Expression Hleihil et al. FIGURE 1 \| Baclofen treatment restored GABAB receptor surface expression and was neuroprotective after OGD stress in vitro. (A) Baclofen treatment after, but not during, OGD increased GABAB receptor cell surface expression. Cultures were treated with baclofen (50 µM) during or after OGD. After 16 h after OGD, neurons were then analyzed for the level of GABAB receptor surface expression using antibodies directed against GABAB2. The incubation time with baclofen was 1 h in the “Bac during OGD” condition and 16 h in the “Bac after OGD” condition. Left, representative images (scale bar, 10 µm). Right: the bar graph depicts the quantiﬁcation of ﬂuorescence intensities. The data represent the mean ± SD of 18–23 neurons per condition from two independent experiments. One-way ANOVA followed by Tukey’s Multiple Comparison test (ns, p > 0.05; , p < 0.001; , p < 0.0001). (B) Treatment with baclofen after, but not during, OGD reduced neuronal loss in vitro. Cultures were treated as in panel (A) and neuronal loss was determined using propidium iodide (red) and NeuN staining (green) 24 h after OGD. Left, representative images (scale bar, 40 µm). Right: the bar graph depicts the quantiﬁcation of ﬂuorescence intensities. The data represent the mean ± SD of 40–41 neurons per condition from three independent experiments. One-way ANOVA followed by Tukey’s Multiple Comparison (ns, p > 0.05; , p < 0.01; **, p < 0.0001). (C) Baclofen treatment normalized GABAB receptor mediated currents in OGD-stressed neurons. Cultured neurons were subjected to OGD and treated or not for 16 h with baclofen. After washing to remove baclofen, baclofen-induced currents were measured for a duration of 10 min using whole cell voltage clamp recordings. Right, representative voltage clamp traces. The data represent the mean ± SD of 6–9 neurons per condition from two independent neuron preparations. One-way ANOVA with Tukey’s Multiple Comparison post hoc test (ns, p > 0.5; , p < 0.01; *, p < 0.001). Statistical Analysis FIGURE 1 \| Baclofen treatment restored GABAB receptor surface expression and was neuroprotective after OGD stress in vitro. (A) Baclofen treatment after, but not during, OGD increased GABAB receptor cell surface expression. Cultures were treated with baclofen (50 µM) during or after OGD. After 16 h after OGD, neurons were then analyzed for the level of GABAB receptor surface expression using antibodies directed against GABAB2. The incubation time with baclofen was 1 h in the “Bac during OGD” condition and 16 h in the “Bac after OGD” condition. Left, representative images (scale bar, 10 µm). Right: the bar graph depicts the quantiﬁcation of ﬂuorescence intensities. The data represent the mean ± SD of 18–23 neurons per condition from two independent experiments. One-way ANOVA followed by Tukey’s Multiple Comparison test (ns, p > 0.05; , p < 0.001; *, p < 0.0001). (B) Treatment with baclofen after, but not during, OGD reduced neuronal loss in vitro. Cultures were treated as in panel (A) and neuronal loss was determined using propidium iodide (red) and NeuN staining (green) 24 h after OGD. Left, representative images (scale bar, 40 µm). Right: the bar graph depicts the quantiﬁcation of ﬂuorescence intensities. The data represent the mean ± SD of 40–41 neurons per condition from three independent experiments. One-way ANOVA followed by Tukey’s Multiple Comparison (ns, p > 0.05; , p < 0.01; **, p < 0.0001). (C) Baclofen treatment normalized GABAB receptor mediated currents in OGD-stressed neurons. Cultured neurons were subjected to OGD and treated or not for 16 h with baclofen. After washing to remove baclofen, baclofen-induced currents were measured for a duration of 10 min using whole cell voltage clamp recordings. Right, representative voltage clamp traces. The data represent the mean ± SD of 6–9 neurons per condition from two independent neuron preparations. One-way ANOVA with Tukey’s Multiple Comparison post hoc test (ns, p > 0.5; , p < 0.01; *, p < 0.001). Baclofen Treatment After Excitotoxic Stress in vitro Restored Cell Surface Expression of GABAB Receptors To investigate the impact of sustained baclofen activation of GABAB receptors on cell surface expression of the receptors under ischemic/excitotoxic conditions, we utilized OGD of primary cortical neurons as an in vitro model. Consistent with previous research (Guetg et al., 2010; Maier et al., 2010, 2014; Terunuma et al., 2010), exposing neurons to excitotoxic September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 5 Baclofen Recovers GABAB Receptor Expression Hleihil et al. and loss of neurons was determined in the neocortex at diﬀerent time points following reperfusion using Nissl and NeuN staining (Figure 3A). Using Nissl staining, loss of neurons became visible on a macroscopic level 3 h after re-establishing reperfusion in the striatum and ventral cortex. It then spreads with time to dorsal areas of the cortex (Figure 3B). Quantiﬁcation of neuronal loss using NeuN staining clearly showed progressing neuronal death in the ventral and dorsal cortex, reaching severe levels in the dorsal cortex considerably later than in the ventral cortex (Figure 3C). Small but signiﬁcant levels of neuronal loss were present in the ventral and dorsal cortex 6 h after reperfusion (Figure 3C). We used this time point to investigate the impact of baclofen on the expression and function of GABAB receptors to minimize the eﬀect of neuronal loss in the following experiments. At this timepoint, we observed a signiﬁcant reduction in GABAB receptor expression in the aﬀected ipsilateral side, in both the dorsal and ventral cortex, as compared to sham operated animals (Figure 4). The magnitude of receptor downregulation exceeded by far the low level of neuronal loss at this time point and, therefore, could not be explained by a reduced number of neurons. Receptor expression was partially recovered following intraperitoneal injection of baclofen immediately after MCAO (Figure 4). Thus, agonist stimulation of GABAB receptors after cerebral ischemia partially restored GABAB receptor expression in vivo. The ineﬀectiveness of baclofen treatment when applied only during OGD might be due to an inappropriate timing of the application or to a too short duration of the application. Finally, we tested whether baclofen treatment restored GABAB receptor function as measured by baclofen-induced K+ currents. OGD strongly reduced baclofen evoked K+ currents in neurons as compared to control neurons (Figure 1C), which is in line with the considerable downregulation of the receptors observed in Figure 1A. Baclofen Treatment After Excitotoxic Stress in vitro Restored Cell Surface Expression of GABAB Receptors Treatment of OGD-stressed neurons with baclofen immediately after OGD for 16 h restored GABAB receptor-mediated currents to a level of unstressed control neurons (Figure 1C). g To explore the pathway underlying the baclofen-induced restoration of cell surface GABAB receptors after OGD, we analyzed the co-localization of the receptors with marker proteins for early endosomes (EEA1) (Mu et al., 1995; Wilson et al., 2000), fast recycling endosomes (Rab4) (van der Sluijs et al., 1992; Daro et al., 1996), slow recycling endosomes (Rab11) (Ullrich et al., 1996) and late endosomes (Rab7) (Feng et al., 1995) using the in situ PLA. OGD did not aﬀect the colocalization of GABAB receptors with EEA1 (Figure 2A), Rab4 positive fast recycling endosomes (Figure 2B) or Rab11 positive slow recycling endosomes (Figure 2C). However, baclofen treatment increased the colocalization of GABAB receptors with Rab4 under control condition and after OGD (Figures 2A,B). In contrast, baclofen treatment strongly reduced the co-localization of the receptors with Rab11 in control as well as in OGD treated neurons (Figure 2C). This indicates a switch from slow recycling Rab11 positive perinuclear endosomes to fast recycling of the receptors from Rab4 positive endosomes. Finally, OGD considerably increased the colocalization of GABAB receptors with Rab7, indicating increased sorting to the lysosomal degradation pathway (Figure 2D). Treatment with baclofen signiﬁcantly reduced the colocalization with Rab7 after OGD, whereas under control conditions baclofen did not aﬀect the colocalization with Rab7 (Figure 2D). The expression levels of endosomal marker proteins were not changed by the treatments except for EEA1 (Figure 2A). Baclofen treatment increased EEA1 expression in control neurons but not in OGD stressed neurons. Hence, the trend of enhanced colocalization of EEA1 and GABAB receptors observed in baclofen-treated control neurons (Figure 2A) might be caused by the upregulation of EEA1. Baclofen Treatment After MCAO Restored GABAB Receptor-Mediated Inhibition and Reduced Neuronal Excitability Next, we analyzed if baclofen treatment after MCAO also restored GABAB receptor-mediated inhibition. GABAB-receptor mediated currents were measured in the dorsal cortex 3–6 h after starting reperfusion using ex vivo patch clamp recordings in acute brain slices obtained from mice subjected to MCAO. In line with the immunohistochemical data, we observed a strong reduction of GABAB receptor-mediated currents in neurons from MCAO-treated mice as compared to sham operated mice (Figure 5A). Injection of baclofen immediately after starting reperfusion restored GABAB receptor-mediated currents to a level of sham operated mice (Figure 5A). g y g These ﬁndings suggest that sustained activation of GABAB receptors after an ischemic insult reduces sorting of GABAB receptors to lysosomal degradation by increasing fast recycling on the expense of slow recycling. We then tested if the restored GABAB receptor-mediated inhibition upon baclofen injection aﬀects neuronal excitability. Compared to sham operated mice, MCAO-treated mice displayed a considerably higher neuronal excitability, as assessed by the number of action potentials elicited by a series of depolarizing current pulses. Injection of baclofen after MCAO, strongly reduced the excitability to the level of sham operated mice (Figure 5B). Baclofen Treatment Partially Restored Downregulated GABAB Receptor Expression After Cerebral Ischemia in vivo To better understand the underlying mechanism by which baclofen reduces neuronal excitability after cerebral ischemia, we measured passive properties of the neurons. After MCAO, the membrane conductance was decreased, as reﬂected by an enhanced input resistance (Figure 5C) and a shift in the I– V curve (Figure 5D), indicating increased excitability of the neurons. Injection of baclofen normalized the input resistance To assess the eﬀect of baclofen on the expression of GABAB receptors in cerebral ischemia, we utilized the MCAO mouse model of stroke. First, we determined the tempo-spatial development of loss of neurons, to select suitable conditions for the following experiments. Mice were subjected to 1 h of MCAO September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 6 Hleihil et al. Baclofen Recovers GABAB Receptor Expression FIGURE 2 \| Sustained baclofen stimulation of GABAB receptors reduced sorting of the receptors to lysosomal degradation after OGD and increased fast recycling. Cultured neurons were subjected (OGD) or not (Ctrl) to OGD for 1 h followed by incubation with baclofen (+Bac). Neurons were analyzed 16 h after OGD for the colocalization of GABAB receptors with endosomal markers by in situ PLA (upper panels) and for expression of the marker proteins (lower panels) using antibodies directed against GABAB2 and EEA1 (A), Rab4 (B), Rab11 (C), and Rab7 (D). (A) Baclofen treatment increased the colocalization of GABAB2 with the early endosome marker EEA1 after OGD. Left, representative images depicting colocalization (upper panels) and EEA1 expression (lower panels) (scale bar, 10 µm). Right: the bar graphs depicts the quantiﬁcation of PLA signals and ﬂuorescence intensities. The data represent the mean ± SD of 28–68 neurons per condition from thee independent experiments for in situ PLA and 14–20 neurons per condition for EEA1 expression. Two-way ANOVA followed by Tukey’s post hoc test (ns, p > 0.05; , p < 0.005; ***, p < 0.0001). (B) Baclofen treatment increased the colocalization of GABAB2 with the fast recycling endosome marker Rab4. Left, representative images (scale bar, 10 µm). Right: the bar graphs depicts the quantiﬁcation of PLA signals and ﬂuorescence intensities of Rab4 expression. The data represent the mean ± SD of 39–46 neurons per condition from thee independent experiments for in situ PLA and 49–70 neurons per condition for Rab4 expression. Baclofen Treatment Partially Restored Downregulated GABAB Receptor Expression After Cerebral Ischemia in vivo Two-way ANOVA followed by Tukey’s post hoc test (ns, p > 0.05; , p < 0.05; **, p < 0.001). (D) Baclofen treatment reduced the OGD-induced colocalization of GABAB receptors with the late endosome marker Rab7. Left, representative images (scale bar, 10 µm). Right: the bar graphs depicts the quantiﬁcation of PLA signals and ﬂuorescence intensities of Rab7 expression. The data represent as mean ± SD of 12–32 neurons per condition from two independent experiments for in situ PLA and 16–25 neurons per condition for Rab7 expression. Two-way ANOVA followed by Tukey’s post hoc test (ns, p < 0.05; , p < 0.05; , p < 0.01; , p < 0.0001). FIGURE 4 \| Cerebral ischemia reduces the expression of GABAB receptor in the neocortex. Mice were subjected to Sham or MCAO surgery and instantly thereafter injected with baclofen (i.p., 25 mg/kg) or saline, followed by 6 h reperfusion. (A) Representative coronal sections depicting the expression of GABAB receptors (scale bar, 1 mm). (A′) Quantiﬁcation of the ﬂuorescence intensities in the ipsilateral dorsal cortex normalized to the corresponding contralateral side. (A′′) Quantiﬁcation of the ﬂuorescence intensities in the ipsilateral ventral cortex normalized to the corresponding contralateral side. The data represent the mean ± SD of slices/mice obtained from: sham = 43/6; MCAO = 39/5; MCAO + Baclofen = 60/8. The data points represent the average ﬂuorescence intensity derived from all slices analyzed per mouse. One-way ANOVA followed by Tukey’s Multiple Comparison test (, p < 0.01; *, p < 0.001; *, p < 0.0001). FIGURE 3 \| Progressing neuronal loss in the cerebral cortex in the mouse MCAO model of ischemic stroke. Mice were subjected to Sham or MCAO surgery followed by 0, 3, 6, 9, 12, and 24 h reperfusion. Brains were then ﬁxated and analyzed for neuronal loss in the cerebral cortex using Nissl (B) and NeuN (C) staining. (A) Scheme of the experimental design. (A′) Schematic drawing of a coronal mouse brain section indicating the areas analyzed in this study (dorsal and ventral cortex). (B) Representative images of Nissl stained brain sections obtained from Sham and MCAO treated mice reperfused for the indicated time intervals before ﬁxation (scale bar, 1 mm). (C) Representative images of NeuN stained brain sections (scale bar, 1 mm). (C′) Quantiﬁcation of ﬂuorescence intensities of NeuN staining in the dorsal cortex normalized to the ﬂuorescence signals of the unaffected contralateral cortex. Baclofen Treatment Partially Restored Downregulated GABAB Receptor Expression After Cerebral Ischemia in vivo The data represent the mean ± SD of 12–25 brain sections obtained from three mice and two independent staining experiments. One-way ANOVA followed by Tukey’s Multiple Comparison post hoc test (ns, p > 0.05; , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001). These results indicate that administration of baclofen directly after re-establishing reperfusion in MCAO-treated mice restored downregulated GABAB receptor-mediated inhibition and reduced elevated excitability of neurons. Baclofen Treatment Partially Restored Downregulated GABAB Receptor Expression After Cerebral Ischemia in vivo Two-way ANOVA followed by Tukey’s post hoc test (ns, p > 0.05; , p < 0.05; , p < 0.01; , p < 0.0001). (C) Baclofen treatment reduced colocalization of GABAB (Continued) FIGURE 2 \| Sustained baclofen stimulation of GABAB receptors reduced sorting of the receptors to lysosomal degradation after OGD and increased fast recycling. Cultured neurons were subjected (OGD) or not (Ctrl) to OGD for 1 h followed by incubation with baclofen (+Bac). Neurons were analyzed 16 h after OGD for the colocalization of GABAB receptors with endosomal markers by in situ PLA (upper panels) and for expression of the marker proteins (lower panels) using antibodies directed against GABAB2 and EEA1 (A), Rab4 (B), Rab11 (C), and Rab7 (D). (A) Baclofen treatment increased the colocalization of GABAB2 with the early endosome marker EEA1 after OGD. Left, representative images depicting colocalization (upper panels) and EEA1 expression (lower panels) (scale bar, 10 µm). Right: the bar graphs depicts the quantiﬁcation of PLA signals and ﬂuorescence intensities. The data represent the mean ± SD of 28–68 neurons per condition from thee independent experiments for in situ PLA and 14–20 neurons per condition for EEA1 expression. Two-way ANOVA followed by Tukey’s post hoc test (ns, p > 0.05; , p < 0.005; ***, p < 0.0001). (B) Baclofen treatment increased the colocalization of GABAB2 with the fast recycling endosome marker Rab4. Left, representative images (scale bar, 10 µm). Right: the bar graphs depicts the quantiﬁcation of PLA signals and ﬂuorescence intensities of Rab4 expression. The data represent the mean ± SD of 39–46 neurons per condition from thee independent experiments for in situ PLA and 49–70 neurons per condition for Rab4 expression. Two-way ANOVA followed by Tukey’s post hoc test (ns, p > 0.05; , p < 0.05; , p < 0.01; *, p < 0.0001). (C) Baclofen treatment reduced colocalization of GABAB (Continued) (Continued) (Continued) Frontiers in Molecular Neuroscience \| www.frontiersin.org September 2021 \| Volume 14 \| Article 726133 7 Hleihil et al. Baclofen Recovers GABAB Receptor Expression FIGURE 2 \| Continued receptors with the slow recycling endosome marker Rab11. Left, representative images (scale bar, 10 µm). Right: the bar graphs depicts the quantiﬁcation of PLA signals and ﬂuorescence intensities of Rab11 expression. The data represent the mean ± SD of 13–26 neurons per condition from three independent experiments for in situ PLA and 32–58 neurons per condition for Rab11 expression. Baclofen Treatment Partially Restored Downregulated GABAB Receptor Expression After Cerebral Ischemia in vivo (C′′) Quantiﬁcation of ﬂuorescence intensities of NeuN staining in the ventral cortex normalized to the ﬂuorescence signals of the unaffected contralateral cortex. The data represent the mean ± SD of 12–25 brain sections obtained from three mice and two independent staining experiments. One-way ANOVA followed by Tukey’s Multiple Comparison post hoc test (ns, p > 0.05; , p < 0.05; , p < 0.01; , p < 0.001; *, p < 0.0001). FIGURE 4 \| Cerebral ischemia reduces the expression of GABAB receptor in the neocortex. Mice were subjected to Sham or MCAO surgery and instantly thereafter injected with baclofen (i.p., 25 mg/kg) or saline, followed by 6 h reperfusion. (A) Representative coronal sections depicting the expression of GABAB receptors (scale bar, 1 mm). (A′) Quantiﬁcation of the ﬂuorescence intensities in the ipsilateral dorsal cortex normalized to the corresponding contralateral side. (A′′) Quantiﬁcation of the ﬂuorescence intensities in the ipsilateral ventral cortex normalized to the corresponding contralateral side. The data represent the mean ± SD of slices/mice obtained from: sham = 43/6; MCAO = 39/5; MCAO + Baclofen = 60/8. The data points represent the average ﬂuorescence intensity derived from all slices analyzed per mouse. One-way ANOVA followed by Tukey’s Multiple Comparison test (, p < 0.01; *, p < 0.001; , p < 0.0001). FIGURE 3 \| Progressing neuronal loss in the cerebral cortex in the mouse MCAO model of ischemic stroke. Mice were subjected to Sham or MCAO surgery followed by 0, 3, 6, 9, 12, and 24 h reperfusion. Brains were then ﬁxated and analyzed for neuronal loss in the cerebral cortex using Nissl (B) and NeuN (C) staining. (A) Scheme of the experimental design. (A′) Schematic drawing of a coronal mouse brain section indicating the areas analyzed in this study (dorsal and ventral cortex). (B) Representative images of Nissl stained brain sections obtained from Sham and MCAO treated mice reperfused for the indicated time intervals before ﬁxation (scale bar, 1 mm). (C) Representative images of NeuN stained brain sections (scale bar, 1 mm). (C′) Quantiﬁcation of ﬂuorescence intensities of NeuN staining in the dorsal cortex normalized to the ﬂuorescence signals of the unaffected contralateral cortex. (C′′) Quantiﬁcation of ﬂuorescence intensities of NeuN staining in the ventral cortex normalized to the ﬂuorescence signals of the unaffected contralateral cortex. Baclofen Treatment Following Cerebral Ischemia Reduced Neuronal Loss This effect was calculated from the slope of I–V curves (D). One-way ANOVA with Tukey’s Multiple Comparison post hoc test (ns, p > 0.05; , p < 0.01; **, p < 0.001). (E) The analysis revealed no change in the resting membrane potential and no change in the neuronal ﬁring threshold in MCAO mice. However, treatment with baclofen depolarized the action potential ﬁring threshold in MCAO mice. One-way ANOVA with Tukey’s Multiple Comparison post hoc test (ns, p > 0.05; , p < 0.05). The data represent the mean ± SEM for the number of cells/mice: sham = 19/6, MCAO = 16/5, R1 = 9/4, Rand = 11/5. FIGURE 5 \| Baclofen treatment rescued the ischemia-induced reduction of GABAB receptor-mediated currents and reduced neuronal hyperexcitability. (A) Baclofen treatment normalized GABAB mediated currents in the ischemic brain. Coronal brain sections obtained from sham or MCAO treated mice were recorded ex vivo within 3–6 h after baclofen or saline injection injection. Mice were sacriﬁzed for slice perparation 1 h after baclofen or saline injection. Baclofen-induced currents were measured for a duration of 15 min using whole cell voltage clamp recordings. Right, representative voltage clamp traces. One-way ANOVA with Tukey’s Multiple Comparison post hoc test (ns, p > 0.5; , p < 0.05; , p < 0.01). (B) Baclofen injection reduced ischemia-induced neuronal hyperexcitability. The experimental conditions were the same as in panel (A) except that recordings were obtained in the current clamp mode. Neurons of MCAO-treated mice show an increased intrinsic excitability reﬂected by an increased number of action potentials elicited by a series of depolarizing current pulses (50 pA/250 ms from resting membrane potential). Repeated measures ANOVA with mixed-effects analysis showed a signiﬁcant interaction among AP counts and treatments [F(1.226,135.4) = 35.23, p < 0.0001). Right, representative current clamp traces elected by 300 pA current pulse. (C–F) Baclofen reduced excitability by modulating intrinsic neuronal properties. (C) While MCAO increased the input resistance, treatment with baclofen reduced the values to sham levels. This effect was calculated from the slope of I–V curves (D). One-way ANOVA with Tukey’s Multiple Comparison post hoc test (ns, p > 0.05; , p < 0.01; *, p < 0.001). (E) The analysis revealed no change in the resting membrane potential and no change in the neuronal ﬁring threshold in MCAO mice. However, treatment with baclofen depolarized the action potential ﬁring threshold in MCAO mice. Baclofen Treatment Following Cerebral Ischemia Reduced Neuronal Loss To test the neuroprotective activity of the baclofen treatment, we quantiﬁed NeuN staining in brain sections derived from MCAO operated mice treated with baclofen or saline immediately after starting reperfusion and sacriﬁced 6 h thereafter (Figure 6). In line with the experiments shown in Figure 3, we observed a small but signiﬁcant level of neuronal loss at this early time point in the dorsal cortex, which was prevented by treatment with baclofen (Figure 6A′). In the more severely aﬀected ventral cortex baclofen likewise exhibited a neuroprotective eﬀect (Figure 6A′′). These and the shifted IV curve back to levels of sham operated mice (Figures 5C,D). Furthermore, we did not observe a signiﬁcant diﬀerence in neuronal resting potential or ﬁring threshold after MCAO (Figures 5E,F). However, administration of baclofen depolarized the ﬁring threshold (Figure 5F), which is known to contribute to the reduction on ﬁring frequency observed in the baclofen injected mice (Deng et al., 2009). September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 8 Hleihil et al. Baclofen Recovers GABAB Receptor Expression FIGURE 5 \| Baclofen treatment rescued the ischemia-induced reduction of GABAB receptor-mediated currents and reduced neuronal hyperexcitability. (A) Baclofen treatment normalized GABAB mediated currents in the ischemic brain. Coronal brain sections obtained from sham or MCAO treated mice were recorded ex vivo within 3–6 h after baclofen or saline injection injection. Mice were sacriﬁzed for slice perparation 1 h after baclofen or saline injection. Baclofen-induced currents were measured for a duration of 15 min using whole cell voltage clamp recordings. Right, representative voltage clamp traces. One-way ANOVA with Tukey’s Multiple Comparison post hoc test (ns, p > 0.5; , p < 0.05; *, p < 0.01). (B) Baclofen injection reduced ischemia-induced neuronal hyperexcitability. The experimental conditions were the same as in panel (A) except that recordings were obtained in the current clamp mode. Neurons of MCAO-treated mice show an increased intrinsic excitability reﬂected by an increased number of action potentials elicited by a series of depolarizing current pulses (50 pA/250 ms from resting membrane potential). Repeated measures ANOVA with mixed-effects analysis showed a signiﬁcant interaction among AP counts and treatments [F(1.226,135.4) = 35.23, p < 0.0001). Right, representative current clamp traces elected by 300 pA current pulse. (C–F) Baclofen reduced excitability by modulating intrinsic neuronal properties. (C) While MCAO increased the input resistance, treatment with baclofen reduced the values to sham levels. Baclofen Treatment Following Cerebral Ischemia Reduced Neuronal Loss One-way ANOVA with Tukey’s Multiple Comparison post hoc test (ns, p > 0.05; , p < 0.05). The data represent the mean ± SEM for the number of cells/mice: sham = 19/6, MCAO = 16/5, R1 = 9/4, Rand = 11/5. September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 9 Baclofen Recovers GABAB Receptor Expression Hleihil et al. FIGURE 6 \| Baclofen treatment after MCAO is neuroprotective. Mice were subjected to MCAO and injected with baclofen or saline followed by 6 h reperfusion. (A) Representative images depicting the expression of NeuN (scale bar, 1 mm). (A′) Quantiﬁcation of the ﬂuorescence intensities in the indicated area in the ipsilateral dorsal cortex normalized to the corresponding contralateral side. (A′′) Quantiﬁcation of ﬂuorescence intensities in the ipsilateral ventral cortex normalized to the corresponding contralateral side. The data represent the mean ± SD of slices/mice obtained from: sham = 47/6; MCAO = 40/5; MCAO + Baclofen = 61/8). The data points represent the average ﬂuorescence intensity derived from all slices analyzed per mouse. One-way ANOVA followed by Tukey’s Multiple Comparison test (ns, p > 0.05; , p < 0.01; , p < 0.001; *, p < 0.0001). Our in vitro experiments on cultured neurons subjected to OGD, to mimic cerebral ischemia, veriﬁed the strong downregulation of GABAB receptors reported previously under excitotoxic conditions provoked by prolonged exposure to NMDA or glutamate (Guetg et al., 2010; Maier et al., 2010; Terunuma et al., 2010; Kantamneni et al., 2014). Treatment of neurons with baclofen after OGD stress indeed recovered GABAB receptor expression at the cell surface by the mechanism proposed by Zhang et al. (2015) based on their ﬁnding on receptors expressed in HEK 293 cells. Interestingly, baclofen treatment after OGD restored downregulated receptor expression close to normal levels. This ﬁnding indicates that under ischemic conditions the supply of new receptors to the cell surface, their internalization, recycling, and degradation mechanisms are still in operation but with altered rates. Our colocalization experiments with endosomal marker proteins for the diﬀerent intracellular traﬃcking pathways indicate OGD-enhanced sorting of GABAB receptors to the lysosomal degradation pathway, which is in line with previous reports using NMDA and glutamate induced excitotoxicity (Maier et al., 2010; Terunuma et al., 2010; Zemoura et al., 2019). Baclofen Treatment Following Cerebral Ischemia Reduced Neuronal Loss Our results show that baclofen treatment after OGD restores cell surface receptor expression by increasing internalization (as indicated by enhanced colocalization with EEA1 positive endosomes) and fast recycling via Rab4 positive endosomes on the expense of slow recycling via Rab11 positive endosomes. This ﬁnding is in accordance with the observation that disruption of GABAB receptor recycling increased lysosomal degradation of the receptors (Grampp et al., 2008; Maier et al., 2010; Zhang et al., 2015). Hence, our experimental data supports a mechanism in which sustained activation of GABAB receptors by baclofen induces fast recycling of the receptors to bypass sorting of the receptors to the lysosomal degradation pathway. FIGURE 6 \| Baclofen treatment after MCAO is neuroprotective. Mice were subjected to MCAO and injected with baclofen or saline followed by 6 h reperfusion. (A) Representative images depicting the expression of NeuN (scale bar, 1 mm). (A′) Quantiﬁcation of the ﬂuorescence intensities in the indicated area in the ipsilateral dorsal cortex normalized to the corresponding contralateral side. (A′′) Quantiﬁcation of ﬂuorescence intensities in the ipsilateral ventral cortex normalized to the corresponding contralateral side. The data represent the mean ± SD of slices/mice obtained from: sham = 47/6; MCAO = 40/5; MCAO + Baclofen = 61/8). The data points represent the average ﬂuorescence intensity derived from all slices analyzed per mouse. One-way ANOVA followed by Tukey’s Multiple Comparison test (ns, p > 0.05; , p < 0.01; *, p < 0.001; **, p < 0.0001). results indicate that baclofen treatment eﬀectively promotes survival of cortical neurons. Employing the MCAO mouse model of cerebral ischemia, we conﬁrmed progressing neuronal loss and diminished GABAB receptor expression in the aﬀected cerebral cortex, which were partially restored after injection of baclofen directly after the ischemic insult. Thus, these data recapitulate our in vitro ﬁndings on cultured neurons subjected to OGD and veriﬁed OGD of cultured neurons as valuable in vitro model for the analysis of molecular mechanisms involved in cerebral ischemia. An upregulation of GABAB receptor expression in animal models of ischemia upon various treatments, including baclofen (Lu et al., 2016), ferulic acid (Cheng et al., 2010), mild hypothermia (Kim et al., 2011), and opposing needling/acupuncture (Xu et al., 2014; Jiang et al., 2016), had been reported, suggesting that upregulated GABAB receptor expression contributed to the neuroprotective eﬀect of the treatments. September 2021 \| Volume 14 \| Article 726133 FUNDING Clinically, baclofen is eﬀective for the treatment of post- ischemic stroke symptoms, i.e., for the relief of severe spasticity and associated pain (Taira et al., 1994; Meythaler et al., 2001; Ivanhoe et al., 2006; Koﬂer et al., 2009; Creamer et al., 2018a,b) and persistent hic-ups (Zhang et al., 2014). There is also indication from animal studies for a potential beneﬁt for the treatment of anxiety (Lu et al., 2016) and sleep disturbances (Hodor et al., 2014) associated with cerebral ischemia. However, the neuroprotective activity of baclofen in acute stroke remains to be established clinically. Considering the multiple beneﬁcial pathways targeted, sustained activation of GABAB receptors might be a promising component for a therapy aimed at limiting progressing excitotoxic neuronal death after an ischemic insult. This study was supported by the Swiss National Science Foundation (grants 31003A_156648 and 31003A_182325 to DB) and the Foundation for Research in Science and the Humanities at the University of Zurich (grant STWF-18-005 to DB). This study was supported by the Swiss National Science Foundation (grants 31003A_156648 and 31003A_182325 to DB) and the Foundation for Research in Science and the Humanities at the University of Zurich (grant STWF-18-005 to DB). DISCUSSION Here we show that sustained activation of GABAB receptors after an ischemic insult restores downregulated GABAB receptor expression and function, which limited excitotoxic neuronal death. This study is based on previous work demonstrating that GABAB receptors are severely downregulated under excitotoxic conditions (Guetg et al., 2010; Maier et al., 2010, 2014; Terunuma et al., 2010; Kim et al., 2011; Kantamneni et al., 2014; Zhu et al., 2015; Huang et al., 2017) and indications from work on GABAB receptors recombinantly expressed in HEK 293 cells that sustained activation of the receptors with baclofen stabilized receptor expression on the cell surface (Zhang et al., 2015). We hypothesized that treatment with baclofen after an ischemic insult may reduce downregulation of the receptors and re-establish GABAB receptor-mediated inhibition. This would explain the well-established neuroprotective activity of baclofen (Lal et al., 1995; Jackson-Friedman et al., 1997; Kulinskii and Mikhel’son, 2000; Babcock et al., 2002; Dave et al., 2005; Zhang et al., 2007; Xu et al., 2008; Cimarosti et al., 2009; Liu et al., 2015) although the receptors are severely downregulated under excitotoxic conditions. As expected, restoration of GABAB receptors also recovered the severely compromised GABAB receptor-mediated inhibition. Although receptor expression was not fully recovered, baclofen treatment restored GABAB receptor-mediated currents and lowered enhanced neuronal excitability to levels of sham operated mice. This was suﬃcient to inhibit ischemia-induced progressing neuronal loss in the less aﬀected dorsal cortex at the time point of testing (6 h after starting reperfusion), whereas there was a smaller neuroprotective eﬀect of baclofen in the more severely September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 10 Hleihil et al. Baclofen Recovers GABAB Receptor Expression ETHICS STATEMENT The animal study was reviewed and approved by Cantonal Veterinary Oﬃce Zurich, Zurich, Switzerland (license ZH152/16 and ZH011/19). DATA AVAILABILITY STATEMENT aﬀected ventral cortex. This result was expected since it is very unlikely that any neuroprotective treatment, even when given immediately after the ischemic insult, can prevent the almost instantly occurring neuronal damage in the ischemic core. aﬀected ventral cortex. This result was expected since it is very unlikely that any neuroprotective treatment, even when given immediately after the ischemic insult, can prevent the almost instantly occurring neuronal damage in the ischemic core. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. One important advantage of targeting GABAB receptors for neuroprotection lays in their regulation of multiple beneﬁcial pathways. The most obvious one is the inhibition of glutamate release (Ouyang et al., 2007), reducing the detrimental overstimulation of all members of the glutamate receptor family, and the activation of postsynaptic GABAB receptors, which inhibits neuronal excitability (Deng et al., 2009; Chalifoux and Carter, 2011a,b). These combined eﬀects reduce Ca2+ overload of the neurons and inhibit multiple mechanisms of glutamate receptor and Ca2+ triggered apoptotic neuronal death. In addition to inhibiting neuronal activity, sustained GABAB receptor activity also promotes neuronal survival via the PI3K/Akt-GSK3β pathway (Tu et al., 2010; Liu et al., 2015; Fu et al., 2016), which is a major neuronal survival cascade (Rai et al., 2019). Finally, baclofen treatment inhibits the upregulation of the pro-apoptotic transcription factor CHOP, which induces apoptotic cell death in response to ER-stress associated with cerebral ischemia (Fu et al., 2016). These combined eﬀects make GABAB receptors a promising target for limiting progressing neuronal death in acute cerebral ischemia. ACKNOWLEDGMENTS We thank Thomas Grampp for excellent technical assistance and Patrick Maier for performing the experiments shown in Figures 1A,B. AUTHOR CONTRIBUTIONS MH, MV, MB, and KB performed the experimental work and analyzed the data. MH and DB designed the study and wrote the manuscript. DB supervised the work and analyzed the data. All authors discussed the results and commented on the manuscript. REFERENCES doi: 10.1038/aps.2010.66 September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 11 Baclofen Recovers GABAB Receptor Expression Hleihil et al. Lu, Y., Li, C. J., Chen, C., Luo, P., Zhou, M., Li, C., et al. (2016). Activation of GABAB2 subunits alleviates chronic cerebral hypoperfusion- induced anxiety-like behaviours: a role for BDNF signalling and Kir3 channels. Neuropharmacology 110, 308–321. doi: 10.1016/j.neuropharm.2016.08.007 Deng, P. Y., Xiao, Z., Yang, C., Rojanathammanee, L., Grisanti, L., Watt, J., et al. (2009). GABAB receptor activation inhibits neuronal excitability and spatial learning in the entorhinal cortex by activating TREK-2 K+ channels. Neuron 63, 230–243. doi: 10.1016/j.neuron.2009.06.022 Feng, Y., Press, B., and Wandinger-Ness, A. (1995). Rab 7: an important regulator of late endocytic membrane traﬃc. J. Cell Biol. 131, 1435–1452. doi: 10.1083/ jcb.131.6.1435 Maier, P. J., Marin, I., Grampp, T., Sommer, A., and Benke, D. (2010). Sustained glutamate receptor activation down-regulates GABAB receptors by shifting the balance from recycling to lysosomal degradation. J. Biol. Chem. 285, 35606– 35614. doi: 10.1074/jbc.M110.142406 Fu, P., Wu, Q., Hu, J., Li, T., and Gao, F. (2016). Baclofen protects primary rat retinal ganglion cells from chemical hypoxia-induced apoptosis through the Akt and PERK pathways. Front. Cell Neurosci. 10:255. doi: 10.3389/fncel.2016. 00255 Maier, P. J., Zemoura, K., Acuna, M. A., Yevenes, G. E., Zeilhofer, H. U., and Benke, D. (2014). Ischemia-like oxygen and glucose deprivation mediates down- regulation of cell surface g-aminobutyric acidB receptors via the endoplasmic reticulum (ER) stress-induced transcription factor CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP). J. Biol. Chem. 289, 12896– 12907. doi: 10.1074/jbc.M114.550517 Gassmann, M., and Bettler, B. (2012). Regulation of neuronal GABAB receptor functions by subunit composition. Nat. Rev. Neurosci. 13, 380–394. doi: 10. 1038/nrn3249 Meythaler, J. M., Guin-Renfroe, S., Brunner, R. C., and Hadley, M. N. (2001). Intrathecal baclofen for spastic hypertonia from stroke. Stroke 32, 2099–2109. doi: 10.1161/hs0901.095682 Grampp, T., Notz, V., Broll, I., Fischer, N., and Benke, D. (2008). Constitutive, agonist-accelerated, recycling and lysosomal degradation of GABAB receptors in cortical neurons. Mol. Cell. Neurosci. 39, 628–637. doi: 10.1016/j.mcn.2008. 09.004 Mu, F. T., Callaghan, J. M., Steele-Mortimer, O., Stenmark, H., Parton, R. G., Campbell, P. L., et al. (1995). EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein ﬂanked by cysteine "ﬁngers" and contains a calmodulin-binding IQ motif. J. Biol. Chem. 270, 13503–13511. doi: 10.1074/jbc.270.22.13503 Guetg, N., Aziz, S. A., Holbro, N., Turecek, R., Rose, T., Seddik, R., et al. REFERENCES (2010). NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1. Proc. Natl. Acad. Sci. U. S. A. 107, 13924–13929. doi: 10.1073/pnas.1000909107 Gurevich, V. V., and Gurevich, E. V. (2019). GPCR signaling regulation: the role of GRKs and arrestins. Front. Pharmacol. 10:125. doi: 10.3389/fphar.2019.00125 Ouyang, C., Guo, L., Lu, Q., Xu, X., and Wang, H. (2007). Enhanced activity of GABA receptors inhibits glutamate release induced by focal cerebral ischemia in rat striatum. Neurosci. Lett .420, 174–178. doi: 10.1016/j.neulet.2007.05.004 Hodor, A., Palchykova, S., Baracchi, F., Noain, D., and Bassetti, C. L. (2014). Baclofen facilitates sleep, neuroplasticity, and recovery after stroke in rats. Ann Clin Transl Neurol 1, 765–777. doi: 10.1002/acn3.115 Rai, S. N., Dilnashin, H., Birla, H., Singh, S. S., Zahra, W., Rathore, A. S., et al. (2019). The role of PI3K/Akt and ERK in neurodegenerative disorders. Neurotox. Res. 35, 775–795. doi: 10.1007/s12640-019-0003-y Huang, L., Li, Q., Wen, R., Yu, Z., Li, N., Ma, L., et al. (2017). Rho-kinase inhibitor prevents acute injury against transient focal cerebral ischemia by enhancing the expression and function of GABA receptors in rats. Eur. J. Pharmacol. 797, 134–142. doi: 10.1016/j.ejphar.2017.01.021 Schaar, K. L., Brenneman, M. M., and Savitz, S. I. (2010). Functional assessments in the rodent stroke model. Exp. Transl. Stroke Med. 2:13. doi: 10.1186/2040- 7378-2-13 Szydlowska, K., and Tymianski, M. (2010). Calcium, ischemia and excitotoxicity. Cell Calcium 47, 122–129. doi: 10.1016/j.ceca.2010.01.003 Ivanhoe, C. B., Francisco, G. E., McGuire, J. R., Subramanian, T., and Grissom, S. P. (2006). Intrathecal baclofen management of poststroke spastic hypertonia: implications for function and quality of life. Arch. Phys. Med. Rehabil. 87, 1509–1515. doi: 10.1016/j.apmr.2006.08.323 Taira, T., Tanikawa, T., Kawamura, H., Iseki, H., and Takakura, K. (1994). Spinal intrathecal baclofen suppresses central pain after a stroke. J. Neurol. Neurosurg. Psychiatry 57, 381–382. doi: 10.1136/jnnp.57.3.381-a Jackson-Friedman, C., Lyden, P. D., Nunez, S., Jin, A., and Zweiﬂer, R. (1997). High dose baclofen is neuroprotective but also causes intracerebral hemorrhage: a quantal bioassay study using the intraluminal suture occlusion method. Exp. Neurol. 147, 346–352. doi: 10.1006/exnr.1997.6637 Terunuma, M., Vargas, K. J., Wilkins, M. E., Ramirez, O. A., Jaureguiberry- Bravo, M., Pangalos, M. N., et al. (2010). Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors. Proc. Natl. Acad. Sci. U. S. A. 107, 13918–13923. doi: 10. 1073/pnas.1000853107 Jiang, Y., Yang, S., Tao, J., Lin, Z., Ye, X., You, Y., et al. (2016). REFERENCES Opposing needling promotes behavior recovery and exerts neuroprotection via the cAMP/PKA/CREB signal transduction pathway in transient MCAO rats. Mol. Med. Rep. 13, 2060–2070. doi: 10.3892/mmr.2016.4773 Tu, H., Xu, C., Zhang, W., Liu, Q., Rondard, P., Pin, J. P., et al. (2010). GABAB receptor activation protects neurons from apoptosis via IGF-1 receptor transactivation. J. Neurosci. 30, 749–759. doi: 10.1523/jneurosci.2343-09.2010 Kantamneni, S., Gonzalez-Gonzalez, I. M., Luo, J., Cimarosti, H., Jacobs, S. C., Jaafari, N., et al. (2014). Diﬀerential regulation of GABAB receptor traﬃcking by diﬀerent modes of N-methyl-D-aspartate (n.d.) receptor signaling. J. Biol. Chem. 289, 6681–6694. doi: 10.1074/jbc.M113.487348 Ullrich, O., Reinsch, S., Urbé, S., Zerial, M., and Parton, R. G. (1996). Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135, 913–924. doi: 10.1083/jcb.135.4.913 Kim, J. Y., Kim, N., Yenari, M. A., and Chang, W. (2011). Mild hypothermia suppresses calcium-sensing receptor (CaSR) induction following forebrain ischemia while increasing GABAB receptor 1 (GABAB-R1) expression. Transl. Stroke Res. 2, 195–201. doi: 10.1007/s12975-011-0082-4 Vaas, M., Enzmann, G., Perinat, T., Siler, U., Reichenbach, J., Licha, K., et al. (2017). Non-invasive near-infrared ﬂuorescence imaging of the neutrophil response in a mouse model of transient cerebral ischaemia. J. Cereb. Blood Flow Metab. 37, 2833–2847. doi: 10.1177/0271678x16676825 Koﬂer, M., Quirbach, E., Schauer, R., Singer, M., and Saltuari, L. (2009). Limitations of intrathecal baclofen for spastic hemiparesis following stroke. Neurorehabil. Neural. Repair 23, 26–31. doi: 10.1177/1545968308317700 van der Sluijs, P., Hull, M., Webster, P., Mâle, P., Goud, B., and Mellman, I. (1992). The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell 70, 729–740. doi: 10.1016/0092-8674(92)90 307-x Kostandy, B. B. (2012). The role of glutamate in neuronal ischemic injury: the role of spark in ﬁre. Neurol. Sci. 33, 223–237. doi: 10.1007/s10072-011-0828-5 Wilkins, M. E., Li, X., and Smart, T. G. (2008). Tracking cell surface GABAB receptors using an α-bungarotoxin tag. J. Biol. Chem. 283, 34745–34752. doi: 10.1074/jbc.M803197200 Kulinskii, V. I., and Mikhel’son, G. V. (2000). Additivity and independence of neuroprotective eﬀects of GABAA and GABAB receptor agonists in complete global cerebral ischemia. Bull Exp. Biol. Med. 130, 772–774. doi: 10.1007/ bf02766091 Wilson, J. M., de Hoop, M., Zorzi, N., Toh, B. H., Dotti, C. G., and Parton, R. G. (2000). EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and ﬁbroblasts. Mol. Biol. Cell. 11, 2657–2671. REFERENCES Choi, D. W. (2020). Excitotoxicity: still hammering the ischemic brain in 2020. Front. Neurosci. 14:579953. doi: 10.3389/fnins.2020.579953 Babcock, A. M., Everingham, A., Paden, C. M., and Kimura, M. (2002). Baclofen is neuroprotective and prevents loss of calcium/calmodulin-dependent protein kinase II immunoreactivity in the ischemic gerbil hippocampus. J. Neurosci. Res. 67, 804–811. doi: 10.1002/jnr.10169 Cimarosti, H., Kantamneni, S., and Henley, J. M. (2009). Ischaemia diﬀerentially regulates GABAB receptor subunits in organotypic hippocampal slice cultures. Neuropharmacology 56, 1088–1096. doi: 10.1016/j.neuropharm.2009.03.007 Creamer, M., Cloud, G., Kossmehl, P., Yochelson, M., Francisco, G. E., Ward, A. B., et al. (2018a). Intrathecal baclofen therapy versus conventional medical management for severe poststroke spasticity: results from a multicentre, randomised, controlled, open-label trial (SISTERS). J. Neurol. Neurosurg. Psychiatry 89, 642–650. doi: 10.1136/jnnp-2017-317021 Bano, D., and Ankarcrona, M. (2018). Beyond the critical point: an overview of excitotoxicity, calcium overload and the downstream consequences. Neurosci. Lett. 663, 79–85. doi: 10.1016/j.neulet.2017.08.048 Buerli, T., Pellegrino, C., Baer, K., Lardi-Studler, B., Chudotvorova, I., Fritschy, J. M., et al. (2007). Eﬃcient transfection of DNA or shRNA vectors into neurons using magnetofection. Nat. Protoc. 2, 3090–3101. doi: 10.1038/nprot.2007.445 Creamer, M., Cloud, G., Kossmehl, P., Yochelson, M., Francisco, G. E., Ward, A. B., et al. (2018b). Eﬀect of intrathecal baclofen on pain and quality of life in poststroke spasticity. Stroke 49, 2129–2137. doi: 10.1161/strokeaha.118.022255 Chalifoux, J. R., and Carter, A. G. (2011a). GABAB receptor modulation of synaptic function. Curr. Opin. Neurobiol. 21, 1–6. doi: 10.1016/j.conb.2011.02.004 Daro, E., van der Sluijs, P., Galli, T., and Mellman, I. (1996). Rab4 and cellubrevin deﬁne diﬀerent early endosome populations on the pathway of transferrin receptor recycling. Proc. Natl. Acad. Sci. U. S. A. 93, 9559–9564. doi: 10.1073/ pnas.93.18.9559 Chalifoux, J. R., and Carter, A. G. (2011b). GABAB receptor modulation of voltage- sensitive calcium channels in spines and dendrites. J. Neurosci. 31, 4221–4232. doi: 10.1523/JNEUROSCI.4561-10.2011 Dave, K. R., Lange-Asschenfeldt, C., Raval, A. P., Prado, R., Busto, R., Saul, I., et al. (2005). Ischemic preconditioning ameliorates excitotoxicity by shifting glutamate/γ-aminobutyric acid release and biosynthesis. J. Neurosci. Res. 82, 665–673. doi: 10.1002/jnr.20674 Cheng, C. Y., Su, S. Y., Tang, N. Y., Ho, T. Y., Lo, W. Y., and Hsieh, C. L. (2010). Ferulic acid inhibits nitric oxide-induced apoptosis by enhancing GABAB1 receptor expression in transient focal cerebral ischemia in rats. Acta Pharmacol. Sin. 31, 889–899. REFERENCES doi: 10.1091/mbc.11.8.2657 Lal, S., Shuaib, A., and Ijaz, S. (1995). Baclofen is cytoprotective to cerebral ischemia in gerbils. Neurochem. Res. 20, 115–119. doi: 10.1007/bf00970534 Xu, J., Li, C., Yin, X. H., and Zhang, G. Y. (2008). Additive neuroprotection of GABAA and GABAB receptor agonists in cerebral ischemic injury via PI- 3K/Akt pathway inhibiting the ASK1-JNK cascade. Neuropharmacology 54, 1029–1040. doi: 10.1016/j.neuropharm.2008.01.014 Lipton, P. (1999). Ischemic cell death in brain neurons. Physiol. Rev. 79, 1431–568. doi: 10.1152/physrev.1999.79.4.1431 Liu, L., Li, C. J., Lu, Y., Zong, X. G., Luo, C., Sun, J., et al. (2015). Baclofen mediates neuroprotection on hippocampal CA1 pyramidal cells through the regulation of autophagy under chronic cerebral hypoperfusion. Sci. Rep. 5:14474. doi: 10.1038/srep14474 Xu, Q., Yang, J., Cao, Y., Zhang, L., Zeng, X., Li, F., et al. (2014). Acupuncture improves locomotor function by enhancing GABA receptor expression in September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 12 Hleihil et al. Baclofen Recovers GABAB Receptor Expression Zhu, X., Hu, H., Li, Z., Lin, R., Mao, J., and Chen, L. (2015). Gua Lou Gui Zhi decoction attenuates poststroke spasticity via the modulation of GABAB receptors. Mol. Med. Rep. 12, 5957–5962. doi: 10.3892/mmr.2015.4207 transient focal cerebral ischemia rats. Neurosci. Lett. 588C, 88–94. doi: 10.1016/ j.neulet.2014.12.057 transient focal cerebral ischemia rats. Neurosci. Lett. 588C, 88–94. doi: 10.1016/ j.neulet.2014.12.057 Zemoura, K., Balakrishnan, K., Grampp, T., and Benke, D. (2019). Ca2+/Calmodulin-dependent protein kinase II (CaMKII) b-dependent phosphorylation of GABAB1 triggers lysosomal degradation of GABAB Receptors via mind bomb-2 (MIB2)-mediated Lys-63-linked ubiquitination. Mol. Neurobiol. 56, 1293–1309. doi: 10.1007/s12035-018-1 142-5 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Zhang, C., Zhang, R., Zhang, S., and Xu, M. (2014). Baclofen for stroke patients with persistent hiccups: a randomized, double- blind, placebo-controlled trial. Trials 15:295. doi: 10.1186/1745-6215- 15-295 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. Zhang, F., Li, C., Wang, R., Han, D., Zhang, Q. G., Zhou, C., et al. (2007). September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org REFERENCES Activation of GABA receptors attenuates neuronal apoptosis through inhibiting the tyrosine phosphorylation of NR2A by Src after cerebral ischemia and reperfusion. Neuroscience 150, 938–949. doi: 10.1016/j.neuroscience.2007.09. 070 Copyright © 2021 Hleihil, Vaas, Bhat, Balakrishnan and Benke. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhang, Z., Zhang, W., Huang, S., Sun, Q., Wang, Y., Hu, Y., et al. (2015). GABAB receptor promotes its own surface expression by recruiting a Rap1- dependent signaling cascade. J. Cell Sci. 128, 2302–2313. doi: 10.1242/jcs.16 7056 September 2021 \| Volume 14 \| Article 726133 Frontiers in Molecular Neuroscience \| www.frontiersin.org 13
https://openalex.org/W2980641810	https://europepmc.org/articles/pmc6779285?pdf=render	English	null	Comparison of different semi-automated cfDNA extraction methods in combination with UMI-based targeted sequencing	Oncotarget	2,019	cc-by	7,450	Comparison of different semi-automated cfDNA extraction methods in combination with UMI-based targeted sequencing p Anna Streubel1, Albrecht Stenzinger2,3, Susann Stephan-Falkenau1, Jens Kollmeier4, Daniel Misch4, Torsten Gerriet Blum4, Torsten Bauer4, Olfert Landt5, Alexander Am Ende5, Peter Schirmacher2, Thomas Mairinger1 and Volker Endris2 1Department of Pathology, Helios Klinikum Emil von Behring, Berlin, Germany 2Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany 3German Cancer Consortium (DKTK), Heidelberg, Germany 4Department of Pneumology, Helios Klinikum Emil von Behring, Berlin, Germany 5TIB Molbiol Syntheselabor GmbH, Berlin, Germany Correspondence to: Volker Endris, email: volker.endris@med.uni-heidelberg.de Keywords: lung cancer; liquid biopsy; cfDNA; molecular diagnostics; T790M Received: May 04, 2019 Accepted: July 17, 2019 Published: October 01, 2019 Copyright: Streubel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: Streubel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT The analysis of circulating cell-free DNA (cfDNA) extracted from peripheral blood can serve as a minimally invasive alternative to tumor tissue biopsies in cases with impaired access to tissue. Its clinical utility has been well demonstrated for EGFR T790M testing in lung cancer patients suffering progress after tyrosine kinase inhibitor treatment. At present, highly sensitive unique molecular identifiers (UMI)-based NGS for liquid biopsy testing is less established compared to single gene assays. However, the critical bottleneck are sufficient cfDNA yields, which are essentially required to obtain meaningful test results. We compared four different cfDNA extraction methods (Qiagen, Promega, Thermo and Stratec) using the same plasma samples in order to evaluate their suitability for further NGS analysis. We managed to draw 60 ml blood from 12 patients each and equally collected 30ml in PAXgene and EDTA tubes at the same time point, sufficient for total of 96 cfDNA extractions. CfDNA concentrations and total amounts were highest for Qiagen and Promega protocols, showing the best read length profiles after sequencing. Known oncogenic driver mutations were identified in 9 out of 12 patients with at least one of the cfDNA extraction methods, again favoring the extraction protocols from Qiagen and Promega. We also uncovered putative sequencing artefacts including known driver genes pointing to a careful consideration for the limit of detection of this methodology. Our study shows that pre-analytical optimization is necessary to achieve the maximum sensitivity of UMI-based sequencing but also highlights the low abundance of tumor-derived cfDNA in lung cancer samples. www.oncotarget.com www.oncotarget.com www.oncotarget.com INTRODUCTION genetic driver mutation responsible for oncogenic progress [1, 2]. To detect and monitor these genetic changes, various molecular methods have been implemented in routine diagnostics, most of them working with PCR- based techniques. In oncology, the continuously evolving concept of precision medicine is mainly based on the development of drugs that are specifically tailored to address the molecular www.oncotarget.com Oncotarget 5690 Up to the very recent present, nucleotides were extracted from fresh or fixed tumor tissue samples. In most cases formalin-fixed and paraffin embedded (FFPE) tumor tissue is available for mutation analysis. Many studies have been carried out to optimize pre-analytical steps and furthermore broaden the spectrum of molecular analyses that can be utilized in this type of material. The main obstacles with FFPE-derived nucleotide acids are their relatively low quality due to fixation-induced degradation and deamination processes that can lead to false-positive sequencing results. While careful considerations of pre- analytical steps minimize these problems, the availability of tumor tissue is restricted to clinical cases where invasive tumor sampling is not limited by the physical fitness of the patient and/or the tumor location. Nevertheless the findings of T790M and its primary mutation in cfDNA gained from plasma remain disappointingly low compared with the tissue bound analysis. One of the causes may be found in the pre- analytical procedures used. To achieve sufficient cfDNA yields, a careful selection of pre-analytical methods is mandatory [6, 7, 8, 16]. This includes not only the choice of blood containers (stabilizing tubes vs. standard EDTA [17]) and transport conditions, but especially also the choice of cfDNA extraction methods. Hence, extraction methods that yield sufficient amounts of cfDNA molecules matching sequencing coverage are paramount to reach theoretical sensitivity levels. However, data investigating the performance and impact of different extraction methods are very limited. In this context one of the very challenging issues is the availability of sufficient sample volume /plasma with NSCLC patients especially [6, 7]. In this study our aim was to i) evaluate and standardize pre-analytical procedures with the ambition to ameliorate the detection rates of relevant genomic aberrations from circulating cfDNA gained from 4ml plasma input per extraction and ii) evaluate a highly-sensitive NGS-based approach that might overcome some of the restrictions of currently used cfDNA analysis strategies. Study concept Although this approach is highly elegant and the mutation looked for is exactly defined, surprisingly the method has not revolutionized the diagnostic procedure. This may be due to the fact that results obtained in many laboratories are disappointing, as especially in NSCLC patients cell-free tumor DNA concentrations are critically low [5] making successful analysis very challenging even when highly sensitive detection methods are applied [9]. Variant allele frequencies (VAF) of mutations often range below 1%, which is below the detection threshold of currently used technologies for mutation analysis using FFPE material such as Sanger sequencing, most real-time PCR or even NGS. To analyse cfDNA several approaches have been developed ranging from single PCR assays to whole genome sequencing [10–12]. Some of these methods reach a theoretical limit of detection (LOD) down to 0.00025-0.01% VAF [10, 13]. For NGS- based approaches this was achieved by introduction of unique molecular identifiers (UMI) to each single DNA molecule allowing deduplication with the downside for the need of ultra-deep sequencing [14, 15]. UMI features some decent advantages compared to standard NGS protocols and compared, to single gene assays UMI- based NGS enables multigene analysis for the detection of resistance mechanisms other than the classical T790M mutation. In our cohort of 12 patients, six patients had initial diagnosis of NSCLC at different stages (stage IIA-IV) without any prior cancer therapy (samples P1-P6, Table 1) and blood was taken before therapy, in case of P1, the only one with a stage II situation, blood was collected after surgery. Patients were selected for this study when molecular testing of corresponding FFPE tissue revealed an activating EGFR (five patients) or KRAS (one patient) mutation (Table 1 and Figure 4). The remaining six patients in this study progressed after first-line tyrosine kinase inhibitor (TKI) therapy targeting an initially identified activating EGFR mutation (samples S1-S6, Table 1). Blood was equally harvested in standard EDTA tubes and PAXgene tubes to determine any different influences of the two blood stabilizing containers on cfDNA extraction and high-sensitive mutation detection. A summary of the study concept is depicted in Figure 1. INTRODUCTION The concept of DNA analysis without the need of invasive surgical procedures has gained attention and holds reasonable advantages, especially in tumor patients where tumor tissue is not easily available. This is particularly true for monitoring of treatment response in malignant tumors, where repeated gaining of tumor tissue is seriously limited. A pioneer in routine use of mutation analysis out of plasma has been the detection of the T790M resistance mutation in EGFR-mutated NSCLC samples. On the background of increasingly sensitive and specific DNA detection methods, cell free DNA (cfDNA) gained attention and the fascinating concept of detection of genetic alterations by analyzing this source of DNA from easily accessible blood plasma came into focus [3–8]. Comparison of cfDNA extraction methods Table 2 summarizes the four protocols: The Stratec protocol provided the highest level of automation operating fully automated with hands-on time of 5 minutes but has a long run time of 160 min, while all other protocols required manual pre-processing of plasma samples prior to samples processing on the robotic machines (Table 2). In case of low sample throughput yet with a need for rapid reporting at the same time one clear technical disadvantage observed with the extraction kits used from Stratec is shown in suboptimal efficiency of reagents coverage if not 12 samples are run simultaneously. A technical disadvantage of 60 min hands-on time show the Thermo and Maxwell protocols compared to Stratec and Qiagen with 5/30 min, respectively. Considering these technical issues, the Qiagen protocol shows a summary of advantages in the least hands-on time combined with a maximum reagent coverage and highest elution volume and number of sample processing flexibility. For this study, elution volumes varied between 36 µl (Qiagen) and 70 µl (Stratec), even though the elution volume can be lowered down to 15 µl using the QiaCube instrument from Qiagen. Comparison of cfDNA extraction methods For our comparison study, we selected four commercially available (semi-)automated cfDNA extraction protocols. All four tested methods allow an input of up to 4 ml of plasma, in 80 of 96 extractions www.oncotarget.com Oncotarget 5691 Table 1: Patient details and therapy regiment ID age gd st FD FFPE collection Mutation (FFPE) blood coll. time from FD (d) blood coll. first-line/second- line P1 81 f IIA 02.05.2017 02.05.2017 EGFR:p. Leu858Arg 10.05.2017 8 - Surgery, no adjuvant chemotherapy P2 74 m IV 08.05.2017 08.05.2017 EGFR:p. Glu746_ Ala750del TP53:p. Leu257Arg PTEN:p. Ser170fs13 12.05.2017 4 before therapy palliative brain- RTX + Afatinib until 10/2017 P3 74 m IIIB 28.04.2017 28.04.2017 EGFR:p. Glu746_ Ala750del TP53:p. Tyr220Cys 19.05.2017 21 after therapy Simultanous radiotherapy/ chemotherapy (Platin/VRB) P4 69 m IV 26.05.2017 26.05.2017 EGFR:p. Glu746_ Ala750del 30.05.2017 4 before therapy Erlotinib ± Ramucirumab in study, since 05/2017 Erlotinib monotherapy P5 78 m IV 31.05.2017 31.05.2017 EGFR:p. Leu747_ T751del 01.06.2017 1 before therapy since 06/2017 Afatinib P6 66 m IV 04.05.2017 04.05.2017 KRAS:p. Gly13Cys STK11:p. Val197fs69 10.05.2017 6 before therapy palliative chemotherapy cisplatin/VRB- >Atezolizumab- >Pembrolizumab- >Tarceva S1 74 m IV 07.11.2016 07.11.2016 EGFR:p. Leu858Arg 16.05.2017 190 after therapy TKI (Erlotinib) until 06/2017, restaging SCLC S2 86 f IV 01.04.2016 01.04.2016 EGFR:p.E746_ A750delinsQP 19.05.2017 413 after therapy Gefitinib until 04/2017, 06-10/2017 Osimertinib S3 81 f IV 10.07.2013 10.07.2013 EGFR:p. Leu858Arg EGFRR:p. T790M 19.05.2017 1409 after therapy 07/2013-05/2017 Erlotinib, 06/2017 Osimertinib S4 68 f IV 04.09.2015 04.09.2015 EGFR:p. Glu746_ Ala750del 20.07.2016 320 after therapy 09/2015-07/2016 Erlotinib, 07/2016-06/2017 Osimertinib, Chemotherapy (continued) Table 1: Patient details and therapy regiment www.oncotarget.com Oncotarget 5692 ID age gd st FD FFPE collection Mutation (FFPE) blood coll. time from FD (d) blood coll. first-line/second- line S5 84 m IIIA 01.11.2010 01.11.2010 EGFR:p. Glu746_ Ala750del 31.07.2017 2464 after therapy neoadjuvant Gefitinib, then radio-/ chemotherapy + surgery + adjuvant chemotherpy S6 83 f IV 23.03.2016 23.03.2016 EGFR:p.L861Q PIK3CA:p. Glu545Lys TP53:p.Ser166* 30.05.2017 433 after therapy 05/16-06/17 Erlotinib, then no further therapy Abbreviations: ID: Sample Identification; m: male; f: female; FD: first diagnosis; RTx: radiotherapy; SCLC: squamous cell carcinoma; VRB: Vinorelbin; TKI: Tyrosine Kinase Inhibitor; blood coll.: blood collection; gd: gender; st: stage. Abbreviations: ID: Sample Identification; m: male; f: female; FD: first diagnosis; RTx: radiotherapy; SCLC: squamous cell carcinoma; VRB: Vinorelbin; TKI: Tyrosine Kinase Inhibitor; blood coll.: blood collection; gd: gender; st: stage. 4ml plasma was available (see Supplementary Table 1). cfDNA quantity In a first step, we compared the total cfDNA concentrations that were obtained by the four protocols. Most samples had cfDNA concentrations independent Figure 1: Workflow overview. From all lung cancer patients, blood was collected on the same day to ensure comparability of the results. Independent of the storage vessel (EDTA or PAX), plasma was separated within one hour after blood collection to ensure optimal results. After cfDNA extraction, NGS libraries were prepared using TagSequencing technology and sequenced on an IonTorrent S5 sequencer. Figure 1: Workflow overview. From all lung cancer patients, blood was collected on the same day to ensure comparability of the results. Independent of the storage vessel (EDTA or PAX), plasma was separated within one hour after blood collection to ensure optimal results. After cfDNA extraction, NGS libraries were prepared using TagSequencing technology and sequenced on an IonTorrent S5 sequencer. Figure 1: Workflow overview. From all lung cancer patients, blood was collected on the same day to ensure comparability of the results. Independent of the storage vessel (EDTA or PAX), plasma was separated within one hour after blood collection to ensure optimal results. After cfDNA extraction, NGS libraries were prepared using TagSequencing technology and sequenced on an IonTorrent S5 sequencer. cfDNA quantity www.oncotarget.com Oncotarget 5693 Table 2: Overview of extraction methods Table 2: Overview of extraction methods manufacturer Promega Thermo Scientific Qiagen Stratec Molecular robot Maxwell® RSC KingFisher™ Flex QIAcube System InviGenius plus product RSC LV ccfDNA Kit, MagMAX™ Cell-Free DNA Isolation Kit QIAamp® MinElute® ccfDNA InviMag® Free Circulating DNA Kit/ IG type bead based bead based filter based bead based sample volume (mL) 1-4; in this study 4 1-5; in this study 4 1-4; in this study 4 4; in this study 4 elution volume (µl) 60 50-100; in this study 50 15-60; in this study: 40 125 recoverable elution volume (µl) ~ 45, in this study 45 ~ 35-80, in this study 45 10-55, in this study: 36 70-80; in this study: 70 level of automation semi semi semi full manual pre processing external proteinase K step, concentration of DNA from the plasma with magnetic particles in 15mL tubes external proteinase K step; manual pipetting of the deep well plates external proteinase K step; concentration of DNA from plasma with magnetic particles in 50 mL tubes none hands-on time (minutes) ~60 ~60 ~30 ~5 automated runtime (minutes) ~35 ~35 ~25 ~160 total runtime (minutes) ~95 ~95 ~55 ~165 of the different extraction protocols below 0.5 ng/µl and a mean total amount of extracted cfDNA of 11.5 ng. (Figure 2A+B). Only two samples (P1 and S5) had higher amounts, ranging up to 282 ng in sample P1. The latter sample P1 derived from a patient whose blood was drawn shortly after surgical tumor resection. The highest DNA quantities were obtained using the Promega Maxwell protocol with a mean cfDNA amount of 24.7 ng using 4 ml of plasma (range 12.6 - 188.4 ng), corresponding to a mean of 6.3 ng/ml plasma (Figure 2B). Thermo and Qiagen yielded similar values, with a mean of 9.4 ng (Thermo; range 2.5-132.5ng, mean 4.0 ng/ml plasma) and 10.8 ng (Qiagen; range 3.1 – 282.8 ng, mean 3.4 ng/ml plasma), respectively. The lowest cfDNA amounts were obtained using the Stratec protocol (mean 8.0 ng, range 1.4 – 53.4 ng, mean 1.7 ng/ml plasma). While difference in cfDNA amounts were not statistically significant between the protocols of Qiagen, Promega and Thermo, the difference of each of these methods compared to the Stratec extraction was (paired t-test: Stratec/Qiagen: p=0.0263; Stratec/Promega: p=0.0031, Stratec/Thermo: p=0.0023). cfDNA quantity Comparing the total extracted cfDNA yields, there was a significant higher cfDNA fraction from plasma stored in EDTA versus PAXgene tubes (paired test: p=0.000097). Due to the very low cfDNA concentrations, length profile assessment by using fragment analysis on a TapeStation was inconclusive. Supplementary Figure 1 illustrates read profiles for sample S1, showing similar patterns of cfDNA from all methods, with typical peaks at approximately 175bp and 353bp. As exemplified with sample P5, the read profiles for the different other cfDNA extractions were too low to draw any conclusions. NGS library preparation In total, 96 NGS libraries were prepared (eight extractions per sample from 12 samples all together). Using the most possible input of 13 µl, the DNA input for all but patient P1 and S5 was below 20 ng. Library concentrations ranged from 22 to 323 pM (Supplementary Figure 1). Six out of 96 libraries failed QC criteria; e.g. the library concentrations were below the optimal concentration of 50pM (P3 Thermo- EDTA; P5 Stratec-PAX; S4 Stratec EDTA+PAX; S4 Promega + PAX). In case of S4, only five out of the eight extraction methods yielded sufficient libraries for sequencing. The mean number of sequenced reads (4 libraries per Ion 530 Chip) was 1.75 million reads (+/- 0.87). With only one exception, at least roughly 1 million reads per sample were analyzable. The mean total number of reads per amplicon was 37741.5x. www.oncotarget.com Oncotarget 5694 The amplicon lengths of the panel vary between 80 bp and 140 bp, with two clusters of 80-94 bp and 105- 139 bp, respectively (Figure 3A). Figure 3B illustrates a typical read length profile obtained from NGS data of the different extraction protocols of case S2. In all cases, there is a party of short reads originating from either primer dimers or short, incomplete sequencing reads. Comparing the distribution patterns, the NGS reads obtained from cfDNA samples derived from Stratec and Thermo extraction protocols show excess of short and incomplete reads. This observation was accompanied by a substantial decline of longer reads when using the Stratec purification method. Identification of known mutations Figure 4 summarizes the sequencing results of the different cfDNA extractions. In 9 out of 11 samples, the oncogenic driver mutations in EGFR or KRAS that were identified from FFPE tissue could also be identified in Oncotarget 5695 www oncotarget com Figure 2: Comparison of total extracted cfDNA amounts. (A) cfDNA amount in ng/ml plasma per sample and extraction method. With the exception of sample P1 and sample S5, the available cfDNA concentrations for further library preparations were below 0.5 ng/µl. (B) cfDNA yields as box-and-whisker plots from the different extraction protocols. The highest yields were obtained from the Promega MaxWell protocol. While the cfDNA amounts from the Qiagen and Thermo protocols were comparable, the Stratec instrumentation yielded the lowest cfDNA amounts. Figure 2: Comparison of total extracted cfDNA amounts. (A) cfDNA amount in ng/ml plasma per sample and extraction method. With the exception of sample P1 and sample S5, the available cfDNA concentrations for further library preparations were below 0.5 ng/µl. (B) cfDNA yields as box-and-whisker plots from the different extraction protocols. The highest yields were obtained from the Promega MaxWell protocol. While the cfDNA amounts from the Qiagen and Thermo protocols were comparable, the Stratec instrumentation yielded the lowest cfDNA amounts. Figure 2: Comparison of total extracted cfDNA amounts. (A) cfDNA amount in ng/ml plasma per sample and extraction method. With the exception of sample P1 and sample S5, the available cfDNA concentrations for further library preparations were below 0.5 ng/µl. (B) cfDNA yields as box-and-whisker plots from the different extraction protocols. The highest yields were obtained from the Promega MaxWell protocol. While the cfDNA amounts from the Qiagen and Thermo protocols were comparable, the Stratec instrumentation yielded the lowest cfDNA amounts. www.oncotarget.com Oncotarget 5695 Figure 3: Read Length Distribution. The Oncomine cfDNA Lung panel amplifies 35 different amplicons, ranging in size between 80 and 139 base pairs. (A) Theoretical size distribution of the Oncomine cfDNA Lung panel. Amplicons with similar sizes were grouped for better visibility. (B) Observed amplicon sizes for sample S2. Colored bars represent the different extraction protocols. Compared to the theoretical size distribution, a group of short fragments representing unincorporated primers is visible. Comparing the different extraction methods, there is a marked difference concerning fragments with short read lengths. Identification of known mutations The libraries prepared from cfDNA extracted using the protocols from Stratec and Thermo present excess shorter reads (unincorporated primers) with a decrease in target specific reads. Figure 3: Read Length Distribution. The Oncomine cfDNA Lung panel amplifies 35 different amplicons, ranging in size between 80 and 139 base pairs. (A) Theoretical size distribution of the Oncomine cfDNA Lung panel. Amplicons with similar sizes were grouped for better visibility. (B) Observed amplicon sizes for sample S2. Colored bars represent the different extraction protocols. Compared to the theoretical size distribution, a group of short fragments representing unincorporated primers is visible. Comparing the different extraction methods, there is a marked difference concerning fragments with short read lengths. The libraries prepared from cfDNA extracted using the protocols from Stratec and Thermo present excess shorter reads (unincorporated primers) with a decrease in target specific reads. Figure 4: Overview of sequencing results. Numbers represent variant allele frequencies. In sample S5, initial sequencing of the FFPE tumor block prior to TKI therapy identified an EGFR exon 19 deletion, which was not detectable in the cfDNA in progress after TKI. Figure 4: Overview of sequencing results. Numbers represent variant allele frequencies. In sample S5, initial sequencing of the FFPE tumor block prior to TKI therapy identified an EGFR exon 19 deletion, which was not detectable in the cfDNA in progress after TKI. www.oncotarget.com Oncotarget 5696 identified by the variant calling pipeline, but were not consistently found throughout the differently extracted cfDNA samples. A closer look for example at an NRAS p.Gly12Asp mutation observed in sample S5 that was found by three different extraction methods (molCOV: 3, 4 and 4 reads) using the IGV browser revealed these calls as false positives based on their presence in short, incomplete reads (compare Supplementary Figure 2 for illustration). Therefore we considered these variants rather as false positive calls instead of true mutations displaying tumor heterogeneity. at least one of the cfDNA extractions (sample P1 is not taken into account in this calculation as blood was taken shortly after surgery). In two cases (samples S3 and P5), no mutations were identified using any of the eight extracted cfDNAs. In some cases, the mutations were not called by the variant caller algorithm due to the very low allele frequency, but were identified by manual inspection of the sequencing reads in the IGV browser or a secondary bioinformatics pipeline. Identification of known mutations FFPE tissue testing of sample S5 identified an EGFR exon 19 deletion at baseline (Table 1). The patient was treated with EGFR kinase inhibitors and radiochemotherapy before onset of disease progression (more detailed description see Table 1). The corresponding blood sample from 2017 now identified an activating NRAS mutation along with two additional mutations in PIK3CA and TP53. These mutations were not examined in the FFPE tissue in 2010. There was unfortunately no original tissue from 2010 left for NGS retesting. Intriguingly, these calls encompassed classical oncogenic driver mutations that can also be found as resistance mutations during EGFR TKI therapy (Supplementary Table 2). For example, in two cases (P3 and S3) low frequency BRAF p.Val600Glu mutations were reported, but only observed in one of the eight analyzed extraction samples. Similarly, a KRAS p.Gly13Ser mutation was called in six different extractions originating from three different individuals. In summary, in 9 out of 11 samples (as mentioned earlier sample P1 was not taken into account), cfDNA was detectable by at least one of the tested extraction methods as judged by detection of oncogenic mutations, correlating to a cfDNA success rate of 82 % (9 out of 11 samples). When considering all samples, tumor-derived ctDNA was detected in 9 out of 12 samples (75%). The difficulty in interpreting these low frequency variant calls can be highlighted by sample S3. The sequencing results of the different cfDNA extractions revealed up to eight mutations with very low molVAFs in the absence of the primary EFGR p.Leu858Arg mutation. While most of these variants were only identified once, two mutations in TP53 (p.Tyr220Cys and p.Arg273His) were found in almost all extractions. Both mutations were not detected by tissue testing. These two TP53 mutations were also observed in samples P3 (p.Tyr220Cys) and S5 (p.Arg273His), but in this case the TP53 p.Tyr220Cys of sample P3 was verified in tissue and the TP53 p.Arg273His was at least found in all sequenced libraries of sample S5. For determination of sensitivity of the different extraction protocols, we only considered those nine samples where cfDNA was present and calculated on basis of all mutations present in the collective. The Qiagen and Promega methods correctly identified 25 out of 26 mutations (combined count of all mutations present in the 9 samples multiplied by 2: EDTA and PAXgene) and 21/22 mutations, respectively, thus achieving a sensitivity of 96 %. Identification of known mutations Using the Thermo Fisher protocol, 19/25 mutations (76 %) were identified, while with the Stratec purification 21/24 mutations (87.5 %) were detected. Figure 5 and Supplementary Table 2 summarize putative false-positive variants. While in most samples no or up to two false-positive variants were found, in three samples (S2, S3 and S5), eight of these dubious variants were observed. As highlighted in Figure 5B, we found a slightly higher number of false-positive calls in plasma samples that were stored in PAXgene tubes compared to EDTA tubes, but the differences were not statistically significant (p=0.229, paired t-test; p=0.3388; Wilcoxon test). Thus, care should be taken when using plasma- stabilizing tubes. DISCUSSION Sufficient extraction of cfDNA from plasma of patients suffering from NSCLC is challenging merely because cfDNA concentrations in this context are seriously low anyway [5, 7]. Therefore, no matter how thorough and abundant extraction methods might be the yield of cfDNA is generally borderline. Consequently, we did not expect striking differences on first insight of results but a close careful second look focusing on the quantity but especially on the quality of cfDNA showed differences concerning mutation detection rates. Furthermore as batch-to-batch variability of plasma drawn at different time points during disease progression limits its comparability due to changes in plasma cfDNA levels, we collected up to 60 ml of whole blood from each patients for 8 extractions out of plasma drawn at one time point. We decided to extract plasma at the same time point for EDTA and PAXgene tubes. Due to the fact that cfDNA concentrations are seriously low and all might contain only a few ctDNA molecules, one may not neglect that any remaining eluted cfDNA that could contain mutated molecules and is excluded from further analysis results into false negative results. One mentionable advantage of the Qiagen minElute extraction method could be that elution volumes can be minimized down to 15 µl meaning that all extracted cfDNA could be used if a sequencing reaction with the Oncomine cfDNA panel only runs once (maximum input of cfDNA: 13 µl). The other extraction methods we evaluated in our study perform a minimum elution volume of 40 µl, 45 µl and 70 µl for the KingFisher instrument, the Maxwell RSC and the Stratec System, respectively. To compare extraction capacities in this study we kept the elution volume of Qiagen to a minimum of 40 µl. The observed overall sensitivity of the liquid biopsy testing in our study was 82 % which is in the typical range reported in the literature, still limiting the capability of liquid biopsy to replace tissue based testing [14, 18]. Comparing the different extraction protocols, the Qiagen minElute assay achieved the highest sensitivity The lower sensitivity of the Stratec and Thermo protocols might be explained by the difference in read length profiles. Our data have shown a decline in target specific reads and a higher amount of short fragments most likely representing primer dimers for both assays. Figure 5: Sequencing artefacts. Numbers represent sum of artefacts identified in each sequencing. Sequencing artefacts The tested cfDNA lung panel uses unique molecular identifiers (molecular barcodes) to facilitate differentiation of single molecules present in the plasma from PCR- generated duplicates. The analysis pipeline removes duplicate reads based on the unique molecular barcode and calls variants based on their molecular allelic frequency. In our test cohort, the molecular variant allele frequencies (molVAF) of the primary mutations ranged from 0.1% - 9.45% (Supplementary Table 1). The molecular allele coverage (molCOV) of the primary mutation, e.g. the number of reads showing the mutation after deduplication ranged from two to 277. Thus, the variant calling pipeline reports mutations that can be identified in at least two different reads with unique molecular barcodes. In order to prove our assumptions on artificially generated mutations we tested some of those samples that provided enough cfDNA after sequencing and showing a KRAS, NRAS or BRAF mutation with a clamped real-time PCR assay. The samples tested were all negative for the suspected mutations in codon 12 and 13 in the KRAS gene, for codon 12, 13 in the NRAS gene and for the V600E mutation in BRAF. We also tested some eluates of P6 and S5 as they were tested positive for KRAS G13C in tissue and for NRAS G13C in all 8 cfDNA eluates with the UMI- based assay, respectively. All eluates tested were positive In our cohort, several variants with low molVAF (0.06 - 3.7%) and molCOV (2-8 molecular reads) were www.oncotarget.com Oncotarget 5697 resultingin95.5%, when only considering plasma samples with detectable amounts of ctDNA). for those mutations in the real-time assay as well. A detailed information of tested eluates and results are found in Supplementary Figure 3 and Supplementary Table 3. One sample was excluded from this calculation as the patient was operated a week before peripheral blood was taken. Hence the high DNA yield observed for that sample together with the fact that the primary EGFR (or any other) mutation could be identified was very likely owed to that circumstance. Sequencing artefacts In summary, the Qiagen minElute shows advantages in quality yield of DNA mainly concerning read length compared to the Stratec and Thermo protocols. We observed a trend that the Qiagen protocol results in a lower number of false-positive artefacts but the sample/mutation number was too small for being statistically relevant. The potential of a minimization of elution volumes down to 15 µl with the Qiagen Assay could be an interesting issue that is worth further investigation. The Oncomine cfDNA Lung panel has a high sensitivity with the advantage of high cfDNA input into one reaction (13 µl) that facilitates detection of oncogenic driver mutations and resistance mechanism from liquid biopsies of lung cancer patients. Pre-analytical steps including the choice of blood tubes and cfDNA extraction method are important to maximize efficiency of liquid biopsy testing. In clinical practice a threshold of 10 allelic reads should be considered to maximize specificity especially when single nucleotide variants (SNV), need to be identified undoubtedly. Due to the low abundance of cfDNA and corresponding low VAF, all methods to detect variants at 0.1% are in risk of identifying false positive variants. We observed minimal but statistically highly significant differences in cfDNA yields between blood samples stored in EDTA or PAXgene tubes (p=0.000097, paired t-test). The study design concentrated primarily on a possible influence of the stabilizing agents on the sequencing results and not their stabilizing properties, which has been shown in previous studies [19]. While we did not record a decline in amplification efficiencies between EDTA and PAX, we noted a difference in the number of putative sequencing artefacts that was statistically not significant likely due to the small test size. A previous report by Yuhua et al reported a higher number of non-EGFR variants observed in samples analyzed using the Oncomine cfDNA Lung assay compared to AmpliSeq generated libraries [20]. The authors also describe oncogenic hotspot variants in KRAS, BRAF or NRAS in samples with primary EGFR mutations that might develop during therapy. Several studies have reported on co-occurring KRAS and EGFR mutations in liquid biopsies that were undetectable in tissue biopsies. Pathway bypass mutations as resistance mechanism have been observed, including for example BRAF p.Val600Glu mutations [21]. However, the biological origin of this mutation is unclear. Deamination processes and BRAF p.Val600Glu mutations in benign naevi are examples that low frequency variants can be present in cfDNA also from healthy individuals [22]. Sequencing artefacts Furthermore, previous studies showed a low concordance level between commercial tissue-based NGS testing and cfDNA analytics [23]. These differences might partially be explained by tumor heterogeneity. This could be the case with sample S5: The originally detected EGFR Exon 19 deletion p.Glu746_Ala750del was not detected in any of the eight extractions but instead we found an NRAS p.Gly13Cys mutation in all extractions. The molecular mutant allelic coverages were all over 100 in all six-sequenced extraction of sample S5. Furthermore this mutation was confirmed by a clamped real-time assay, which was performed on four remaining cfDNA eluates of this patient (Supplementary Figure 3, Supplementary Table 3). Whether the NRAS mutation derives from a new lesion or is as a matter of fact a result of possible tumor heterogeneity can be hardly assessed confidently from blood as one cannot determine the location where the mutated fraction in the cfDNA came from. Unfortunately we were not able to gain any more tissue to further explore A.S. and V.E. conducted the experiments, analyzed results and wrote the manuscript. T.M. and Al.S. supervised the manuscript. A.S. collected samples and blood and extracted cfDNA from all samples with four different extraction methods. J.K; D.M. and T.G.B. provided clinical data, S.S.-F. provided histopathological data, A.E. and O.L. run real-time PCR tests DISCUSSION Note the slightly higher count of artefacts in PAXgene versus EDTA tubes, but differences are not statistically significant (p=0.266, students t-test). S-E: Stratec-EDTA, S-P: Stratec-PAXgene, Q-E: Qiagen-EDTA, Q-P: Qiagen-PAXgene, P-E: Promega-EDTA, P-P: Promega-PAXgene, T-E: Thermo-EDTA, T-P: Thermo-PAXgene. Figure 5: Sequencing artefacts. Numbers represent sum of artefacts identified in each sequencing. Note the slightly higher count of artefacts in PAXgene versus EDTA tubes, but differences are not statistically significant (p=0.266, students t-test). S-E: Stratec-EDTA, S-P: Stratec-PAXgene, Q-E: Qiagen-EDTA, Q-P: Qiagen-PAXgene, P-E: Promega-EDTA, P-P: Promega-PAXgene, T-E: Thermo-EDTA, T-P: Thermo-PAXgene. www.oncotarget.com Oncotarget 5698 Of note, in sample S2 both extractions from Thermo detected the primary activating EGFR exon 19 deletion but failed to detect the clinically relevant EGFR p.T790M mutation (read coverage 16806x / 24303x for PAXgene and EDTA, respectively). this issue. On the other hand, the random distribution of discordant variants in our comparative analysis with eight independent sequencings from identical plasma samples, we believe that in the other samples showing these hotspot mutations are rather false positives considering also their low molecular allele coverage and frequency (Supplementary Figure 4). NGS library preparation For this study, we used the Oncomine Lung cfDNA panel (Thermo Scientific, Waltham, USA) which has a theoretical limit of detection of 0.1% VAF if using at least 20 ng cfDNA input. The panel comprises 35 amplicons covering clinically actionable hotspot mutations from 11 genes using the proprietary TagSequencing technology. From each extraction, NGS sequencing libraries using the Oncomine cfDNA Lung panel were prepared, resulting in 96 libraries overall. We used the maximum possible input volume of 13 µl for NGS library generation using the TagSequencing protocol. Patient cohort During April 2017 and August 2017, peripheral whole blood was collected from 12 non-small cell lung cancer patients during ongoing treatment, five before initiation of first line systemic therapy (Samples P2-P5), one after post-surgery (P1) and six patients after first line systemic therapy (S1-S6). All patients harboured an activating mutation either in the EGFR or KRAS gene (Table 1). The activating mutation had already been analysed from FFPE tissue during routine diagnostics prior to blood sampling using targeted sequencing with the AmpliSeq Colon Lung Panel v2 on the Ion Torrent S5XL instrument (Thermo Scientific, Waltham, USA). Only Sample S5 was analyzed by pyrosequencing as NGS was not practiced in our institute at the time point of first diagnosis in 2010 and no more tissue or DNA was left to retest the latter with NGS methods. Informed consent was obtained from each patient with protocols approved by an ethical committee. www.oncotarget.com Oncotarget 5699 Plasma samples After two further rounds of purification using AmpureXP beads, the final library is quantified using the Ion Library quantification kit (Thermo Scientific, Waltham, USA). In total, 6 out of 96 libraries did not match quality criteria and were excluded from further analysis. On average, 4-5 libraries were pooled on an Ion 530 chip. Following template preparation on an Ion Chef, the chip was sequenced on an Ion S5XL sequencer using 200 bp chemistry and 500 flows. As batch-to-batch variability of plasma drawn at different time points during disease progression can occur and may limit its comparability due to changes in plasma cfDNA levels, we collected 60 ml blood at one time point from each patient. Two 30 ml batches of whole blood were taken and collected in EDTA or PAXgene tubes (Qiagen, Hilden, Germany), respectively. Blood samples were immediately centrifuged at 1300x g for 13 min [16]. Plasma was transferred to new tubes and centrifuged for 12 min at 16.000x g, plasma drawn from PAXgene tubes were centrifuged at room temperature whereas EDTA plasma was centrifuged at 4°C [16]. Clear supernatants were stored short term at -20°C or extracted immediately. Mutation analysis Sequence alignment and variant calling was performed using plugins in Torrent Suite version 5.6 and IonReporter 5.2 / 5.6. Data was analyzed with the IGV browser. Real-time PCR testing Frozen plasma samples were thawed and purified by filtration (Minisart Syringe filter, Sartorius, Göttingen, Germany). cfDNA was extracted from 4 ml plasma by using each of the following methods: InviMag® Free Circulating DNA Kit, (Stratec, Berlin, Germany), minElute ccfDNA extraction kit (Qiagen, Hilden, Germany), Maxwell RSC ccfDNA extraction kit (Promega, Madison, USA) and MagMax cfDNA extraction (Thermo Scientific, Waltham, USA) on a KingFisher instrument according to the instruction of the manufacturer. Elution volumes ranged between 36 and 70 µl (for detailed description see Table 2). cfDNA quantification was performed by a fluorescent assay using the Qubit 3.0 Fluorometer (Thermo Scientific, Waltham, USA). DNA extracts (2 µl) were tested with the beta version of LightMix kit 40-0654-64 NRAS-KRAS, comprising of a multiplex pre-amplification of the gene regions followed by clamped-probe wild type sequence suppressing amplification of the four codon regions 12/13, 59/61, 117 and 146 in NRAS and KRAS, and identification of mutations by running a melting curve, using a Roche (Mannheim, Germany) cobas z 480 analyzer. KRAS mutation results were confirmed using LightMix kit 40-0416-09 KRAS 12/13, BRAF mutations were identified with the LightMix kit 40-0406-96 BRAF, following the kit instructions. CONFLICTS OF INTEREST Qualitative assessment of cfDNA was performed on a TapeStation 2200 (Agilent Technologies, Santa Clara, USA) using up to 2 µl cfDNA on a high-sensitivity D1000 tape according to manufacturer instructions. V.E. is a consultant/advisory board member for ThermoFisher and received lecture fees from AstraZeneca and Novartis. Al.S. is a consultant/advisory board member of Bayer, AstraZeneca, Bristol-Myers Squibb, Novartis, ThermoFisher and Illumina, and received speaker’s honoraria from Bayer, BMS, MSD, Roche, Illumina, AstraZeneca, Novartis and ThermoFisher, Takeda as well as research funding from Chugai and BMS. P.S. received advisory board honoraria from Pfizer, Roche, Novartis and AstraZeneca as well as speaker’s honoraria and research funding from Roche, AstraZeneca and Novartis. O.L. is the owner of TIB Molbiol. from circulating tumor DNA. Nat Med. 2014; 20:430–35. https://doi.org/10.1038/nm.3511. [PubMed] and molecular subtypes. Nat Rev Clin Oncol. 2015; 12:511– 26. https://doi.org/10.1038/nrclinonc.2015.90. [PubMed] and molecular subtypes. Nat Rev Clin Oncol. 2015; 12:511– 26. https://doi.org/10.1038/nrclinonc.2015.90. [PubMed] 13. Zonta E, Garlan F, Pécuchet N, Perez-Toralla K, Caen O, Milbury C, Didelot A, Fabre E, Blons H, Laurent- Puig P, Taly V. Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations. PLoS One. 2016; 11:e0159094. https://doi.org/10.1371/journal.pone.0159094. [PubMed] 3. Diaz LA Jr, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol. 2014; 32:579–86. https://doi.org/10.1200/JCO.2012.45.2011. [PubMed] 4. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011; 11:426–37. https://doi.org/10.1038/nrc3066. [PubMed] 14. Vollbrecht C, Lehmann A, Lenze D, Hummel M. Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients. Oncotarget. 2018; 9:18529–39. https://doi.org/10.18632/oncotarget.24908. [PubMed] 5. Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014; 6:224ra224– 224ra224. https://doi.org/10.1126/scitranslmed.3007094. [PubMed] 15. Li BT, Janku F, Jung B, Hou C, Madwani K, Alden R, Razavi P, Reis-Filho JS, Shen R, Isbell JM, Blocker AW, Eattock N, Gnerre S, et al. Ultra-deep next-generation sequencing of plasma cell-free DNA in patients with advanced lung cancers: results from the Actionable Genome Consortium. Ann Oncol. 2019; 30:597–603. https://doi.org/10.1093/annonc/mdz046. [PubMed] 6. van Dessel LF, Vitale SR, Helmijr JC, Wilting SM, van der Vlugt-Daane M, Oomen-de Hoop E, Sleijfer S, Martens JW, Jansen MP, Lolkema MP. High-throughput isolation of circulating tumor DNA: a comparison of automated platforms. Mol Oncol. 2019; 13:392–402. https://doi.org/10.1002/1878-0261.12415. [PubMed] 7. van Ginkel JH, van den Broek DA, van Kuik J, Linders D, de Weger R, Willems SM, Huibers MM. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics. Cancer Med. 2017; 6:2297–307. https://doi.org/10.1002/cam4.1184. [PubMed] 16. Sorber L, Zwaenepoel K, Deschoolmeester V, Roeyen G, Lardon F, Rolfo C, Pauwels P. A Comparison of Cell- Free DNA Isolation Kits: Isolation and Quantification of Cell-Free DNA in Plasma. J Mol Diagn. 2017; 19:162–68. https://doi.org/10.1016/j.jmoldx.2016.09.009. [PubMed] 17. Risberg B, Tsui DW, Biggs H, Ruiz-Valdepenas Martin de Almagro A, Dawson SJ, Hodgkin C, Jones L, Parkinson C, Piskorz A, Marass F, Chandrananda D, Moore E, Morris J, et al. Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients. J Mol Diagn. REFERENCES 1. Santarpia M, Altavilla G, Salazar MF, Magri I, Pettineo G, Benecchi S, Rosell R. Tyrosine kinase inhibitors for non-small-cell lung cancer: finding patients who will be responsive. Expert Rev Respir Med. 2011; 5:413–24. https://doi.org/10.1586/ers.11.27. [PubMed] This library preparation method incorporates unique identifiers (molecular barcodes) in a two-cycle PCR reaction allowing deduplication of sequenced reads for increased sensitivity. Following purification with AmPureXP Beads (Agilent, Santa Clara, USA), a second PCR (18 cycles) is used for library preparation. 2. Thomas A, Liu SV, Subramaniam DS, Giaccone G. Refining the treatment of NSCLC according to histological www.oncotarget.com Oncotarget 5700 from circulating tumor DNA. Nat Med. 2014; 20:430–35. https://doi.org/10.1038/nm.3511. [PubMed] 23. Chae YK, Davis AA, Carneiro BA, Chandra S, Mohindra N, Kalyan A, Kaplan J, Matsangou M, Pai S, Costa R, J, Salem G, Pohida T, Heenan P, et al. High frequency of BRAF mutations in nevi. Nat Genet. 2003; 33:19–20. https://doi.org/10.1038/ng1054. [PubMed] from circulating tumor DNA. Nat Med. 2014; 20:430–35. https://doi.org/10.1038/nm.3511. [PubMed] 2018; 20:883–92. https://doi.org/10.1016/j.jmoldx.2018.07.005. [PubMed] 8. Cook L, Starr K, Boonyaratanakornkit J, Hayden R, Sam SS, Caliendo AM. Does Size Matter? Comparison of Extraction Yields for Different-Sized DNA Fragments by Seven Different Routine and Four New Circulating Cell-Free Extraction Methods. J Clin Microbiol. 2018; 56:e01061–18. https://doi.org/10.1128/JCM.01061-18. [PubMed] 18. Oxnard GR, Thress KS, Alden RS, Lawrance R, Paweletz CP, Cantarini M, Yang JC, Barrett JC, Jänne PA. Association Between Plasma Genotyping and Outcomes of Treatment With Osimertinib (AZD9291) in Advanced Non- Small-Cell Lung Cancer. J Clin Oncol. 2016; 34:3375–82. https://doi.org/10.1200/JCO.2016.66.7162. [PubMed] 9. Volckmar AL, Sültmann H, Riediger A, Fioretos T, Schirmacher P, Endris V, Stenzinger A, Dietz S. A field guide for cancer diagnostics using cell-free DNA: from principles to practice and clinical applications. Genes Chromosomes Cancer. 2018; 57:123–39. https://doi.org/10.1002/gcc.22517. [PubMed] 19. Alidousty C, Brandes D, Heydt C, Wagener S, Wittersheim M, Schäfer SC, Holz B, Merkelbach-Bruse S, Büttner R, Fassunke J, Schultheis AM. Comparison of Blood Collection Tubes from Three Different Manufacturers for the Collection of Cell-Free DNA for Liquid Biopsy Mutation Testing. J Mol Diagn. 2017; 19:801–04. https://doi.org/10.1016/j.jmoldx.2017.06.004. [PubMed] 10. Newman AM, Lovejoy AF, Klass DM, Kurtz DM, Chabon JJ, Scherer F, Stehr H, Liu CL, Bratman SV, Say C, Zhou L, Carter JN, West RB, et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat Biotechnol. 2016; 34:547–55. https://doi.org/10.1038/nbt.3520. [PubMed] 11. Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, Parkinson C, Chin SF, Kingsbury Z, Wong AS, Marass F, Humphray S, Hadfield J, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 2013; 497:108–12. https://doi.org/10.1038/nature12065. [PubMed] 20. Yuhua L, Yee JM, Min T. Targeted Next-generation Sequencing of Circulating Biomarkers in Non-Small Cell Lung Cancer (NSCLC). IRCSet Conference. http://ircset. org/anand/2017papers/IRC-SET_2017_paper_S4-3.pdf. 21. Huang L, Fu L. Mechanisms of resistance to EGFR tyrosine kinase inhibitors. Acta Pharm Sin B. 2015; 5:390–401. https://doi.org/10.1016/j.apsb.2015.07.001. [PubMed] 12. Thierry AR, Mouliere F, El Messaoudi S, Mollevi C, Lopez-Crapez E, Rolet F, Gillet B, Gongora C, Dechelotte P, Robert B, Del Rio M, Lamy PJ, Bibeau F, et al. Clinical validation of the detection of KRAS and BRAF mutations 22. Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM, Moses TY, Hostetter G, Wagner U, Kakareka www.oncotarget.com Oncotarget 5701 J, Salem G, Pohida T, Heenan P, et al. High frequency of BRAF mutations in nevi. Nat Genet. 2003; 33:19–20. https://doi.org/10.1038/ng1054. [PubMed] 23. Chae YK, Davis AA, Carneiro BA, Chandra S, Mohindra N, Kalyan A, Kaplan J, Matsangou M, Pai S, Costa R, Jovanovic B, Cristofanilli M, Platanias LC, Giles FJ. Concordance between genomic alterations assessed by next-generation sequencing in tumor tissue or circulating cell-free DNA. Oncotarget. 2016; 7:65364–73. https://doi.org/10.18632/oncotarget.11692. [PubMed] Jovanovic B, Cristofanilli M, Platanias LC, Giles FJ. Concordance between genomic alterations assessed by next-generation sequencing in tumor tissue or circulating cell-free DNA. Oncotarget. 2016; 7:65364–73. https://doi.org/10.18632/oncotarget.11692. [PubMed] www.oncotarget.com Oncotarget 5702
https://openalex.org/W2791170282	https://www.frontiersin.org/articles/10.3389/fchem.2018.00072/pdf	English	null	Thermal Expansion and Magnetostriction Measurements at Cryogenic Temperature Using the Strain Gauge Method	Frontiers in chemistry	2,018	cc-by	4,251	ORIGINAL RESEARCH published: 20 March 2018 doi: 10.3389/fchem.2018.00072 Thermal expansion and magnetostriction, the strain responses of a material to temperature and a magnetic ﬁeld, especially properties at low temperature, are extremely useful to study electronic and phononic properties, phase transitions, quantum criticality, and other interesting phenomena in cryogenic engineering and materials science. However, traditional dilatometers cannot provide magnetic ﬁeld and ultra-low temperature (<77 K) environment easily. This paper describes the design and test results of thermal expansion and magnetostriction at cryogenic temperature using the strain gauge method based on a Physical Properties Measurements System (PPMS). The interfacing software and automation were developed using LabVIEW. The sample temperature range can be tuned continuously between 1.8 and 400 K. With this PPMS-aided measuring system, we can observe temperature and magnetic ﬁeld dependence of the linear thermal expansion of different solid materials easily and accurately. Edited by: Jun Chen, University of Science and Technology Beijing, China Edited by: Jun Chen, University of Science and Technology Beijing, China University of Science and Technology Beijing, China Reviewed by: Jun Yan, Beijing Jiaotong University, China Kun Lin, University of Science and Technology Beijing, China Ranjan Mittal, Bhabha Atomic Research Centre, India Correspondence: Rongjin Huang huangrongjin@mail.ipc.ac.cn Laifeng Li laifengli@mail.ipc.ac.cn Keywords: thermal expansion, magnetostriction, strain gauge method, cryogenic temperature, PPMS Specialty section: This article was submitted to Physical Chemistry and Chemical Physics, a section of the journal Frontiers in Chemistry Received: 30 January 2018 Accepted: 06 March 2018 Published: 20 March 2018 INTRODUCTION Cryogenic engineering has been extensively used in the ﬁelds of aerospace industry, superconducting technology, and large scientiﬁc instruments, etc. The physical performances of materials directly aﬀect the safety and service time of the cryogenic system, among of which thermal expansion and magnetostriction, the strain responses of a material to temperature and a magnetic ﬁeld, are the key thermodynamic properties closely related to speciﬁc heat and magnetization. Thus, accurate measurement of thermal expansion at low temperatures is necessary to ensure the security and reliability of the cryogenic systems. Also, these properties are very useful to investigate electronic and phononic properties, phase transitions, quantum criticality, and other interesting physical phenomena in cryogenic engineering and materials science (Ventura and Risegari, 2008; Inoue et al., 2014; Iwakami et al., 2014). Wei Wang 1,2, Huiming Liu 2, Rongjin Huang 2,3, Yuqiang Zhao 2, Chuangjun Huang 2, Shibin Guo 2, Yi Shan 2 and Laifeng Li 1,2,3* Wei Wang 1,2, Huiming Liu 2, Rongjin Huang 2,3, Yuqiang Zhao 2, Chuangjun Huang 2, Shibin Guo 2, Yi Shan 2 and Laifeng Li 1,2,3 1 Institute of Advanced Metal Materials, School of Materials Science and Engineering, Tianjin University, Tianjin, China, 2 State Key Laboratory of Technologies in Space Cryogenic Propellants, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, China, 3 Department of Science and Technology of Liquid Metal Material, School of Future Technology, University of Chinese Academy of Sciences, Beijing, China Thermal Expansion and Magnetostriction Measurements at Cryogenic Temperature Using the Strain Gauge Method Wei Wang 1,2, Huiming Liu 2, Rongjin Huang 2,3, Yuqiang Zhao 2, Chuangjun Huang 2, Shibin Guo 2, Yi Shan 2 and Laifeng Li 1,2,3 Citation: (2a) (2b) Subtracting Equation (2b) from (2a), and rearranging: αs = εS −εr 1T + αr (3) (3) Fortunately, Quantum Design Physical Properties Measurements System has been widely used for measurements of thermodynamic properties, such as speciﬁc heat, magnetization, electrical resistance, and thermal conductivity. In this report, we present the thermal expansion and magnetostriction measurements using the strain gauge method installed in a PPMS in the temperature range of 1.8–400 K and magnetic ﬁelds up to 14 T. The interfacing software and automation were developed using LabVIEW. Since the variation of the sensitivity coeﬃcient of strain gauges at low temperatures is a key factor aﬀecting the measurement accuracy, it was veriﬁed in the temperature range of 2–300 K by comparing the measured thermal expansion data of oxygen-free copper with the source data from National Institute of Standards and Technology (NIST), USA. Thus, the CTE of test sample can be obtained with the accurately measured εS −εr and 1T, as well as the known CTE of reference sample αr. In this paper, we use fused silica of high purity (>99.99%) as the reference sample in the measurement of thermal expansion due to its very low and almost temperature- independent mean linear CTE (5.5 × 10 −7/K) (Kirby and Hahn, 1971). EXPERIMENTAL PROCEDURES AND METHODS The schematic view of the measurement system is shown in Figure 1, which consists of a PPMS, nanovoltmeters, and a LabVIEW PC. Also, using this PPMS-aided measuring system, we observed temperature and magnetic ﬁeld dependence of the linear thermal expansion of NaZn13-type LaFe13−xAlx compounds, which absolutely expands the practical application scope of this Multi- function testing system. With the help of LabVIEW, we can design experiments using our own measurement electronics when working on the general purpose platforms of the PPMS (Quantum Design Inc., 2002, 2013). Firstly, nanovoltmeters (KEITHLEY 2182A, USA) can monitor the electrical signals of samples in PPMS via a GPIB interface simultaneously. And then a LabVIEW computer attached to nanovoltmeters also via a GPIB interface. The QDInstrument VIs and QDInstrument.dll are installed on this computer and communicate over .NET. In brief, the QDInstrument program is a translator with a .NET interface for communication with the LabVIEW VI and an OLE interface for communication with MultiVu. The DLL is conﬁgured to address the QDInstrument_server.exe program running on the MultiVu computer. This is done via connecting an Ethernet cable directly between the two computers in the absence of a LAN. The server program in turn communicates over OLE with the MultiVu application. Finally, by controlling both external devices as well as PPMS’s state (temperature and magnetic ﬁeld) on a separate LabVIEW computer from MultiVu, we can observe the temperature and magnetic ﬁeld dependence of the linear thermal expansion of diﬀerent solid materials easily and accurately. Citation: Wang W, Liu H, Huang R, Zhao Y, Huang C, Guo S, Shan Y and Li L (2018) Thermal Expansion and Magnetostriction Measurements at Cryogenic Temperature Using the Strain Gauge Method. Front. Chem. 6:72. doi: 10.3389/fchem.2018.00072 The approaches to estimate the thermal expansion of solid material can be classiﬁed, according to an extensometer, into quartz dilatometer, interferometer, optical lever, mechanical lever, magnetic meter, pointer meter, and capacitance micrometer methods, strain gauges, etc. (Clark, 1968; James et al., 2001; Kanagaraj and Pattanayak, 2003). The quartz dilatometer March 2018 \| Volume 6 \| Article 72 Frontiers in Chemistry \| www.frontiersin.org 1 Thermal Expansion and Magnetostriction Measurements Wang et al. method is frequently used at low temperatures, because of its high accuracy (Corruccini and Gniewck, 1961; Deng and Xu, 2003; ASTM, 2006).However, it is expensive and complex to operate, and the sample size requirements are quite high (Tang et al., 2014). For a test specimen of large length, the accuracy may be aﬀected by the potential temperature gradient along it, which then requires a cryostat to have high temperature uniformity. method is frequently used at low temperatures, because of its high accuracy (Corruccini and Gniewck, 1961; Deng and Xu, 2003; ASTM, 2006).However, it is expensive and complex to operate, and the sample size requirements are quite high (Tang et al., 2014). For a test specimen of large length, the accuracy may be aﬀected by the potential temperature gradient along it, which then requires a cryostat to have high temperature uniformity. factor of strain gauge, αs −αg is the diﬀerence in coeﬃcient of thermal expansion (CTE) between the tested sample and gauge, respectively, and 1T is the temperature change from arbitrary initial reference temperature. For two same types of strain gauges installed on test sample (αs) and reference sample (αr), respectively, the apparent strains can be expressed as: εs = βg Fg 1T + (αs −αg) 1T (2a) εr = βg Fg 1T + (αr −αg) 1T (2b) The strain gauge method is also widely used to measure the thermal expansion of material at low temperatures (Liu et al., 2012, 2014). Compared with the quartz dilatometer method, the strain gauge method can measure multiple samples at the same time and has lower requirements of the test specimen size. However, traditional dilatometers cannot provide magnetic ﬁeld environment easily and only been used in relative higher temperature range (>77 K) (Kaufman, 1963; Hannah and Reed, 1992). Frontiers in Chemistry \| www.frontiersin.org STRAIN GAUGE METHOD It is well known that a strain gauge can measure the thermal strain on the surface of a test specimen to reﬂect the linear thermal expansion of material because it can transform the strain into the electric signal with a high precision. The strain caused by temperature ﬂuctuation is deﬁned as thermal output. Without a mechanical force imposed on the test specimen, the temperature change can bring about a length variation both in the test sample and in the strain gauge, which then cause the resistivity variation of the strain gauge correspondingly. And the strain variation induced by the net temperature can be expressed as follows (Poore and Kesterson, 1978; Walsh, 1997; Micro-Measurements, 2007): ε = βg Fg 1T + (αs −αg) 1T (1) (1) The sample puck, as demonstrated in Figure 2, is redesigned to meet the special requirement of thermal expansion measurement. The whole sample puck is emerged into sample chamber of PPMS to cool the test sample. The temperature and where ε is the apparent strain of strain gauge, βg is the thermal coeﬃcient of resistivity of the grid material, Fg is the gauge March 2018 \| Volume 6 \| Article 72 Frontiers in Chemistry \| www.frontiersin.org 2 Thermal Expansion and Magnetostriction Measurements Wang et al. FIGURE 1 \| Schematic of measurement system for thermal expansion of solid materials. FIGURE 2 \| Schematic of the sample chamber. magnetic ﬁeld can be adjusted by the PPMS in the range of 1.8– 400 K and ±14 T. The dimensions of the test sample can be tuned from 3 × 3 × 3 to 8 × 8 × 8 mm. Its relatively small dimensions can reduce the diﬃculty in machining and the inﬂuence of the non uniform temperature ﬁeld on the measurement accuracy test sample and reference sample are placed on the sample puck stress-free. Two identical strain gauges are bonded on the center of the test sample surface and the reference sample surface, respectively, using a high performance adhesive M-Bond 610. This adhesive is capable of forming thin hard gluelines for FIGURE 1 \| Schematic of measurement system for thermal expansion of solid materials. FIGURE 1 \| Schematic of measurement system for thermal expansion of solid materials. FIGURE 1 \| Schematic of measurement system for thermal expansion of solid materials. FIGURE 2 \| Schematic of the sample chamber. FIGURE 2 \| Schematic of the sample chamber. Frontiers in Chemistry \| www.frontiersin.org STRAIN GAUGE METHOD test sample and reference sample are placed on the sample puck stress-free. Two identical strain gauges are bonded on the center of the test sample surface and the reference sample surface, respectively, using a high performance adhesive M-Bond 610. This adhesive is capable of forming thin, hard gluelines for maximum ﬁdelity in transferring strains from the sample surface to the gauge. magnetic ﬁeld can be adjusted by the PPMS in the range of 1.8– 400 K and ±14 T. The dimensions of the test sample can be tuned from 3 × 3 × 3 to 8 × 8 × 8 mm. Its relatively small dimensions can reduce the diﬃculty in machining and the inﬂuence of the non-uniform temperature ﬁeld on the measurement accuracy. The reference sample with the same dimensions is made from fused silica of 99.99% purity. To measure the thermal expansion, March 2018 \| Volume 6 \| Article 72 Frontiers in Chemistry \| www.frontiersin.org 3 Thermal Expansion and Magnetostriction Measurements Wang et al. NaZn13-type compounds LaFe13−xAlx have aroused extensive attention (Fujieda et al., 2002; Shen et al., 2009; Li et al., 2014; Wang et al., 2015). The MCE of this materials is chieﬂy due to the itinerant-electron metamagnetic (IEM) transition, i.e., a magnetic-ﬁeld-induced phase transition from the paramagnetic state to the ferromagnetic state. The NTE eﬀect is a thermo- physical property and attributed to the magnetovolume eﬀect (MVE). Nevertheless, very few report explores the relationship between the change of volume expansion and external magnetic ﬁeld, i.e., magnetostriction, in LaFe13−xAlx compounds. The Ni-Cr resistive elements BB120-2AA250 (11) Karma foil strain gauges (Zhonghang Electronic Measuring Instruments Co. Ltd., China) were used. The nominal resistance is (119.9 ± 0.1) , and the nominal sensitivity coeﬃcient is 1.74 ± 0.01. When the measurement starts, the automated program controls the temperatures and magnetic ﬁeld of the PPMS at the diﬀerent rates. And the thermal output of the strain gauges and the temperature are recorded by nanovoltmeters. Then the LabVIEW computer can save the temperature and magnetic ﬁeld dependence of the linear thermal expansion of tested sample. g 3 p Generally, the large magnetostriction originates from the interaction between atom magnetic moments. LaFe13−xAlx compounds (1.04 < x < 7.02) share the same NaZn13- type crystalline structure while showing diﬀerent magnetic states (antiferromagnetic state, ferromagnetic state and paramagnetic state). Speciﬁcly, LaFe13−xAlx (1.0< x <1.8) show an antiferromagnetic state at low temperature. STRAIN GAUGE METHOD And the antiferromagnetic ordering is very vulnerable and can be turned into the ferromagnetic state easily when bringing an external magnetic ﬁeld. Diﬀerent magnetic states have diﬀerent magnetic moments. So a large magnetostriction LaFe13−xAlx of compounds is caused by these magnetic phase transition. In order to better understanding the magnetostrictive property, as displayed in Figure 4, the linear thermal expansion data (1L/L 300 K) of LaFe13−xAlx (x = 1.4, 1.6, and 1.8) compounds were measured using this PPMS-aided measuring system over a temperature range of 10–300 K ﬁrstly (Zhao et al., 2017). For LaFe13−xAlx (x = 1.4 and 1.6) below the Neel temperature, it shows that the CTE is very constant across a wide temperature range (10–200K). However, for the sample LaFe11.2Al1.8, 1L/L rises with decreasing temperature in the whole temperature range examined. Speciﬁcally, 1L/L grows gradually around room temperature, then increases rapidly, and again grows slowly with decreasing temperature, indicating that NTE occurs in the whole range measured RESULTS AND DISCUSSION The accuracy of the CTE measurement mainly depends on the accuracy of the strain gauges, the nanovoltmeters and the procedure of the gauges installation. To verify the accuracy of this test apparatus and method, the thermal expansion of oxygen-free copper are measured in the temperature range of 2–300 K on this apparatus and quartz dilatometer (Linseis L75), respectively. Also, the Comparisons between experimental and published values of thermal expansion of copper are displayed in Figure 3 (Kirby and Hahn, 1969). The results of thermal expansion of copper matched closely with reference data, and the maximum error for instantaneous coeﬃcients is 1.1 × 10−6 K−1, which is smaller than that testing using quartz dilatometer. At the same time, to validate the reliability of the measurement, experiments are repeated from 2 – 300 K. We can see that there is no signiﬁcant diﬀerence of the thermal expansions (<2%) between the two tests, which shows that the whole measurement system is applicable in the temperature range of 2–300 K. Also, this method is available for other special structure materials such as low symmetry samples and orientation preferred samples with relatively smooth and nonporous surfaces. In the last several years, due to the giant magnetocaloric eﬀect (MCE) and the intriguing negative thermal expansion (NTE), FIGURE 4 \| Temperature dependence of linear thermal expansions 1L/L (reference temperature: 300K) of LaFe13−xAlx. FIGURE 4 \| Temperature dependence of linear thermal expansions 1L/L (reference temperature: 300K) of LaFe13−xAlx. FIGURE 3 \| Experimental and published values of thermal expansion of copper. FIGURE 4 \| Temperature dependence of linear thermal expansions 1L/L (reference temperature: 300K) of LaFe13−xAlx. FIGURE 4 \| Temperature dependence of linear thermal expansions 1L/L (reference temperature: 300K) of LaFe13−xAlx. FIGURE 4 \| Temperature dependence of linear thermal expansions 1L/L (reference temperature: 300K) of LaFe13−xAlx. FIGURE 3 \| Experimental and published values of thermal expansion of copper. Frontiers in Chemistry \| www.frontiersin.org March 2018 \| Volume 6 \| Article 72 4 Thermal Expansion and Magnetostriction Measurements Wang et al. FIGURE 5 \| Temperature dependence of the magnetostriction λ(T) in LaFe11.2Al1.8 at different magnetic ﬁelds. FIGURE 6 \| Magnetic ﬁeld dependence of the magnetostriction λ(H) of LaFe11.2Al1.8 compounds at different temperatures. FIGURE 5 \| Temperature dependence of the magnetostriction λ(T) in LaFe11.2Al1.8 at different magnetic ﬁelds. FIGURE 6 \| Magnetic ﬁeld dependence of the magnetostriction λ(H) of LaFe11.2Al1.8 compounds at different temperatures. RESULTS AND DISCUSSION FIGURE 6 \| Magnetic ﬁeld dependence of the magnetostriction λ(H) of LaFe11.2Al1.8 compounds at different temperatures. FIGURE 5 \| Temperature dependence of the magnetostriction λ(T) in LaFe11.2Al1.8 at different magnetic ﬁelds. FIGURE 6 \| Magnetic ﬁeld dependence of the magnetostriction λ(H) of LaFe11.2Al1.8 compounds at different temperatures. (2–300 K). It is worth noting that the 1L/L vs. T curves are diﬀerent from one another, which demonstrates that the NTE properties are aﬀected by the partial substitution of Fe by Al sensitively. However, when magnetic ﬁeld exceeds 4T, the magnetostriction increases slowly and becomes lower than that in 200 K. And after that, the magnetostriction is rapidly decreased with temperature decline from 100 to 10 K. The results indicate that the magnetostriction of this sample is very sensitive and the giant magnetostriction happens in a relatively narrow temperature range. Also it is worth mentioning that the linear magnetostriction of this material is isotropic. Because the magnetostriction measurements have been conducted along the parallel and perpendicular directions to the magnetic ﬁeld separately and the anisotropic magnetostriction is negligible. Figure 5 gives the temperature dependence of the linear magnetostriction λ(T) of LaFe11.8Al1.8 compounds between 10 and 300 K. It is noted that all the magnetostrictions of the sample in diﬀerent magnetic ﬁelds exhibit a sudden increase with the decrease of the temperature and then, decrease rapidly with following temperature decline. In addition to this, it is apparent that the temperature with giant magnetostriction shifts to higher temperature as the magnetic ﬁeld increases. Importantly, giant linear magnetostriction values higher than 1,000 ppm are observed when the magnetic ﬁeld exceeds 4T in a relatively wide temperature range. Especially, it can reach up to 1,900ppm at 12T. Interestingly, the peak value of magnetostriction of the LaFe11.2Al1.8 compound is larger than the maximum of the typical magnetostriction material, Terfenol-D alloy (Grössinger et al., 2014). CONCLUSION In summary, an experimental setup with a PPMS based on the strain gauge method has been developed to measure the thermal expansion and magnetostriction of solid materials at low temperatures (1.8–400 K). The relatively small dimensions of the test sample can save the liquid helium and reduce the impact of the non-uniform temperature ﬁeld on measurement accuracy. The measurement accuracy was veriﬁed in the temperature range of 2–300 K by comparing the measured thermal expansion data of oxygen-free copper with the source data from NIST. Also, using this PPMS-aided measuring system, we observed temperature and magnetic ﬁeld dependence of the linear thermal expansion of NaZn13-type LaFe13−xAlx compounds. Such Multi-function testing system with a broad measurement temperature range will open up an intriguing research frontier for exploring potential magnetostrictive and NTE materials. To further explore the magnetostrictive property, the magnetic ﬁeld dependence of the magnetostriction λ(H) under diﬀerent temperatures for LaFe11.2Al1.8 sample have been investigated in Figure 6. Apparently, with the increase of the magnetic ﬁeld, the λ(H) raises gently at 300 K and reaches to 340 ppm under 12T ﬁnally, suggesting the magnetostrictive eﬀect of this material is extremely weak at room temperature. But when the temperature decreases, the λ(H) increases rapidly with increasing magnetic ﬁeld. And when the temperature comes up to 200 K, the λ(H) is almost increase linearly with the increase of magnetic ﬁeld. Besides, when the temperature falls below 150K, it is observed that below 4T, the magnetostriction increases quickly with the increase of the magnetic ﬁeld. Frontiers in Chemistry \| www.frontiersin.org REFERENCES Liu, H., Gong, L., Xu, D., Huang, C., Zhang, M., Xu, P., et al. (2014). Top loading cryogen-free apparatus for low temperature thermophysical properties measurement. Cryogenics 62, 11–13. doi: 10.1016/j.cryogenics.2014.02.020 ASTM (2006). E228-17 Standard Test Method for Linear Thermal Expansion of Solid Materials with a Push-rod Dilatometer. American Society for Testing Materials, 10. Liu, H., Xu, D., Xu, P., Huang, R., Xu, X., Li, L., et al. (2012). An apparatus for measurements of thermal conductivity and thermal expansion based on GM cryocooler. J. Phys. Conf. Ser. 400:052017. doi: 10.1088/1742-6596/400/5/052017 Clark, A. (1968). Low temperature thermal expansion of some metallic alloys. Cryogenics 8, 282–289. doi: 10.1016/S0011-2275(68)80003-7 Corruccini, R. J., and Gniewck, J. J. (1961). Thermal Expansion of Technical Solids at Low Temperatures: a Compilation From the Literature. Washington, DC: National Bureau of Standards. Micro-Measurements (2007). Measurement of thermal expansion coeﬃcient using strain gages. Tech. Note TN-513-1, 119–129. Available online at: http:// blhnobel.se/docs/11063/tn5131tn.pdf Poore, M., and Kesterson, K. (1978). Measuring the thermal expansion of solids with strain gages. J. Test. Eval. 6, 98–102. doi: 10.1520/JTE10926J Deng, D., and Xu, L. (2003). Measurements of thermal expansion coeﬃcient of phenolic foam at low temperatures. Cryogenics 43, 465–468. doi: 10.1016/S0011-2275(03)00122-X Quantum Design Inc. (2002). Interfacing 3rd Party Instruments to the PPMS Software Environment. Application Note 1070-210, 4 p. Fujieda, S., Fujita, A., and Fukamichi, K. (2002). Large magnetocaloric eﬀect in La(FexSi1−x)13 itinerant-electron metamagnetic compounds. Appl. Phys. Lett. 81,1276–1278. doi: 10.1063/1.1498148 Quantum Design Inc. (2013). Interfacing LabVIEW Programs with PPMS, DynaCool, Versalab and MPMS 3 Systems. Application Note 1070-210, 5 p. Grössinger, R., Turtelli, R. S., and Mehmood, N. (2014). “Materials with high magnetostriction,” in IOP Conference Series: Materials Science and Engineering (Islamabad: IOP Publishing). Shen, B., Sun, J., Hu, F., Zhang, H., and Cheng, Z. (2009). Recent progress in exploring magnetocaloric materials. Adv. Mater. 21, 45–64. doi: 10.1002/adma.200901072 Hannah, R., and Reed, S. (1992). Strain Gage Users’ Handbook. London: Elsevier Applied Science. Tang, K., Sha, L., Li, Y. J., Jin, T., and Liu, S. J. (2014). Measurement of thermal expansion at low temperatures using the strain gage method. J. Zhejiang Univ. Sci. A. 15, 323–330. doi: 10.1631/jzus.A1400051 Inoue, D., Kaido, D., Yoshikawa, Y., Minegishi, M., Matsumoto, K., and Abe, S. (2014). Thermal expansion and magnetostriction measurements using a high sensitive capacitive dilatometer at millikelvin temperatures. J. Phys. Conf. Ser. 568:032001. doi: 10.1088/1742-6596/568/3/032001 Ventura, G., and Risegari, L. (2008). The Art of Cryogenics: Low-Temperature Experimental Techniques. AUTHOR CONTRIBUTIONS WW and HL: Designed experiments; YZ, SG, and YS: Carried out experiments; HL and CH: Analyzed experimental results; WW, RH, and LL: Wrote the manuscript. March 2018 \| Volume 6 \| Article 72 5 Thermal Expansion and Magnetostriction Measurements Wang et al. FUNDING Fusion Science Program (2015GB121001), Beijing Nova program (Z161100004916114), Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-JSC042), and the Fund of the State Key Laboratory of Technologies in Space Cryogenic Propellants (SKLTSCP1701, SKLTSCP1601). Fusion Science Program (2015GB121001), Beijing Nova program (Z161100004916114), Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-JSC042), and the Fund of the State Key Laboratory of Technologies in Space Cryogenic Propellants (SKLTSCP1701, SKLTSCP1601). This work was supported by the National Natural Science Foundation of China (Grant Nos. 51577185, 51427806, 51522705, 51401224), the National Magnetic Conﬁnement REFERENCES Oxford: Elsevier Ltd. Walsh, R. P. (1997). “Use of strain gages for low temperature thermal expansion measurements,” in Proceedings of the 16th ICEC/ICMC (Kitakyushu: Elsevier Science), 661–6644. Iwakami, O., Kawata, N., Takeshita, M., Yao, Y., Abe, S., and Matsumoto, K. (2014). Thermal expansion and magnetostriction measurements using a Quantum Design Physical Property Measurement System. J. Phys. 568:032002. doi: 10.1088/1742-6596/568/3/032002 Wang, W., Huang, R., Li, W., Tan, J., Zhao, Y., Li, S., et al. (2015). Zero thermal expansion in NaZn13-type La(Fe, Si)13 compounds. Phys. Chem. Chem. Phys. 17, 2352–2356. doi: 10.1039/C4CP04672B James, J., Spittle, J., Brown, S., and Evans, R. (2001). A review of measurement techniques for the thermal expansion coeﬃcient of metals and alloys at elevated temperatures. Meas. Sci. Technol. 12, R1–R15. doi: 10.1088/0957-0233/ 12/3/201 Zhao, Y., Huang, R., Li, S., Liu, H., Wang, W., Jiang, X., et al. (2017). Giant isotropic magnetostriction in NaZn13-type LaFe13−xAlx compounds. Appl. Phys. Lett. 110:011906. doi: 10.1063/1.4973476 Kanagaraj, S., and Pattanayak, S. (2003). Measurement of the thermal expansion of metal and FRPs. Cryogenics 43, 399–424. doi: 10.1016/S0011-2275(03) 00096-1 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Kaufman, A. (1963). Investigation of strain gages for use at cryogenic temperatures. Exp. Mech. 3, 177–183. doi: 10.1007/BF02325791 The reviewer KL and handling Editor declared their shared aﬃliation. Kirby, R., and Hahn, T. (1969). Standard Reference Material 736. Copper Thermal Expansion. Washington, DC: National Bureau of Standards. 10 p. Kirby, R., and Hahn, T. (1971). NIST Standard Reference Material 739, Fused-silica Thermal Expansion. . Washington, DC: National Bureau of Standards. Copyright © 2018 Wang, Liu, Huang, Zhao, Huang, Guo, Shan and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original 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. Li, W., Huang, R., Wang, W., Tan, J., Zhao, Y., Li, S., et al. (2014). Enhanced negative thermal expansion in La1−xPrxFe10.7Co0.8Si1.5 compounds by doping the magnetic rare-earth element praseodymium. Inorg. Chem. 53, 5869–5873. REFERENCES doi: 10.1021/ic500801b March 2018 \| Volume 6 \| Article 72 Frontiers in Chemistry \| www.frontiersin.org 6
https://openalex.org/W4313361525	https://www.mdpi.com/2073-4433/14/1/68/pdf?version=1673524062	English	null	In-Depth Examination of Machine Learning Models for the Prediction of Ground Temperature at Various Depths	Atmosphere	2,022	cc-by	15,534	Jong-Won Yang 1 and Khongorzul Dashdondov 2,* 1 National Program of Excellence in Software, Chungbuk National University, Cheongju 28644, Republic of Korea 2 Department of Computer Engineering, School of Electrical and Computer Engineering, Chungbuk National University, Cheongju 28644, Republic of Korea * Correspondence: khongor@chungbuk.ac.kr; Tel.: +82-043-261-3270 1 National Program of Excellence in Software, Chungbuk National University, 1 National Program of Excellence in Software, Chungbuk National University, Cheongju 28644, Republic of Korea 2 Department of Computer Engineering, School of Electrical and Computer Engineering, Chungbuk National University, Cheongju 28644, Republic of Korea * Correspondence: khongor@chungbuk.ac.kr; Tel.: +82-043-261-3270 Cheongju 28644, Republic of Korea 2 Department of Computer Engineering, School of Electrical and Computer Engineering, Chungbuk National University, Cheongju 28644, Republic of Korea * Correspondence: khongor@chungbuk.ac.kr; Tel.: +82-043-261-3270 Abstract: Ground temperature (GT) or soil temperature (ST) is simply the measurement of the warmness of the soil. Even though GT plays a meaningful role in agricultural production, the direct method of measuring the GT is time-consuming, expensive, and requires human effort. The foremost objective of this study is to build machine learning (ML) models for hourly GT prediction at different depths (5, 10, 20, and 30 cm) with the optimum hyperparameter tuning with less complexity. The present study utilizes a statistical model (multiple linear regression (MLR)) and four different ML models (support vector regression (SVR), random forest regression (RFR), multi-layered perceptron (MLP), and XGBoost (XGB)) for predicting GT. Overall, 13 independent variables and 5 GTs with different depths as response variables were collected from a meteorological station at an interval of 1 h between 1 January 2017 and 1 July 2021. In addition, two different input datasets named M1 (selected number of parameters) and M2 (collected dataset with all variables) were used to assess the model. The current study employed the Spearman rank correlation coefﬁcient approach to extract the best features and used it as the M1 dataset; in addition, the present study adopted regression imputation for solving the missing data issues. From the results, the XGB model outperformed the other standard ML-based models in any depth GT prediction (MAE = 1.063; RMSE = 1.679; R2 = 0.978 for GT; MAE = 0.887; RMSE = 1.263; R2 = 0.979 for GT_5; MAE = 0.741; RMSE = 1.025; R2 = 0.985 for GT_10; MAE = 0.416; RMSE = 0.551; R2 = 0.995 for GT_20; MAE = 0.280; RMSE = 0.367; R2 = 0.997 for GT_20). Academic Editor: Stephan Havemann Received: 19 November 2022 Revised: 14 December 2022 Accepted: 22 December 2022 Published: 29 December 2022 Article Article Jong-Won Yang 1 and Khongorzul Dashdondov 2,* Therefore, the present study developed a simpler, less-complex, faster, and more versatile model to predict the GT at different depths for a short-term prediction with a minimum number of predictor attributes. atmosphere atmosphere Citation: Yang, J.-W.; Dashdondov, K. In-Depth Examination of Machine Learning Models for the Prediction of Ground Temperature at Various Depths. Atmosphere 2023, 14, 68. https://doi.org/10.3390/ atmos14010068 Keywords: ground temperature prediction; ML prediction; XGBoost model; Spearman rank correlation coefﬁcient Keywords: ground temperature prediction; ML prediction; XGBoost model; Spearman rank correlation coefﬁcient 1. Introduction The unabated increase in greenhouse gas emissions is raising the temperature of the Earth. The consequences include melting glaciers, increased precipitation, extreme weather events, and shifting seasons. Combined with global population and income growth, climate change poses a global threat to food security [1,2]. Climate change, in particular, has exacerbated the effects on agriculture in recent decades, owing to changes in the frequency and severity of droughts and ﬂoods [2,3]. Climate change will have a wide-ranging impact on the global food equation, both on supply and demand and on food systems at the local level, where small farm communities frequently rely on local and self-production. In addition, it signiﬁcantly impacts global, regional, and local food production. Climate change is attributed to an increase in greenhouse gases, such as carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O), and is caused by human activities such as the use of fossil fuels for energy production, agricultural activities, and deforestation. Temperature atmosphere atmosphere atmosphere Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/atmosphere Atmosphere 2023, 14, 68. https://doi.org/10.3390/atmos14010068 Atmosphere 2023, 14, 68 2 of 30 rise due to global warming is expected to continue, rising from 1.8 ◦C to 4.0 ◦C by the end of the century [4]. A recent systematic review of changes in crop yields of major crops grown in Africa and South Asia due to climate change discovered that average crop yields in both regions could fall by 8% by the 2050s [5,6]. Climate change is expected to reduce yields by −17% (wheat), −5% (maize), −15% (sorghum), and −10% (millet) in Africa, and by −16% (maize) and −11% (sorghum) in South Asia [5,6]. The temperature of the Earth’s soil is one of the signiﬁcant agricultural factors affected by global warming; the characteristics of the Earth’s soil are critical in Earth sciences such as agriculture, forestry, geology, and land-atmosphere interactions [7]. g gy p Soil temperature (ST) or ground temperature (GT) is simply the measurement of the warmness of the soil. Soil absorbs and stores heat, acting as an energy driver during the day and a heat source at the soil surface at night. In terms of annual energy transfer, during the warm season, the soil stores energy and releases it into the air during the colder months of the year [8,9]. GT impacts the rate of nitriﬁcation; also, it inﬂuences the soil moisture content, aeration, and heat energy balance between the atmosphere and land surface. The GT is the factor that drives the germination of seeds; in addition, it directly affects plant growth since GT regulates the soil’s physical and chemical properties. In practice, this soil property can seriously inﬂuence nutrient uptake, respiration within the soil’s evaporation and transpiration processes, root and plant growth, and microorganism activity [10–13]. In contrast, several vital processes, such as dehumidiﬁcation and the concentration of physical, chemical, and microbiological processes in soil media, are also tremendously governed by the thermos—physical properties of the soil; thereby, knowledge of GT is highly relevant in cultivation-based decision-making tasks. Most soil organisms function best at an optimum soil temperature, which aids in generating quality and an improved production quantity. Atmosphere 2023, 14, 68 3 of 30 3 of 30 Statistical and time series-based models are established through the relationship be- tween GT and climatological variables. The previous statistical model which was used for GT prediction correlates the interrelationship between air temperature, relative hu- midity, wind speed, solar radiation, rainfall, atmospherics, and sunshine duration and the GT [17–24]. Even though most statistical models adopt linearity between the indepen- dent and dependent variables, the GT is nonlinear to the other climatological variables, so such models are not optimal for predicting future GT. Other widely used models are time series-based models known as Box–Jenkins models for GT, which work by predict- ing future values from past historical time-based values. Among the other time series- based statistical models, the auto-regressive moving average (ARMA) model and the auto-regressive integrated moving average (ARIMA) model are extensively used for future prediction [25–27]. Previous studies’ [26,28] results show that the temperature series is not sufﬁciently static for these models. So, the reliability of these models is still a question for various depth predictions. p p Machine learning (ML), a subcategory of artiﬁcial intelligence (AI), has been compre- hensively employed to solve nonlinear problems for the last two decades. To solve the GT prediction scenario, several researchers have used neural network-based frameworks. Unlike physical models, ML models adopt the trends of input and output parameters through their self-learning competence. Artiﬁcial neural network (ANN), classiﬁcation and regression trees (CART), random forest regression (RFR), support vector regression (SVR), multiple linear regression modeling, boosted regression tree (BRT), long short-term mem- ory network (LSTM), gated recurrent unit network (GRU), etc., are some models often used not only used for GT prediction but also for rainfall-runoff, climate prediction, and indoor microclimates [1,11,29,30]. In a previous study, Ref. [19] utilized an ANN model to estimate daily and annual GT, and the results showed that the ANN model could sufﬁciently predict the GT. Likewise, another study [10] developed an ANN model used to predict monthly soil temperature by using various meteorological variables of the previous month for the city of Adana, Turkey. That study used seven atmospheric variables to predict the GT at various depths by using the three-layered feed-forward-based ANN structure and network to predict the GT with satisfactory results. Later, Ref. Metrological variables have an exerted inﬂuence on GT; thus, GT and distribution at different depths are manipulated by external climatic factors such as atmospheric air temperature, relative humidity, wind speed, solar radiation, rainfall, atmospherics, and sunshine duration. Furthermore, the soil temperature is an important factor in some other ﬁelds, such as water resources and hydrologic engineering. Besides that, in the ﬁeld of atmospheric science, changes in GT have obvious effects on the decomposition of organic matter, resulting in an increase in carbon dioxide (CO2) in the atmosphere [14]. As a result, a model which is capable of accurately forecasting GT is in high demand. Measuring GT only from the surface does not complete the forecasting models because the nature and temperature of the soil vary according to the speciﬁc depth. Previous studies have mentioned that a soil depth of 5 cm plays a signiﬁcant role in seed germination, growth, and nutrient uptake [15]. At a depth of 10 cm, the soil’s nature also governs the growth of microorganisms and their transmission to plants; similarly, the soil at a depth of 20 cm ensures the growth of the roots of plants and the absorption of water and nutrients by the roots [16]. Therefore, predicting GT changes at different depths is indispensable for ensuring the proper growth of plants. There are generally two methods used to measure GT. They are as follows: y (1) Direct measuring method (method of manually inserting high-precision soil tem- perature sensors into the soil surface and at the required depths). y (1) Direct measuring method (method of manually inserting high-precision soil tem- perature sensors into the soil surface and at the required depths). (2) Indirect method—predictive modeling method (giving atmospheric parameter input to various prediction models and training the models with historical values at the required depths and times). Even though the direct measuring method is more reliable, such measurements are not only cumbersome but also require a vast number of human resources and an abundance of time. However, the predictive modeling methods are accessible. If the models achieve high accuracy with minimal error in their predictions, they can be used as a navigation for temperature changes. Various kinds of modeling techniques such as statistical modeling, time series modeling, energy balance models, and computational models used for predict- ing GT have become vastly available in recent years [17–20]. The following components comprehensively explain modeling concepts and methods. [31] conducted a comparative study between ANN, an adaptive neuro-fuzzy inference system (ANFIS), and gene expression programming (GEP) for predicting GT at four different depths (5, 10, 50, and 100 cm) at 31 studied stations in Iran. In this process, the geographical information (latitude, longitude, and altitude) and periodicity component (the number of months) were applied as inputs in the used models. The results showed that the utilization of the ANFIS model enhanced the prediction accuracy of the soil temperature for all 31 stations. After the evolution of the vast number of machine learning algorithms, several studies used SVR, RFR, and other machine-learning models such as genetic algorithm (GA) and a gradient-boosting decision tree (GBDT) for predicting the GT. Recently, deep learning- (DL) based recurrent neural network (RNN) models were used to predict short- and long-term data. For instance, Ref. [32] employed an autore- gressive integrated moving average (FARIMA) model to predict the GT, and the model’s performance compared with gene expression programming (GEP) FFBPNN. The results indicate FARIMA outperformed the FFBPNN and GEP methods in terms of prediction accuracy; however, FARIMA’s predictions are relatively inadequate for acute GT values. Previous literature [33] utilized RNN-DL-based LSTM, GRU, ANN, and extreme learning machine models (ELM) to predict GT at 5, 10, and 15 cm depths. The results show that the model predictions are not signiﬁcantly different between the RNN models. Even though the DL-RNN-based models still produce better prediction, there is some limitation with the time series-based RNN models acclaimed from the previous studies [14,33,34]. Those limitations are (1) that the time series-based RNN models struggle to predict the missing information over a long time series, and (2) that these kinds of time series models are good enough to produce more accuracy in long-time predictions with more signiﬁcant historical inputs, but are ﬂawed in the case of short-time predictions with fewer data. Such Atmosphere 2023, 14, 68 4 of 30 limitations mentioned above have led the researchers adopt simple ML models to predict the GT at different depths in the short term. Since there is a knowledge gap in utilizing ML models for predicting GT at different depths, the current study is intended to build an optimal model with less complexity in short-term prediction. The present study utilizes a statistical model (multiple linear regression (MLR)) and four different ML models (SVR, RFR, MLP, and XGBoost (XGB)) for predicting GT. In addition, numerous metrological parameters are available from the weather stations. The use of more input parameters increases the complexity of the model. Using the proper input parameters creates room to learn more quickly and accurately about the models. Unlike statistical models, selecting the suitable parameter and inputting it into the ML models increases accuracy and training time. The primary objectives of this study are as follows: (1) Building ML learning models for hourly GT prediction at different depths (5 cm, 10 cm, 20 cm, and 30 cm) with the optimum hyperparameter tuning with less complexity. (2) Comparing the accuracy of all the models using two different datasets: (1) selected number of parameters and (2) collected dataset with all variables. (1) Building ML learning models for hourly GT prediction at different depths (5 cm, 10 cm, 20 cm, and 30 cm) with the optimum hyperparameter tuning with less complexity. (1) Building ML learning models for hourly GT prediction at different depths (5 cm, 10 cm, 20 cm, and 30 cm) with the optimum hyperparameter tuning with less complexity. (2) Comparing the accuracy of all the models using two different datasets: (1) selected number of parameters and (2) collected dataset with all variables. (2) Comparing the accuracy of all the models using two different datasets: (1) selected number of parameters and (2) collected dataset with all variables. p (3) Comparing the statistical and ML model performance for the prediction of GT. p (3) Comparing the statistical and ML model performance for the prediction of GT. 2. Materials and Methods 2.1. Site Description and Data Collection 2.1. Site Description and Data Collection The current study examines the GT of Busan, South Korea’s second-most populous metropolitan city after Seoul, with over 3.4 million inhabitants. South Korea is in the temperate climate zone of the mid-latitude (34–43◦N) geographical area, with distinct seasons of spring, summer, autumn, and winter. The metrological data were collected from the Busan metrological station (35◦06′19.0′′ N, 129◦01′55.8′′ E). Busan’s daily mean temperature is 15.0 ◦C, average high temperature is 19.2 ◦C, average low temperature is 11.7 ◦C, average relative humidity is 63.3%, and average precipitation is 1576.7 mm as recorded from the last 30 years’ metrological data. The data collected from 1 January 2017 to 1 July 2021 (4.7 years) were used for modeling and validation. Because climate change has recently had a signiﬁcant impact on agriculture, recent data with short intervals were chosen for the current study. Overall, 13 independent variables and 5 GTs with different depths (GT, GT_5, GT_10, GT_20, and GT_30) as response variables were collected at an interval of 1 h. Figure 1 depicts the location of the Busan metrological station, and Table 1 depicts the collected parameters with a basic description. Figure 1. The location and geographical information of the metrological station in Busan (Highlighted in map), South Korea (35◦06′19.0′′ N, 129◦01′55.8′′ E). Figure 1. The location and geographical information of the metrological station in Busan (Highlighted in map), South Korea (35◦06′19.0′′ N, 129◦01′55.8′′ E). 5 of 30 Atmosphere 2023, 14, 68 Table 1. Statistical summary of the GT associated parameters. 2.1. Site Description and Data Collection S.No Variable Name Nomenclature Unit Skewness Kurtosis Mean SD Min Max 1 Temperature Te (◦C) −0.25 −0.64 15.04 8.42 −12.2 36.1 2 Precipitation Pr (mm) 21.27 708.72 2.03 1.47 0 78.8 3 Wind speed WS (m/s) 1.09 2.59 3.16 1.76 0 18.1 4 Wind direction WD (Azimuth) −0.04 −1.41 181.44 111.16 0 360 5 Humidity Hu (%) −0.15 −0.85 62.29 20.87 7 100 6 Vapor pressure VP (hPa) 0.59 −0.75 12.9 8.6 0.6 36.3 7 Dew point temperature DT (◦C) −0.42 −0.66 7.15 11.68 −29 27.3 8 Local atmospheric pressure AP (hPa) −0.18 −0.05 1007.5 7.41 952.2 1027.1 9 Barometric pressure BP (hPa) −0.16 −0.12 1015.8 7.66 959.7 1036 10 Sunshine SS (h) −0.27 −0.8 0.55 0.33 0 1 11 Solar radiation SR (MJ/m2) 0.83 1.25 1.17 0.74 0 4.77 12 Mid-lower cloud cover Cl (decile) 0.78 −1.05 2.82 3.54 0 10 13 Visibility Vi (10 m) 1.37 1.62 2019.5 1178 14 5000 14 Ground temperature GT (◦C) 0.44 0.29 17.18 11.68 −10 66.3 15 5 cm underground temperature GT_5 (◦C) 0.1 −0.84 16.78 8.97 −4.4 44.7 16 10 cm underground temperature GT_10 (◦C) 0.04 −1.03 16.61 8.43 −0.5 40.7 17 20 cm underground temperature GT_20 (◦C) 0.02 −1.16 16.72 7.84 1.5 35.4 18 30 cm underground temperature GT_30 (◦C) 0.02 −1.21 16.68 7.46 2.4 32.6 Table 1. Statistical summary of the GT associated parameters. 2.2. Used Data Graphical representation of GT, GT_5, GT_10, GT_20, and GT_30 prediction using machine- learning algorithms. Figure 3. Graphical representation of GT, GT_5, GT_10, GT_20, and GT_30 prediction using machine- learning algorithms. Figure 3. Graphical representation of GT, GT_5, GT_10, GT_20, and GT_30 prediction using machine- learning algorithms. 2.2. Used Data As mentioned above, a dataset consisting of 13 independent variables and 5 dependent variables was collected from the metrological stations. Since it is an open dataset, missing values should also be considered before using the data for training/testing. In order to avoid such issues, exploratory data analysis (EDA) procedures were followed [10,11,29]. EDA is an approach to summarize the data by taking their main characteristics and visualizing them with proper representations. EDA focuses more narrowly on checking assumptions required for model ﬁtting and hypothesis testing, and handling missing values and making transformations of variables as needed [35]. Later, the method of cleaning the missing values is described in this study. The basic description of the dataset is shown in Table 1 in a detailed manner with mean, standard deviation, minimum, maximum, kurtosis, and skewness known through the basic interpretation of the data. Figure 2 explains the graphical description of the GT using data from various depths. g g As mentioned in the objective for this study, two different datasets were used for training/testing the model. To achieve the desired accuracy with less computational cost, a reference for selecting the parameters to be recorded must be generated. Selecting an appropriate input source is a key in the ML technique to obtain superlative productivity from the algorithms [1,11]. As a result, the current study employs the Spearman rank correlation coefﬁcient approach to extract the best features, a widely used method for investigating relationships between attributes. The heat correlation map using the Spear- man rank correlation coefﬁcient for the GT, GT_5, GT_10, GT_20, and GT_30 is shown in Figure 3. According to the correlation map, highly correlated variables have been selected for the ﬁrst dataset, so that the current study used two datasets for GT-modeling purposes, named M1 (“Te, VP, DT, AP, BR, and SR” as the independent variables) and M2 (collected all independent variables). Atmosphere 2023, 14, 68 6 of 30 Figure 2. Variations in GT at 5 cm, 10 cm, 20 cm, and 30 cm. Figure 3. Graphical representation of GT, GT_5, GT_10, GT_20, and GT_30 prediction using machine- learning algorithms. 2 3 M d l D l t Figure 2. Variations in GT at 5 cm, 10 cm, 20 cm, and 30 cm. Figure 2. Variations in GT at 5 cm, 10 cm, 20 cm, and 30 cm. Figure 3. 2.3.2. Multi-Layered Perceptron 2.3.2. Multi-Layered Perceptron The multi-layer perceptron (MLP) is a fundamental, classical type of neural network model for overcoming linear models, because linear models can extract attributes’ relation- ships [10]. MLP models have been developed to solve nonlinearity problems by combining back-propagation (BP) techniques with perceptron. Because the MLP has a three-layer structure (an input layer, a hidden layer, and an output layer), weights have been generated for each layer. The weights are updated until the optimal results are given to the output layer, which is the main advantage of BP techniques [38,39]. Finally, the weighted sum of the input layer is converted as output using a nonlinear activation function. This proce- dure entails lowering the output layer’s prediction error. The neural network technique is essential for data classiﬁcation and regression because it can adapt and adjust to the data regardless of the primary mode’s special functional or distributional requirements. The MLP with BP can be expressed as follows [38,39]: yp= f0 " n ∑ j=1 wkjfh m ∑ j=1 wjixi+wjb ! +wkb # (2) (2) where yp is the output (predicted); n is the number of output neurons; f0 is the activation function for the output neuron; wkj is the connecting neuron of the hidden and output layers’ weight; fh is the activation function of the hidden neuron; m is the number of hidden neurons; wji is the weight for the connecting neuron of input and hidden layers; xi is the input variable; wjb is the bias for the hidden neuron; and wkb is the bias for the output neuron. As mentioned earlier, during BP training, the bias and weights were generated until the threshold levels were reached. Such threshold-level training methods reduce the evaluation metrics of the predicted values. So, the updated weight can be expressed as [38,39] W∗ X= WX −a(∂Error ∂WX ) (3) (3) where WX* is the updated weight, WX is the old weight, a is learning rate, and ∂Error is the derivative of error with respect to the weight. The threshold method to stop the training used in this study is root means square error (RMSE), which is also called an error function. According to the model, training should be stopped if minimum RMSE is found during the training time. That error function can be expressed as E = p ∑ p=1 Ep = p ∑ p=1 . 2.3.2. Multi-Layered Perceptron n ∑ k−1 yp −ya 2 (4) (4) where E is the error of the input patterns and Ep is the square difference between the actual value and predicted value. Ya is the actual observed data, whereas yp is predicted data. 2.3.1. Multiple Linear Regression 2.3.1. Multiple Linear Regression Multiple linear regression (MLR) is a more extended version of simple linear regression (SLR). It is popular among other statistical models because of its candid nature, calculation interpretability, and understanding of outlines or anomalies within the dependent vari- ables [36,37]. These models are widely used in various ﬁelds, including business foresting, power consumption, energy consumption prediction, weather forecasting, and so on. MLR models make the following assumptions: g p MLR can be mathematically expressed by the following formula [36,37]: Y = a0+a1X1+a2X2+ . . . + aiXi + ε (1) (1) Y = a0+a1X1+a2X2+ . . . + aiXi + ε where Y is the dependent variable; a0 is the intercept or constant of the model; X1, X2, . . . , Xi are the independent variables; a1, a2, . . . , ai are the regression coefﬁcients; and the ε is the noise or random error of the model. 7 of 30 7 of 30 Atmosphere 2023, 14, 68 2.3.4. Support Vector Regression Vapnik proposed support vector regression (SVR) as a subset of support vector ma- chines (SVMs) in 1992 [43,44]. Because SVMs are statistical, root-based algorithms, they were initially used only to solve classiﬁcation-related problems. The general concept be- hind SVMs is to map the original data, X, into a high-dimensional feature space, F, using a nonlinear mapping function and then build an optimal hyperplane in the new space. During that time, SVMs had some important hyperparameters such as kernel (which aids in the discovery of a hyperplane in higher dimensional space), hyperplane (the middle line that separates the classes), and a decision boundary (minimum and maximum boundary lines). SVR was later developed as a regression-solver by Drucker and Vapnik in 1996 using the same concept as SVMs [43,44]. The structural risk minimization (SRM) principle is used to train the SVR, which uses a hypothesis space of linear functions in a high-dimensional feature space. A nonlinear mapping function maps the SVR input into a high-dimensional feature space. That is why SVR is so popular among ML models for solving regression problems. If y is the output variable, then the SVR prediction process can be expressed as follows [44–46]: y = f(x)= ω.ϕ(x)+b (6) (6) where x is the input of the datasets; ω and b are the parameter vectors; and ϕ(x) is the mapping function assumed by the SVR. Here, y can have unlimited prediction possibilities if it has a multidimensional dataset. So, a limitation for the constraints proposed to determine the optimization issues [44–46] could be expressed as Minimize : 1 2 ω2+C n ∑ i=1 (ξi + ξ∗ i ) Subject to    yi −(ω.ϕ(x)+b) ≤ε+ξi (ω.ϕ(x)+b) −yi ≤ε+ξ∗ i ξi, ξ∗ i ≥0, i = 1, . . . , n (7) (7) (7) where ε is the minimum and maximum margin line/sensitivity zone of the hyperplane; ξ and ξi* are the slack variables that measure the training errors which are subjected to ε; and C is the positive constant. To minimize the error between the sensitive zones of the hyperplane, the slack variables were utilized. 2.3.3. Random Forest Regression Random forest regression is a supervised learning algorithm that performs regression using the ensemble learning method. A technique that combines predictions from multiple machine-learning algorithms to make a more accurate prediction than a single model is known as ensemble learning [1,30]. Similarly, RFR is an ensemble learning technique that consists of aggregating multiple decision trees (DT), resulting in a lower variance compared to single decision trees. The out-of-bags (OOB) error is used to estimate the generalization error of RFR for training points that are not contained in the bootstrap training sets (about one-third of the points are left out in each bootstrap training set). Because OOB is indis- tinguishable from N-fold cross-validation, the process of OOB estimation is responsible for its non-overﬁtting nature. Random forest is based on the bagging technique, which aids in the optimization of the algorithm’s performance. It performs well “out-of-the-box,” requiring no hyperparameter tuning, and outperforms linear algorithms, making it a viable Atmosphere 2023, 14, 68 8 of 30 8 of 30 option [40–42]. Furthermore, random forest is relatively fast and robust, and it can display feature importance, which can be quite useful. The expression of RFR can be represented as following equation [40–42]: option [40–42]. Furthermore, random forest is relatively fast and robust, and it can display feature importance, which can be quite useful. The expression of RFR can be represented as following equation [40–42]: Y = 1 M M ∑ i=1 H(Ti) where H(Ti) from DTR (5) (5) where M is the total trees; Y is the final output; H (Ti) is a sample in the training set; and Ti is the one sample among all the n subsets which encompasses the total amount of training data. 2.3.4. Support Vector Regression The sensitive zones can also be expressed using Lagrange multipliers, and the optimization techniques to solve the dual nonlinear problem can be rewritten as the following equation [44–46]: min : 1 2 n ∑ i=1 n ∑ j=1 (ai −a∗ i ) aj −a∗ j K+ε n ∑ i=1 (ai+a∗ i ) − n ∑ i=1 yi(ai −a∗ i ) Subject to    n ∑ i=1 (ai −a∗ i )= 0 0 ≤ai, a∗ i ≤C, i = 1, . . . , n (8) (8) where ai and ai* are the Lagrange multipliers which are subject to ε, and K is the kernel function. The kernel function uses the kernel trick to solve nonlinear problems using a linear classiﬁer. Generally, linear, radial basis function (RBF), polynomial, and sigmoid are the used kernel functions of SVR models [44–46]. The current study chose RBF as the kernel Atmosphere 2023, 14, 68 9 of 30 function to optimize SVR during simulation after a random test of other kernel functions. The RBF kernel function can be expressed as the following equation: function to optimize SVR during simulation after a random test of other kernel functions. The RBF kernel function can be expressed as the following equation: K(I, j)= exp(−γ xi −xj 2 (9) (9) (9) where, γ is referred to as the structural parameter of the RBF kernel function. Finally, the decision function of SVR can be expressed as [45,46] where, γ is referred to as the structural parameter of the RBF kernel function. Finally, the decision function of SVR can be expressed as [45,46] f(xi) = n ∑ i=1 (ai −a∗ i )K(xi, xk)+b (10) (10) 2.3.5. Extreme Gradient Boosting Extreme gradient boosting (XGBoost) is a kind of boosting algorithm proposed by Chen and Guestrin in 2011 and later optimized by several researchers [47–49]. XGBoost functions based on boosting-tree algorithms. Unlike the DT, RFR, and other boosting-tree- based algorithms, XGB combines the results of the weak learner group and the strong learner group, then produces cumulative results. Only the ﬁrst derivative information is used in traditional boosting-tree models. It is difﬁcult to implement distributed training when training the nth tree because the residual of the previous n −1 trees is used. XGBoost performs a second-order Taylor expansion on the loss function and automatically uses the CPU’s multithreading for parallel computing [48,49]. First, the initial learner is ﬁtted to the interior environment of input data. The second model is then ﬁtted with the residual of the ﬁrst learner in order to improve the previous learner’s weak learning ability. Until the threshold is satisﬁed, the accumulation process is repeated. The algorithm’s ﬁnal predictive result is calculated by adding the predictions of all learners. Since XGB algorithms use optimized resources and optimal speed calculation through parallel computing ability, they are fast learners and successors of boosting-family accuracy. As mentioned above, the ﬁrst step of prediction is expressed as follows [40,47–49]: f (t) i = t ∑ k=1 fk(xi) = f (t−1) i + fi(xi) (11) (11) where fi(xi) is the learner in step t, f (t) i and f (t−1) i are the predicted values in steps t and t −1, and xi is the input observation. In order to prevent the over-ﬁtting of the training data from affecting the calculation speed of the model, the XGB model uses the following equation to evaluate the model [40,47,48]: Objt = n ∑ k=1 l y− i , yi + t ∑ k=1 Ω( fi) (12) (12) where l represents the loss function, n represents the amount of input data, and Ωrepresents the regular term, which is deﬁned as follows [40,47,48]: Ω( f ) = γT + 1 2λ\|\|ω \|\|2 (13) Ω( f ) = γT + 1 2λ\|\|ω \|\|2 (13) (13) where ω represents the leaf score, λ is the hyperparameter of the regular term, and γ represents the minimum loss that the leaf node needs to split. 2.4. Modeling Concept 2.4. Modeling Concept 2.4. Modeling Concept The current part explains the thought framework of the current study; the graphical representation of the study is shown in Figure 3 as a step-by-step ﬂow chart. Firstly, the metrological data for every 1 h was collected and stored. Overall, 18 variables, including the dependent variables, were stored and underwent preprocessing/EDA analysis. As the next step, the missing data for every attribute were checked. In general, missing data Atmosphere 2023, 14, 68 10 of 30 10 of 30 are highly disputed for most ML modeling ﬁelds in metrological studies [50–52]. Various researchers have already conducted work on solving the missing data. Most of the studies remove the missing data and model their datasets; such models are not reliable enough when it comes to modeling. Some statistical theories, such as linear interpolation, data imputation, the k-nearest neighbor algorithm, etc., are available to resolve the missing data dispute [50–52]. p The current study adopts regression imputation, which is familiar and more competent for solving the missing data dispute. Regression imputation is similar to single linear regression, but can also be used for the multiple missing variables. If data are missing at random (MAR), regression imputation, also known as conditional mean imputation, replaces each missing value with a predicted value based on a regression model [50]. A general regression procedure is divided into two stages: ﬁrst, a regression model is constructed using all of the available complete observations, and then missing values are estimated using the constructed regression models. The current study uses the “sklearn” (python-based library) to impute the missing data. Both independent data and dependent data are imputed separately in order to avoid the pre-training errors. Next, the distribution of every attribute is considered through skewness and kurtosis. As mentioned earlier, since the current study intends to state and art of the ML model’s performance, the Spearman rank correlation test was examined to choose the best features. The heat correlation map of every response variable is shown in Figures 4 and 5. Figure 4. (A) Pearson heat correlation map of GT; (B) Pearson heat correlation map of GT_5. Figure 4. (A) Pearson heat correlation map of GT; (B) Pearson heat correlation map of GT_5. The next step of preprocessing is the normalization of the data considered. Data normalization is a standard procedure if attributes are in different ranges. A different range of variables complicates the learning of any ML algorithm [1,11,37]. 2.4. Modeling Concept The results Atmosphere 2023, 14, 68 12 of 30 12 of 30 were observed; as expected, the ﬁne-tuned model results overcame the default models in all the predictions. Thus, the current study chose the tuned models for modeling GT predictions. The list of tuned hyperparameters is shown clearly in Table 2. Finally, the model training and testing results were evaluated using three evaluation metrics: mean absolute error (MAE), root mean square error (RMSE), and coefﬁcient of determination (R2) methods. Those are evaluation metrics used often to understand model performance [1,11,37,39]. were observed; as expected, the ﬁne-tuned model results overcame the default models in all the predictions. Thus, the current study chose the tuned models for modeling GT predictions. The list of tuned hyperparameters is shown clearly in Table 2. Finally, the model training and testing results were evaluated using three evaluation metrics: mean absolute error (MAE), root mean square error (RMSE), and coefﬁcient of determination (R2) methods. Those are evaluation metrics used often to understand model performance [1,11,37,39]. MAE =∑n i=1\|yi−pi\| n (15) RMSE = s ∑n i=1 (y i−pi)2 n (16) R2 = 1 − ∑n i =1(yi −pi)2 ∑n i =1 yi −1 n ∑n i =1 yi 2 (17) (15) (16) (17) Here, i is the variable; N is number of non-missing data points; yi is actual observed data; and pi is predicted data. Here, i is the variable; N is number of non-missing data points; yi is actual observed data; and pi is predicted data. Table 2. The range of critical hyperparameters tuned during the prediction. Table 2. The range of critical hyperparameters tuned during the prediction. Table 2. The range of critical hyperparameters tuned during the prediction. 2.4. Modeling Concept Algorithms Hyperparameters Distribution (Range) Multiple linear regression (MLR) - - Multilayered perceptron (MLP) Number of hidden layers * Ud (1, 4) Number of hidden neurons Ud (1, 200) Learning rate Adaptive Solver Adam Activation function Relu Support vector regression (SVR) Kernel Radial-basis function C Ud (1, 1000) Gamma 1 Epsilon 0.1 Random forest regression (RFR) Number of trees Ud (10, 250) Minimum number of observations in a leaf Ud (1, 30) Number of variables used in each split Ud (1, 4) Maximum tree depth Ud (1, 100) XGBoost (XGB) Max depth of a tree Ud (1, 10) Learning rate Ud (0.05, 0.1) Sample ratio of training data 2 Sample ratio of features 2 Alpha 0.2 Number of estimators Ud (100, 1000) * Ud stands for uniform discrete random distribution from a to b. The models used for this study, except MLR, were developed in a python environment (Version 3.7), and the MLR model was developed in IBM SPSS Statistics IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY, USA. The graphs were drawn in Originpro (Version 9, OriginLab, Northampton, MA, USA); likewise, all other statistical analysis was conducted in IBM SPSS Statistics (version 26, IBM, Armonk, NY, USA). 2.4. Modeling Concept In order to achieve better efﬁciency and higher accuracy, mapping the data to a speciﬁc range will avoid the consequences aforementioned. Minimum-maximum (min-max) normalization is a common normalization technique used in machine learning which may rescale the variables from the range of −1 to 1 or 0 to 1. The current study employed a min-max scalar with the range of −1 (minimum) to 1 (maximum) to rescaling; this could be mathematically expressed by [1,11,37] xnor = 2 ∗(x −x min) (x max −xmin) −1 (14) (14) Atmosphere 2023, 14, 68 11 of 30 11 of 30 where xnor is the normalized data, Xmax is the maximum of original data, xmin is the minimum of original data, and x is the original data. Next, training and testing data were partitioned to feed the training data for the ML algorithms. Still, there is no proper deﬁnition for the splitting of training and testing data. Most often, three ratios are commonly followed by ML practitioners: 70:30 (training: validation), 80:20, or 90:10 for building ML models. The current study chooses 70% data for training and 30% for validation. Figure 5. (A) Pearson heat correlation map of GT_10; (B) Pearson heat correlation map of GT_ (C) Pearson heat correlation map of GT_30. Figure 5. (A) Pearson heat correlation map of GT_10; (B) Pearson heat correlation map of GT_20; (C) Pearson heat correlation map of GT_30. In the next step, utilizing the maximum efﬁciency of any ML model starts with tuning its internal parameters, which is called hyperparameter tuning. Every ML algorithm has its internal structure; for instance, MLP has hidden layers, hidden neurons, learning rates, solvers, and activation functions within its architecture [1,40,42,53]. Such a phenomenon directly inﬂuences model performances. Therefore, ﬁnding the optimal hyperparameters according to the given input data feed increases the model’s performance [1,40,42,53]. The current study ﬁne-tuned every model with its hyperparameter according to our dataset so the maximum efﬁciency of the model could be utilized. Initially, every model was tested with a 70% training dataset without hyperparameters (default values) and validated with 30% of the validation dataset. Later, a “grid search” method was used to ﬁnd out the optimal hyperparameters, then ﬁne-tuned according to the best parameters; every model was trained with the optimized model and validated with the same datasets. 3.1. Dataset Performance In this section, the testing results of the M1 and M2 datasets are compared to make the performance analysis. Since one of the objectives of the current study is to ﬁnd out the optimal input dataset to avoid the complexity of the model, the current study analyzed the performance of available datasets. The highest performance between M1 and M2 testing data was obtained from the XGB in GT temperature prediction (MAE = 1.063; RMSE = 1.679; R2 = 0.978) while using the M1 dataset. For GT prediction, all the models outperformed using the M1 dataset rather than M2 datasets. The lowest performance was obtained in MLR (MAE = 3.205; RMSE = 4.131; R2 = 0.872). The performance results of all the models for various depth GT predictions are shown in Table 3. The overall results show that ML-based models performed better than the MLR model while using M1 and M2 datasets. When comparing the performance between the two datasets, the M1 dataset has minimum error and maximum correlation over the M2 dataset. For instance, the XGB result for MAE is 6.92% lower, for RMSE is 5.19% lower, and for R2 is 0.20% higher using the M1 dataset than the M2 dataset. Likewise, the least result model’s (MLR) MAE is 3.23% lower, RMSE is 2.09% lower, and R2 is 0.69% higher while using the M1 dataset rather than the M2 dataset. The highest difference was found using the SVR model (MAE is 13.41% lower, RMSE is 9.96% lower, and R2 is 1.63% higher) while using the M1 dataset for GT prediction. g g p For the GT_5 predictions, the performance of the models followed a similar pattern to the GT results. As with GT results, the highest performance between M1 and M2 testing data was obtained from the XGB in GT_5 predictions (MAE = 0.887; RMSE = 1.263; R2 = 0.979) when using M1 dataset. The highest-ranking model XGB’s MAE is 10.94% lower, RMSE is 11.80% lower, and R2 is 0.51% higher when compared with the M2 dataset performance, followed by the RFR (MAE is 8.78% lower, RMSE is 12.84% lower, and R2 is 1.04% higher) model performance using M1 dataset. The MLR model has a difference in MAE that is 3.47% lower, RMSE that is 4.68% lower, and R2 that is 0.88% higher using M1 datasets with the least prediction rank, like the GT prediction results. 3. Results The results included dataset performance, model comparison, and model performance. The evaluation results were categorized by dataset and model performance during the training and validation phases. In part-named dataset performance, the results obtained using M1 and M2 datasets were deliberated. For model performance, the percentage Atmosphere 2023, 14, 68 13 of 30 13 of 30 difference in all models’ results was discussed, and the percentage difference between the models was discussed in the model performance part. difference in all models’ results was discussed, and the percentage difference between the models was discussed in the model performance part. 3.1. Dataset Performance Unlike GT prediction, the biggest percentage results were obtained in MLP’s prediction, with MAE 14.78% lower, RMSE 14.33% lower, and R2 1.59% higher. For the GT_10 prediction results, the performance of the models follows a similar pattern to the GT_5 results. The highest-ranking model XGB’s MAE is 12.41% lower, RMSE is 14.15% lower, and R2 is 0.61% higher when compared with the M2 dataset performance, followed by the RFR (MAE is 8.00% lower, RMSE is 9.80% lower, and R2 is 0.62% higher) model performance using M1 dataset. The MLR model has a difference in MAE of 8.15% lower, RMSE of 9.80% lower, and R2 of 1.43% higher using M1 datasets, with the lowest prediction rank of the GT prediction results. As with GT_5 predictions, the most signiﬁcant percentage results were obtained in MLP’s prediction, with MAE 22.17% lower, RMSE is 20.50% lower, and R2 is 2.92% higher between M1 and M2 datasets. g Unlike the GT, GT_5, and GT_10 results, the GT_20 prediction results obtained higher performance using the M2 dataset rather than M1. As with the other predictions, the XGB outperformed other models, but the M2 dataset produced a better prediction than the M1 prediction’s MAE, except the MAE values. Other error metrics were lower in the M1 dataset. The percentage difference between XGB results are as follows: MAE is 0.24% lower, RMSE is 1.551% lower, and R2 is 0.10% higher in M2. Except for XGB’s MAE performance, the performance ranking pattern is similar to the GT_10 predictions. For example, the second rankings were obtained in RFR (MAE is 10.59% higher, RMSE is 11.14% higher, and R2 is 0.30% lower in M1 compared with M2), whereas the lowest performance was obtained during MLR prediction (MAE is 11.46% lower, RMSE is 10.37% lower, and R2 is 2.61% higher in M1). The highest percentage difference was found in MLP predictions (MAE is 26.77% lower, RMSE is 26.26% lower, and R2 is 4.51% higher) as for the GT, GT_5, and GT_10 predictions. Atmosphere 2023, 14, 68 14 of 30 14 of 30 Table 3. Training and testing evaluation metrics of the models in modeling GT, GT_5, GT_10, GT_20, and GT_30 with M1 and M2 datasets. 3.1. Dataset Performance GT M1 M2 Models Training Testing Training Testing MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MLR 3.204 4.151 0.874 3.205 4.131 0.872 3.316 4.238 0.869 3.312 4.219 0.866 MLP 1.616 2.420 0.957 1.668 2.475 0.954 1.865 2.764 0.944 1.813 2.699 0.945 RFR 4.158 5.991 1.0 1.234 2.006 0.969 0.531 0.869 0.994 1.372 2.170 0.964 SVR 1.791 2.971 0.935 1.815 2.955 0.934 1.865 2.764 0.944 2.096 3.282 0.919 XGB 0.669 0.915 0.993 1.063 1.679 0.978 0.805 1.109 0.991 1.142 1.771 0.976 GT_5 M1 M2 Models Training Testing Training Testing MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MLR 1.916 2.501 0.922 1.917 2.505 0.920 1.988 2.618 0.915 1.986 2.628 0.912 MLP 1.360 1.807 0.959 1.390 1.854 0.956 1.654 2.168 0.941 1.631 2.164 0.941 RFR 3.942 5.443 1.0 1.080 1.555 0.969 0.453 0.704 0.993 1.184 1.784 0.959 SVR 1.457 2.011 0.949 1.480 2.031 0.948 1.654 2.168 0.941 1.672 2.258 0.935 XGB 0.594 0.789 0.992 0.887 1.263 0.979 0.772 1.078 0.985 0.996 1.432 0.974 GT_10 M1 M2 Models Training Testing Training Testing MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MLR 1.832 2.332 0.923 1.825 2.315 0.923 1.982 2.513 0.911 1.987 2.510 0.910 MLP 1.352 1.755 0.956 1.380 1.803 0.953 1.768 2.275 0.927 1.773 2.268 0.926 RFR 3.786 5.197 1.0 0.954 1.381 0.972 0.395 0.601 0.994 1.037 1.531 0.966 SVR 1.417 1.894 0.949 1.429 1.900 0.948 1.768 2.275 0.927 1.790 2.322 0.923 XGB 0.511 0.674 0.993 0.741 1.025 0.985 0.655 0.906 0.988 0.846 1.194 0.979 GT_20 M1 M2 Models Training Testing Training Testing MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MLR 1.893 2.405 0.906 1.900 2.395 0.905 2.136 2.670 0.884 2.146 2.672 0.882 MLP 1.298 1.684 0.954 1.343 1.735 0.950 1.882 2.418 0.905 1.834 2.353 0.909 RFR 3.784 5.083 1.0 0.658 1.008 0.983 0.230 0.411 0.997 0.595 0.907 0.986 SVR 1.379 1.843 0.944 1.402 1.848 0.943 1.801 2.814 0.906 1.910 2.474 0.899 XGB 0.286 0.368 0.997 0.416 0.551 0.995 0.316 0.362 0.997 0.415 0.560 0.994 GT_30 M1 M2 Models Training Testing Training Testing MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MAE RMSE R2 MLR 1.969 2.503 0.887 1.985 2.501 0.886 2.195 2.746 0.864 2.208 2.753 0.862 MLP 1.341 1.761 0.944 1.399 1.828 0.939 1.812 2.370 0.899 1.875 2.412 0.894 RFR 3.716 4.949 1.0 0.491 0.857 0.986 0.134 0.253 0.998 0.348 0.637 0.992 SVR 1.421 1.916 0.934 1.457 1.935 0.932 1.928 2.545 0.883 1.946 2.548 0.882 XGB 0.189 0.243 0.998 0.280 0.367 0.997 0.165 0.215 0.999 0.222 0.289 0.998 Table 3. 3.2. Model Performance Based on the dataset performance outcomes, the M1 input dataset assists in achieving superior performance from all models for GT predictions except GT_30 predictions. Overall prediction results of both training time and testing time are satisﬁed in the XGB model. The performance of the ﬁve models for GT, GT_5, GT_10, GT_20, and GT_30 is shown in Figures 6–10. g For the GT prediction training time, XGB results are comparatively higher (MAE = 0.669, RMSE = 0.915, and R2 = 0.993) followed by the MLP (MAE = 1.616, RMSE = 2.42, and R2 = 0.957). Other than those models, SVR and MLR performed third and fourth, respectively. Surprisingly, the RFR regression model evidently shows over-ﬁt during the testing time, not only in GT prediction but also in the rest of the predictions (refer to Table 3). Even though RFR performed follow-through of XGB models during the testing time, the reliability is under suspicion due to the over-ﬁt during the train- ing time. Because validation assessment is essential, as mentioned above, the current study considered testing results as the model’s performance. Thus, during the time of testing, XGB outperformed other models (MAE = 1.063, RMSE = 1.679, and R2 = 0.978). RFR’s performance followed that of XGB (MAE = 16.086% high, RMSE = 19.475% high, and R2 = 0.920% low), and the third, fourth, and ﬁfth performances were from MLP (MAE = 56.914% high, RMSE = 47.409% high, and R2 = 2.453% low), SVR (MAE = 70.743% high, RMSE = 75.997% high, and R2 = 4.5% low), and MLR (MAE = 201.505% high, RMSE = 146.039% high, and R2 = 10.838% low), accordingly. The MLR model suffered to produce a more competitive outcome than the ML-based models for the GT predictions as from the results. The comparison results between the actual values and predicted values are clearly shown in Figure 6. By GT prediction results, the GT_5 followed an akin performance pattern. XGB per- formance outperformed during the training and testing time of other models. The top performance was achieved from the XGB model for the GT_5 predictions during the train- ing time (MAE = 0.594; RMSE = 0.789; R2 = 0.992) and testing time as well (MAE = 0.887; RMSE = 1.263; R2 = 0.979). However, according to the training results of GT_5 predictions, MLP, SVR, and MLR retained the second, third, and fourth positions, respectively. 3.1. Dataset Performance Training and testing evaluation metrics of the models in modeling GT, GT_5, GT_10, GT_20, and GT_30 with M1 and M2 datasets. GT Atmosphere 2023, 14, 68 15 of 30 15 of 30 Contradictory to the other GT predictions, the GT_30 predictions was outperformed when using the M2 dataset. Most models predict the GT_30 with more accuracy when using the M2 dataset. Take, for example, that XGB (MAE is 26.13% lower, RMSE is 26.99% lower, and R2 is 0.10% higher) and RFR (MAE is 41.09% lower, RMSE is 34.54% lower, and R2 is 0.60% higher) outperformed when the input is the M2 dataset. Interestingly, the MLR performance pattern was similar to the other depth predictions. The M1 input dataset ﬁts the MLR model to get the optimum output (MAE is 10.10% lower, RMSE is 9.15% lower, and R2 is 2.78% higher); however, the performance of the MLR model with M1 or M2 datasets remains the lowest prediction when compared with the ML-based models. Even though the MLP (MAE is 25.39% lower, RMSE is 24.21% lower, and R2 is 5.03% higher) and the SVR (MAE is 25.13% lower, RMSE is 24.06% lower, and R2 is 5.60% higher) models predict the GT_30 effectively using the M1 dataset, the highest prediction was attained by XGB, followed by RFR. y Summarization of all models’ prediction performance during the training phase and validation phase with both M1 and M2 datasets is shown comprehensively in Table 3. 3.2. Model Performance Ac- cording to the testing results, RFR, MLP, SVR, and MLR attained second (21.758% higher MAE, 23.119% higher RMSE, and 1.021% lesser R2), third (56.708% higher MAE, 46.793% higher RMSE, and 2.349% lesser R2), fourth (66.856% higher MAE, 60.807% higher RMSE, and 3.167% lesser R2), and ﬁfth (116.122% higher MAE, 98.337% higher RMSE, and 6.026% lesser R2), respectively (refer to Table 3). As mentioned above, the XGB model attained su- perior performance during the GT_10 predictions. The results of GT_10 prediction training and testing time were comprehensively explained in Table 3. According to that, the XGB performance overcame the MLP (second), SVR (third), and MLR (fourth) performances, Atmosphere 2023, 14, 68 16 of 30 respectively, during the training time. The difference obtained when compared to the XGB model performance were RFR (28.745% higher MAE, 34.731% higher RMSE, and 1.320% lesser R2), MLP (86.234% higher MAE, 75.902% higher RMSE, and 3.248% lesser R2), SVR (92.847% higher MAE, 85.365% higher RMSE, and 3.756% lesser R2), and MLR (146.289% higher MAE, 125.854% higher RMSE, and 6.295% lesser R2). respectively, during the training time. The difference obtained when compared to the XGB model performance were RFR (28.745% higher MAE, 34.731% higher RMSE, and 1.320% lesser R2), MLP (86.234% higher MAE, 75.902% higher RMSE, and 3.248% lesser R2), SVR (92.847% higher MAE, 85.365% higher RMSE, and 3.756% lesser R2), and MLR (146.289% higher MAE, 125.854% higher RMSE, and 6.295% lesser R2). higher MAE, 125.854% higher RMSE, and 6.295% lesser R ). Figure 6. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT. Figure 6. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT. 17 of 30 17 of 30 Atmosphere 2023, 14, 68 Figure 7. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT_5. Figure 7. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT_5. Atmosphere 2023, 14, 68 18 of 30 18 of 30 Figure 8. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT_10. Figure 8. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT_10. 19 of 30 19 of 30 Atmosphere 2023, 14, 68 Figure 9. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT_20. Figure 9. 3.2. Model Performance Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT_20. 20 of 30 Atmosphere 2023, 14, 68 Figure 10. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT_30. Figure 10. Comparison of observed and predicted values using MLR, MLP, SVR, and XGB for GT_30. When examining the results of GT_20 predictions, GBR’s results of training time (MAE = 0.286, RMSE = 0.368, and R2 = 0.997) and testing time (MAE = 0.416, RMSE = 0.551, and R2 = 0.995) were superior, whereas MLP, SVR, and MLR carried out the second, third, and fourth positions. During the testing time, RFR (58.173% higher MAE, 82.94% higher RMSE, and 1.20% lesser R2), MLP (222.837% higher MAE, 214.882% higher RMSE, and 4.523% lesser R2), SVR (237.01% higher MAE, 235.39% higher RMSE, and 5.226% lesser R2), and MLR (356.731% higher MAE, 334.664% higher RMSE, and 9.045% lesser R2) were placed in the second, third, fourth, and ﬁfth positions, respectively. As with other results, the GT_30 prediction results also prove that the models operate in the same way. However, the difference between the superior performing model and the others is developed when the GT depth increases. The GBR executes better results than SVR (75.357% higher MAE, 133.515% higher RMSE, and 1.10% lesser R2), MLP (399.643% higher MAE, 398.09% higher RMSE, and 5.81% lesser R2), SVR (420.357% higher MAE, 427.248% higher RMSE, and 6.5419% lesser R2), and MLR (608.929% higher MAE, 581.471% higher RMSE, and 11.134% lesser When examining the results of GT_20 predictions, GBR’s results of training time (MAE = 0.286, RMSE = 0.368, and R2 = 0.997) and testing time (MAE = 0.416, RMSE = 0.551, and R2 = 0.995) were superior, whereas MLP, SVR, and MLR carried out the second, third, and fourth positions. During the testing time, RFR (58.173% higher MAE, 82.94% higher RMSE, and 1.20% lesser R2), MLP (222.837% higher MAE, 214.882% higher RMSE, and 4.523% lesser R2), SVR (237.01% higher MAE, 235.39% higher RMSE, and 5.226% lesser R2), and MLR (356.731% higher MAE, 334.664% higher RMSE, and 9.045% lesser R2) were placed in the second, third, fourth, and ﬁfth positions, respectively. As with other results, the GT_30 prediction results also prove that the models operate in the same way. However, the difference between the superior performing model and the others is developed when the GT depth increases. 3.2. Model Performance The GBR executes better results than SVR (75.357% higher MAE, 133.515% higher RMSE, and 1.10% lesser R2), MLP (399.643% higher MAE, 398.09% higher RMSE, and 5.81% lesser R2), SVR (420.357% higher MAE, 427.248% higher RMSE, and 6.5419% lesser R2), and MLR (608.929% higher MAE, 581.471% higher RMSE, and 11.134% lesser Atmosphere 2023, 14, 68 21 of 30 21 of 30 R2) during the treating time. During the training time, MLP, SVR, and MLR accomplish second, third, and fourth performance positions, respectively. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for all GT predictions were shown in Figures 11–15. In addition, the comparison of evaluation metric’s results between the training and testing time were displayed in Figures 16 and 17. The comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for all GT predictions were displayed in Figures 18 and 19. Figure 11. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT predictions. Figure 12. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_5 predictions. Figure 11. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT predictions. Figure 11. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT predictions. Figure 11. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT predictions. Figure 12. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_5 predictions. Figure 12. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_5 predictions. Figure 12. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_5 predictions. 22 of 30 Atmosphere 2023, 14, 68 Figure 13. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_10 predictions. Figure 14. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_20 predictions. Figure 13. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT 10 predictions. Figure 13. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT 10 predictions. _ p Figure 14. 3.2. Model Performance The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_20 predictions. Figure 14. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_20 predictions. 23 of 30 Atmosphere 2023, 14, 68 Figure 15. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_30 predictions. Figure 16 (A) The evaluation metrics results of MLR MLP SVR RFR and XGB for GT prediction; Figure 15. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_30 predictions. Figure 15. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT 30 di ti Figure 15. The scatterplots of predicted and actual values using MLR, MLP, SVR, RFR, and XGB for GT_30 predictions. Figure 16. (A) The evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT prediction; (B) the evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT_5 predictions. Figure 16. (A) The evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT prediction; (B) the evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT_5 predictions. Atmosphere 2023, 14, 68 24 of 30 24 of 30 Figure 17. (A) The evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT_10 pred (B) the evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT_20 predictions; evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT_30 prediction. Figure 17. (A) The evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT_10 predictions; (B) the evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT_20 predictions; (C) the evaluation metrics results of MLR, MLP, SVR, RFR, and XGB for GT_30 prediction. 25 of 30 Atmosphere 2023, 14, 68 Figure 18. (A) The comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT predictions; (B) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_5 predictions; (C) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_10 predictions. Figure 19. 4. Discussion Ground or soil temperature (GT) and plant growth are immeasurably associated since the photosynthesis, membrane stability, water and nitriﬁcation process, metabolites, and diversity of soil microbial communities are inﬂuenced precisely by the GT [11,13,21]. However, the critical aspects for exceptional ﬁnal quality and quantity of production, such as seed germination, root-growth orientation, root and shoot development, branching, leaf development, and ﬂowering, were predominant dependencies of GT [9,11,13]. Even though GT plays a meaningful role in agricultural production, the direct method of measuring the GT is time-consuming, expensive, and requires human effort. Therefore, the current study uses computational methods to model the GT at different depths. The current study analyzes the performance of prediction models in terms of evaluation metrics with two different datasets. As a result, the M1 dataset helps to model GT more precisely than the M2 except in GT_30 predictions. A previous study [11] conducted a study to model GT at different depths (5, 10, 50, and 100 cm) on a monthly basis with different independent variables as in the present study. That study used advanced ML models such as CART, GMDH, ELM, and ANN models for predicting GT, and the results show that the extreme learning machine (ELM) outperformed the others. Whereas GT at 5, 10, and 50 cm depth predictions was accomplished using one dataset as input, the prediction of GT at 100 cm depth was modeled using different datasets. Such study has a notable variation in evaluation metrics from one dataset (RMSE = 3.211; NSE = 0.749; R2 = 0.901) to the other (RMSE = 5.149; NSE = 0.356; R2 = 0.459), whereas the present study has no notable ﬂuctuation (MAE is 0.24% lower, RMSE is 1.551% lower, and R2 is 0.10% higher in M2 than M1). Modeling multiple dependent variables with a single dataset is a straightforward and evident approach to reducing computational time, cost, and complexity. The present study considers such notations and endorses the M1 as the optimal dataset for predicting GT. p p g The results show that the mathematical model’s (MLR model) performance is ﬁnite during the whole prediction time compared to the computational models (ML model). For instance, the RMSE difference between observed and predicted for the all-computational model was 0.2–2.4 ◦C, whereas MLR has a difference of 4.1 ◦C. 3.2. Model Performance (A) The comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_20 predictions; (B) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_30 prediction. Figure 18. (A) The comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT predictions; (B) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_5 predictions; (C) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_10 predictions. Figure 18. (A) The comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT predictions; (B) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_5 predictions; (C) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_10 predictions. Figure 19. (A) The comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_20 predictions; (B) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_30 prediction. Figure 19. (A) The comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_20 predictions; (B) the comparison of observed and predicted values of 50 random samples to understand clearly using the XGB model for GT_30 prediction. Atmosphere 2023, 14, 68 26 of 30 4. Discussion When compared with the results (RMSE = 1.229, MAE = 1.510, R2 = 0.955 for 5 CM; RMSE = 0.879, MAE = 0.686, R2 = 0.974 for 10 CM; RMSE = 0.735, MAE = 0.541, R2 = 0.978 for 30 CM), as similar to the previously mentioned study, our model performed better in terms of R2, which is an essential metric to evaluate data extrapolation. Overall, the present study developed a simple, non-complex, faster, and more versatile model to predict the GT at different depths for a short-term prediction with a minimum number of predictor attributes. 4. Discussion Even though the MAE and RMSE are less in the previous studies, the R2 results are better in our proposed model (RMSE = 1.263, MAE = 0.887, R2 = 0.979 for 5 CM: RMSE = 1.025, MAE = 0.741, R2 = 0.985 for 10 CM). In addition, LSTM, ELM, and GRU models come in under the RNN models, which are required, higher graphical processors than the ML models. Generally, those models are popular for voice recognition, natural language processing, and in time series modeling. On the other hand, the previous study [14] utilized EEMD-CNN models, which are more convoluted with deep neural networks to predict the GT in 5, 10, and 30 CM. When compared with the results (RMSE = 1.229, MAE = 1.510, R2 = 0.955 for 5 CM; RMSE = 0.879, MAE = 0.686, R2 = 0.974 for 10 CM; RMSE = 0.735, MAE = 0.541, R2 = 0.978 for 30 CM), as similar to the previously mentioned study, our model performed better in terms of R2, which is an essential metric to evaluate data extrapolation. Overall, the present study developed a simple, non-complex, faster, and more versatile model to predict the GT at different depths for a short-term prediction with a minimum number of predictor attributes. expiration of a plant’s life span [9]. Many types of research [11,33,36] have been conducted to predict the soil temperature in long-term intervals, such as one day, one week, or monthly averages; such cases could be considered as a reference for precautions to avoid extreme GT. If plants suffer from extreme temperatures for a particular period, they may drastically lose moisture absorption capacity, since the soil moisture may decrease by 85% due to evap- oration and transpiration [9,32]. The current research tries to amend the shortcomings of previous studies so that we propose a short-term (1 h) prediction model. Few studies have been conducted to predict the different GT depths using ML models in short-term intervals. For instance, a previous study [30] modeled GT in 2 cm, 5 cm, 10 cm, and 20 cm depths with 30 min intervals. That study used relative humidity, air temperature, global solar radiation, wind speed, and vapor pressure deﬁcit (VPD) as inputs for prediction using ELM, GRNN, BPNN, and RFR. 4. Discussion The ﬁndings show that ELM performs well during all the predictions (2 CM RMSE = 1.74 MAE = 1.37; 5 CM RMSE = 1.85 MAE = 1.44; 10 CM RMSE = 2.05 MAE = 1.60; and 20 CM RMSE = 2.47 MAE = 1.91), which is comparatively less than our proposed model (5 CM RMSE = 1.263 MAE = 0.887; 10 CM RMSE = 1.025 MAE = 0.741; 20 CM RMSE = 0.551 MAE = 0.416). However, the proposed model overcomes that lit- erature not only in terms of error metrics, but also that the referenced literature formed a weather station to model GT, whereas the present literature used government weather data, which are cost-efﬁcient and easy to collect. Even though there are errors in the public dataset, they could be corrected using the preprocessing method. Likewise, a study [33] was conducted to predict GT in 3 different depths (5, 10, and 15 cm) for two different areas where the data were collected from the metrological station as in the present study. That study employed BPNN, LSTM, ELM, GRU, and an optimized GRU model for a 6-h prediction. The literature concluded that their proposed optimized GRU model performed well during the entire prediction. They concluded with the proposed model performing better with the evidence of ﬁndings through evolution metrics (RMSE = 1.1012, MAE = 0.7586, R2 = 0.9638 for 5 CM; RMSE = 0.7658, MAE = 0.5554, R2 = 0.9756 for 10 CM; RMSE = 0.6926, MAE = 0.5435, R2 = 0.9799 for 15 CM). Even though the MAE and RMSE are less in the previous studies, the R2 results are better in our proposed model (RMSE = 1.263, MAE = 0.887, R2 = 0.979 for 5 CM: RMSE = 1.025, MAE = 0.741, R2 = 0.985 for 10 CM). In addition, LSTM, ELM, and GRU models come in under the RNN models, which are required, higher graphical processors than the ML models. Generally, those models are popular for voice recognition, natural language processing, and in time series modeling. On the other hand, the previous study [14] utilized EEMD-CNN models, which are more convoluted with deep neural networks to predict the GT in 5, 10, and 30 CM. 4. Discussion Even though the mathe- matical models are straightforward, accessible, and simple to design, they are limited to a scenario of extrapolating beyond the range of data. Such deviations could be solved using computational models since those models are adaptive to the scene due to the essential nature of learning through weights, so the computational models have superiority over standard statistical models. The optimization techniques also induce the prediction values to ﬁt more closely to the observed values. As mentioned in Section 2.4 all other models except MLR have been optimized using hyperparameter tuning (ﬁne-tuning) using the grid-search method. The actual results of native algorithms have been noted, and the same algorithms are subjected to optimal hyperparameter searching then re-trained using the ﬁne-tuned parameters. The current study observed that the model performance was improved after optimization most of the time. The error results also evidenced a marginal improvement in results after the optimization for overall prediction, which concurs with the previous literature. Mostly, the overﬁtting problems were solved during ﬁne-tuning, but RFR over-ﬁtted during the present examination. Even though the RFR’s nature leads to overﬁtting, different optimization techniques can solve the issue because more author- itative optimization techniques have been developed recently. Since the objective of the present study is to develop a reliable and straightforward model for GT prediction, the computational time was considered for the main examination; so, the present study used standard optimization techniques. The reason to increase the accuracy when the sensor goes deep into the soil surface is that ﬂuctuations in temperature on top of the soil are frequent. However, according to the Korean metrological society, multiple sensors would have been placed and collected the data. So, the ﬁnal sensory value of the topsoil surface is elusive, whereas the ﬂuctuations are lesser when the sensor goes into the deep soil. Extreme GT is catastrophic for soil microorganism development and a plant’s life. Particularly at 32 ◦C, the plant’s growth will face slowdown, whereas 60 ◦C leads to the Atmosphere 2023, 14, 68 27 of 30 27 of 30 expiration of a plant’s life span [9]. Many types of research [11,33,36] have been conducted to predict the soil temperature in long-term intervals, such as one day, one week, or monthly averages; such cases could be considered as a reference for precautions to avoid extreme GT. 4. Discussion If plants suffer from extreme temperatures for a particular period, they may drastically lose moisture absorption capacity, since the soil moisture may decrease by 85% due to evap- oration and transpiration [9,32]. The current research tries to amend the shortcomings of previous studies so that we propose a short-term (1 h) prediction model. Few studies have been conducted to predict the different GT depths using ML models in short-term intervals. For instance, a previous study [30] modeled GT in 2 cm, 5 cm, 10 cm, and 20 cm depths with 30 min intervals. That study used relative humidity, air temperature, global solar radiation, wind speed, and vapor pressure deﬁcit (VPD) as inputs for prediction using ELM, GRNN, BPNN, and RFR. The ﬁndings show that ELM performs well during all the predictions (2 CM RMSE = 1.74 MAE = 1.37; 5 CM RMSE = 1.85 MAE = 1.44; 10 CM RMSE = 2.05 MAE = 1.60; and 20 CM RMSE = 2.47 MAE = 1.91), which is comparatively less than our proposed model (5 CM RMSE = 1.263 MAE = 0.887; 10 CM RMSE = 1.025 MAE = 0.741; 20 CM RMSE = 0.551 MAE = 0.416). However, the proposed model overcomes that lit- erature not only in terms of error metrics, but also that the referenced literature formed a weather station to model GT, whereas the present literature used government weather data, which are cost-efﬁcient and easy to collect. Even though there are errors in the public dataset, they could be corrected using the preprocessing method. Likewise, a study [33] was conducted to predict GT in 3 different depths (5, 10, and 15 cm) for two different areas where the data were collected from the metrological station as in the present study. That study employed BPNN, LSTM, ELM, GRU, and an optimized GRU model for a 6-h prediction. The literature concluded that their proposed optimized GRU model performed well during the entire prediction. They concluded with the proposed model performing better with the evidence of ﬁndings through evolution metrics (RMSE = 1.1012, MAE = 0.7586, R2 = 0.9638 for 5 CM; RMSE = 0.7658, MAE = 0.5554, R2 = 0.9756 for 10 CM; RMSE = 0.6926, MAE = 0.5435, R2 = 0.9799 for 15 CM). 5. Conclusions Thanks to recent technological breakthroughs, agricultural output may be made more sustainable in the face of food insecurity caused by climate change and global warming. GT is a critical feature for plant development and output; modeling GT may lead to increased productivity and improved irrigation methods. The current study predicts and analyses numerous models and concludes with the important points listed below: • The XGB model outperforms the other standard ML-based models in any depth GT prediction. Furthermore, the model’s dependability is superior to that of the others. p p y p • In order to develop an optimum model, the input dataset is critical to improving the efficiency of the output. Finding the proper input parameters utilizing feature importance Atmosphere 2023, 14, 68 28 of 30 will enhance the model’s maximum efficiency, yet providing a more significant number of input parameters increases model complexity and computational time. will enhance the model’s maximum efficiency, yet providing a more significant number of input parameters increases model complexity and computational time. p p p y p • Tuning the hyperparameters (ﬁne-tuning) of the computational model can signiﬁcantly enhance efﬁciency and help overcome overﬁtting challenges. • Tuning the hyperparameters (ﬁne-tuning) of the computational model can signiﬁcantly enhance efﬁciency and help overcome overﬁtting challenges. y p g g • GT modeling has limitations in that GT is not a universal number and can be volatile related to soil color, shape, vegetation cover, physical and chemical characteristics, and location. As a result, developing a worldwide model to anticipate GT is insufﬁcient. • GT modeling has limitations in that GT is not a universal number and can be volatile related to soil color, shape, vegetation cover, physical and chemical characteristics, and location. As a result, developing a worldwide model to anticipate GT is insufﬁcient. Th t i t hi hli ht d b b i f t t di p g p The constraints highlighted above can be overcome in future studies. Author Contributions: J.-W.Y. conceived and designed the experiments; J.-W.Y. performed the experiments; J.-W.Y. analyzed the data and discussed the results; J.-W.Y. wrote the paper; K.D. supervised, checked, gave comments, and approved this work. All authors have read and agreed to the published version of the manuscript. 5. Conclusions Funding: This research was ﬁnancially supported by the Ministry of Trade, Industry, and Energy (MOTIE) of Korea under the “Regional Specialized Industry Development Program” (R&D, P0002072) supervised by the Korea Institute for Advancement of Technology (KIAT). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Arulmozhi, E.; Moon, B.E.; Basak, J.K.; Sihalath, T.; Park, J.; Kim, H.T. Machine Learning-Based Microclimate Model for Indoor Air Temperature and Relative Humidity Prediction in a Swine Building. Animals 2021, 11, 222. [CrossRef] [PubMed] 2. Gornall, J.; Betts, R.; Burke, E.; Clark, R.; Camp, J.; Willett, K.; Wiltshire, A. Implications of Climate Change for Agricultural Productivity in the Early Twenty-First Century. Philos. Trans. R. Soc. B Biol. Sci. 2010, 365, 2973–2989. [CrossRef] [PubMed] 3. Arulmozhi, E.; Bhujel, A.; Moon, B.E.; Kim, H.T. The Application of Cameras in Precision Pig Farming: An Overview for Swine-Keeping Professionals. Animals 2021, 11, 2343. [CrossRef] 4. Hoegh-Guldberg, O.; Jacob, D.; Taylor, M. Impacts of 1.5◦C of Global Warming on Natural and Human Systems. Spec. Rep. Intergov. Panel Clim. Chang. 2018, 175–181. 5. Knox, J.; Hess, T.; Daccache, A.; Wheeler, T. Climate Change Impacts on Crop Productivity in Africa and South Asia. Environ. Res. Lett. 2012, 7, 34032. [CrossRef] D.; Iizumi, T. Evidence of Crop Production Losses in West Africa Due to Historical Global Warming in Two ep. 2019, 9, 12834. [CrossRef] [PubMed] 6. Sultan, B.; Defrance, D.; Iizumi, T. Evidence of Crop Production Losses in West Africa Due to Histo Crop Models. Sci. Rep. 2019, 9, 12834. [CrossRef] [PubMed] 7. Jia, G.; Shevliakova, E.; Artaxo, P.; De Noblet-Ducoudré, N.; Houghton, R.; Anderegg, W.; Bernier, P.; Carlo Espinoza, J.; Semenov, S.; Xu, X.; et al. Land-Climate Interactions Coordinating. IPCC Rep. 2019, 131–248. g p 8. Gupta, D.; Chowdhury, A.; Rahaman, M.S. Soil Temperature Prediction under Limited Data Condition. Int. J. Curr. Microbiol. Appl. Sci. 2019, 8, 102–112. [CrossRef] 9. Sabri, N.S.A.; Zakaria, Z.; Mohamad, S.E.; Jaafar, A.B.; Hara, H. Importance of Soil Temperature for the Growth of Temperate Crops under a Tropical Climate and Functional Role of Soil Microbial Diversity. Microbes Environ. 2018, 33, 144–150. [CrossRef] 10. Bilgili, M. The Use of Artiﬁcial Neural Networks for Forecasting the Monthly Mean Soil Temperatures in Adana, Turkey. Turkish J. Agric. For. 2011, 35, 83–93. [CrossRef] .; Ahmed, A.N.; Mert, C.; Fai, C.M.; Kim, S.; Kim, N.W.; El-Shaﬁe, A. Advanced Machine Learning Model for curacy of Soil Temperature at Different Depths. PLoS ONE 2020, 15, e0231055. [CrossRef] [PubMed] 11. Alizamir, M.; Kisi, O.; Ahmed, A.N.; Mert, C.; Fai, C.M.; Kim, S.; Kim, N.W.; El-Shaﬁe, A. Advanced Ma Better Prediction Accuracy of Soil Temperature at Different Depths. PLoS ONE 2020, 15, e0231055. [Cro 12. 17. Enrique, G.S.; Braud, I.; Jean-Louis, T.; Michel, V.; Pierre, B.; Jean-Christophe, C. Modelling Heat and Water Exchanges of Fallow Land Covered with Plant-Residue Mulch. Agric. For. Meteorol. 1999, 97, 151–169. [CrossRef] 17. Enrique, G.S.; Braud, I.; Jean-Louis, T.; Michel, V.; Pierre, B.; Jean-Christophe, C. Modelling Heat an Land Covered with Plant-Residue Mulch. Agric. For. Meteorol. 1999, 97, 151–169. [CrossRef] References Hanson, C.L.; Marks, D.; Van Vactor, S.S. Long-Term Climate Database, Reynolds Creek Experimental Watershed, Idaho, United States. Water Resour. Res. 2001, 37, 2839–2841. [CrossRef] [ ] 13. Jahanfar, A.; Drake, J.; Sleep, B.; Gharabaghi, B. A Modiﬁed FAO Evapotranspiration Model for Reﬁned Water Budget Analysis for Green Roof Systems. Ecol. Eng. 2018, 119, 45–53. [CrossRef] y g 14. Hao, H.; Yu, F.; Li, Q. Soil Temperature Prediction Using Convolutional Neural Network Based on Ensemble Empirical Mode Decomposition. IEEE Access 2020, 9, 4084–4096. [CrossRef] 15. Tian, Y.; Guan, B.; Zhou, D.; Yu, J.; Li, G.; Lou, Y. Responses of Seed Germination, Seedling Growth, and Seed Yield Traits to Seed Pretreatment in Maize (Zea mays L.). Sci. World J. 2014, 2014, 834630. [CrossRef] 16. Onwuka, B. Effects of Soil Temperature on Some Soil Properties and Plant Growth. Adv. Plants Agric. Res. 2018, 8, 34–37. [CrossRef] 29 of 30 Atmosphere 2023, 14, 68 18. Kang, S.; Kim, S.; Oh, S.; Lee, D. Predicting Spatial and Temporal Patterns of Soil Temperature Based on Topography, Surface Cover and Air Temperature. For. Ecol. Manag. 2000, 136, 173–184. [CrossRef] p g 19. Mihalakakou, G. On Estimating Soil Surface Temperature Proﬁles. Energy Build. 2002, 34, 251–259. [CrossRef] 20 Koçak K ; Saylan L ; Eitzinger J Nonlinear Prediction of Near-Surface Temperature via Univariate and Multivariate Time Series 19. Mihalakakou, G. On Estimating Soil Surface Temperature Proﬁles. Energy Build. 2002, 34, 251 259. [Cr 20. Koçak, K.; ¸Saylan, L.; Eitzinger, J. Nonlinear Prediction of Near-Surface Temperature via Univariate and Embedding. Ecol. Modell. 2004, 173, 1–7. [CrossRef] g 21. Gaumont-Guay, D.; Black, T.A.; Grifﬁs, T.J.; Barr, A.G.; Jassal, R.S.; Nesic, Z. Interpreting the Dependence of Soil Respiration on Soil Temperature and Water Content in a Boreal Aspen Stand. Agric. For. Meteorol. 2006, 140, 220–235. [CrossRef] 21. Gaumont-Guay, D.; Black, T.A.; Grifﬁs, T.J.; Barr, A.G.; Jassal, R.S.; Nesic, Z. Interpreting the Dependen Soil Temperature and Water Content in a Boreal Aspen Stand. Agric. For. Meteorol. 2006, 140, 220–235. 22. Gao, Z.; Bian, L.; Hu, Y.; Wang, L.; Fan, J. Determination of Soil Temperature in an Arid Region. J. Arid Environ. 2007, 71, 157–168. [CrossRef] ia, F.; Lykoudis, S.; Tsiros, I.; Alvertos, N.; Akylas, E.; Garofalakis, I. Ground Temperature Estimations Us i l d S i E i i l A h S l E 2009 83 211 219 [C R f] 23. References Mehdizadeh, S.; Behmanesh, J.; Khalili, K. Evaluating the Performance of Artiﬁcial Intelligence Methods for Estimation of Monthly Mean Soil Temperature without Using Meteorological Data. Environ. Earth Sci. 2017, 76, 325. [CrossRef] 32. Mehdizadeh, S.; Ahmadi, F.; Kozekalani Sales, A. Modelling Daily Soil Temperature at Different Depths via the Classical and g 31. Mehdizadeh, S.; Behmanesh, J.; Khalili, K. Evaluating the Performance of Artiﬁcial Intelligence Methods for Estimation of Monthly Mean Soil Temperature without Using Meteorological Data. Environ. Earth Sci. 2017, 76, 325. [CrossRef] 32. Mehdizadeh, S.; Ahmadi, F.; Kozekalani Sales, A. Modelling Daily Soil Temperature at Different Depths via the Classical and Hybrid Models. Meteorol. Appl. 2020, 27, e1941. [CrossRef] 33. Wang, X.; Li, W.; Li, Q. A New Embedded Estimation Model for Soil Temperature Prediction. Sci. Prog [CrossRef] 34. Feigl, M.; Lebiedzinski, K.; Herrnegger, M.; Schulz, K. Machine-Learning Methods for Stream Water Temperature Prediction. Hydrol. Earth Syst. Sci. 2021, 25, 2951–2977. [CrossRef] 35. Sahoo, K.; Samal, A.K.; Pramanik, J.; Pani, S.K. Exploratory Data Analysis Using Python. Int. J. Innov. Technol. Explor. Eng. 2019, 8, 4727–4735. [CrossRef] 36. Bilgili, M. Prediction of Soil Temperature Using Regression and Artiﬁcial Neural Network Models. Meteorol. Atmos. Phys. 2010, 110, 59–70. [CrossRef] 37. Imanian, H.; Cobo, J.H.; Payeur, P.; Shirkhani, H.; Mohammadian, A. A Comprehensive Study of Artiﬁcial Intelligence Applica- tions for Soil Temperature Prediction. Preprints 2022, 2022020101. [CrossRef] 38. Elanchezhian, A.; Basak, J.K.; Park, J.; Khan, F.; Okyere, F.G.; Lee, Y.; Bhujel, A.; Lee, D.; Sihalath, T.; Kim, H.T. Evaluating Different Models Used for Predicting the Indoor Microclimatic Parameters of a Greenhouse. Appl. Ecol. Environ. Res. 2020, 18, 2141–2161. [CrossRef] 39. Taki, M.; Ajabshirchi, Y.; Ranjbar, S.F.; Matloobi, M. Application of Neural Networks and Multiple Regression Models in Greenhouse Climate Estimation. Agric. Eng. Int. CIGR J. 2016, 18, 29–43. g g J. Prediction of Outdoor Air Temperature and Humidity Using Xgboost. IOP Conf. Ser. Earth Environ. Sci rossRef] 40. Ma, X.; Fang, C.; Ji, J. Prediction of Outdoor Air Temperature and Humidity Using Xgboost. IOP Con 2020, 427, 12013. [CrossRef] , , [ ] 41. Vassallo, D.; Krishnamurthy, R.; Sherman, T.; Fernando, H.J.S. Analysis of Random Forest Modeling Strategies for Multi-Step Wind Speed Forecasting. Energies 2020, 13, 5488. [CrossRef] p g g 42. Walker, S.; Khan, W.; Katic, K.; Maassen, W.; Zeiler, W. Accuracy of Different Machine Learning Algorithms and Added-Value of Predicting Aggregated-Level Energy Performance of Commercial Buildings. Energy Build. References Droulia, F.; Lykoudis, S.; Tsiros, I.; Alvertos, N.; Akylas, E.; Garofalakis, I. Ground Temperature Estimations Using Simpliﬁed Analytical and Semi-Empirical Approaches. Sol. Energy 2009, 83, 211–219. [CrossRef] ia, F.; Lykoudis, S.; Tsiros, I.; Alvertos, N.; Akylas, E.; Garofalakis, I. Ground Temperature Estimations Us ytical and Semi-Empirical Approaches. Sol. Energy 2009, 83, 211–219. [CrossRef] is, S.; Tsiros, I.; Alvertos, N.; Akylas, E.; Garofalakis, I. Ground Temperature Estimations Using Simpliﬁed -Empirical Approaches. Sol. Energy 2009, 83, 211–219. [CrossRef] l, J.; McGowan, G. Soil Temperature Calculation for Burial Site Analysis. Forensic Sci. Int. 2009, 191, 104–109. 24. Prangnell, J.; McGowan, G. Soil Temperature Calculation for Burial Site Analysis. Forensic Sci. Int. 2 25. Adhikari, R.; Agrawal, R. An Introductory Study on Time Series Modeling and Forecasting Ratnadip Adhikari R. K. Agrawal. arXiv 2013, arXiv:1302.6613. 26. Shirvani, A.; Moradi, F.; Moosavi, A.A. Time Series Modelling of Increased Soil Temperature Anomalies during Long Period. Int. Agrophysics 2015, 29, 509–515. [CrossRef] g p y 27. Kotu, V.; Deshpande, B. Chapter 12. Time Series Forecasting, 2nd ed.; Morgan Kaufmann: Burlington, 27. Kotu, V.; Deshpande, B. Chapter 12. Time Series Forecasting, 2nd ed.; Morgan Kaufmann: Burlington, MA, USA, 2019; pp. 395–445. 28. Patowary, A.N. Monthly Temperature Prediction Based on Arima Model: A Case Study in Dibrugarh Station of Assam, India. Int. J. Adv. Res. Comput. Sci. 2017, 8, 292–298. [CrossRef] 28. Patowary, A.N. Monthly Temperature Prediction Based on Arima Model: A Case Study in Dibrugarh Station of Assam, India. Int. J. Adv. Res. Comput. Sci. 2017, 8, 292–298. [CrossRef] J p 29. Samadianfard, S.; Ghorbani, M.A.; Mohammadi, B. Forecasting Soil Temperature at Multiple-Depth with a Hybrid Artiﬁcial Neural Network Model Coupled-Hybrid Fireﬂy Optimizer Algorithm. Inf. Process. Agric. 2018, 5, 465–476. [CrossRef] p 29. Samadianfard, S.; Ghorbani, M.A.; Mohammadi, B. Forecasting Soil Temperature at Multiple-Depth 29. Samadianfard, S.; Ghorbani, M.A.; Mohammadi, B. Forecasting Soil Temperature at Multiple-De Neural Network Model Coupled-Hybrid Fireﬂy Optimizer Algorithm. Inf. Process. Agric. 2018, 5, 4 p y y p g f g 30. Feng, Y.; Cui, N.; Hao, W.; Gao, L.; Gong, D. Estimation of Soil Temperature from Meteorological D Learning Models Geoderma 2019 338 67 77 [CrossRef] 30. Feng, Y.; Cui, N.; Hao, W.; Gao, L.; Gong, D. Estimation of Soil Temperature from Meteorological Data Using Different Machine Learning Models. Geoderma 2019, 338, 67–77. [CrossRef] g g p g Learning Models. Geoderma 2019, 338, 67–77. [CrossRef] g [ ] 31. 47. Chen, T.; Guestrin, C. XGBoost: A Scalable Tree Boosting System. In Proceedings of the 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, San Francisco, CA, USA, 13–17 August 2016; pp. 785–794. [CrossRef] 48. Ibrahem Ahmed Osman, A.; Najah Ahmed, A.; Chow, M.F.; Feng Huang, Y.; El-Shaﬁe, A. Extreme Gradient Boosting (Xgboost) Model to Predict the Groundwater Levels in Selangor Malaysia. Ain. Shams Eng. J. 2021, 12, 1545–1556. [CrossRef] g y g 49. Dashdondov, K.; Song, M.H. Factorial Analysis for Gas Leakage Risk Predictions from a Vehicle-Based Methane Survey. Appl. Sci. 2022, 12, 115. [CrossRef] 50. Vasker Sharma Imputing Missing Data in Hydrology Using Machine Learning Models. Int. J. Eng. Res. 2021, 10, 78–82. [CrossRef] 51. Yoon, H.; Jun, S.C.; Hyun, Y.; Bae, G.O.; Lee, K.K. A Comparative Study of Artiﬁcial Neural Networks and Support Vector Machines for Predicting Groundwater Levels in a Coastal Aquifer. J. Hydrol. 2011, 396, 128–138. [CrossRef] 50. Vasker Sharma Imputing Missing Data in Hydrology Using Machine Learning Models. Int. J. Eng. Res. 2021, 10, 78–82. [CrossRef] 51. Yoon, H.; Jun, S.C.; Hyun, Y.; Bae, G.O.; Lee, K.K. A Comparative Study of Artiﬁcial Neural Networks and Support Vector Machines for Predicting Groundwater Levels in a Coastal Aquifer. J. Hydrol. 2011, 396, 128–138. [CrossRef] 52. Che, Z.; Purushotham, S.; Cho, K.; Sontag, D.; Liu, Y. Recurrent Neural Networks for Multivariate Time Series with Missing Values. Sci. Rep. 2018, 8, 6085. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. p [ ] 53. Sattari, M.T.; Avram, A.; Apaydin, H.; Matei, O. Soil Temperature Estimation with Meteorological Parameters by Using Tree-Based Hybrid Data Mining Models. Mathematics 2020, 8, 1407. [CrossRef] 51. Yoon, H.; Jun, S.C.; Hyun, Y.; Bae, G.O.; Lee, K.K. A Comparative Study of Artiﬁcial Neural Networks and Support Vector Machines for Predicting Groundwater Levels in a Coastal Aquifer. J. Hydrol. 2011, 396, 128–138. [CrossRef] 52. Che, Z.; Purushotham, S.; Cho, K.; Sontag, D.; Liu, Y. Recurrent Neural Networks for Multivariate Time Series with Missing Values. Sci. Rep. 2018, 8, 6085. [CrossRef] Machines for Predicting Groundwater Levels in a Coastal Aquifer. J. Hydrol. 2011, 396, 128 138. [CrossRef] 52. Che, Z.; Purushotham, S.; Cho, K.; Sontag, D.; Liu, Y. Recurrent Neural Networks for Multivariate Time Series with Missing Values. Sci. Rep. 2018, 8, 6085. [CrossRef] References 2020, 209, 109705. [CrossRef] 42. Walker, S.; Khan, W.; Katic, K.; Maassen, W.; Zeiler, W. Accuracy of Different Machine Learning Algorithms and Added-Value of 42. Walker, S.; Khan, W.; Katic, K.; Maassen, W.; Zeiler, W. Accuracy of Different Machine Learning Algorithms and Added-Value of Predicting Aggregated-Level Energy Performance of Commercial Buildings. Energy Build. 2020, 209, 109705. [CrossRef] 43. Drucker, H.; Burges, C.J.C.; Kaufman, L.; Smola, A.; Vapnik, V. Support Vector Regression Machines. In Advances in Neural I f ti P i S t M M C J d M P t h T Ed MIT P C b id MA USA 1996 9 Predicting Aggregated-Level Energy Performance of Commercial Buildings. Energy Build. 2020, 209, 109705. [CrossRef] 43. Drucker, H.; Burges, C.J.C.; Kaufman, L.; Smola, A.; Vapnik, V. Support Vector Regression Machines. In Advances in Neural Information Processing Systems; Mozer, M.C., Jordan, M., Petsche, T., Eds.; MIT Press: Cambridge, MA, USA, 1996; p. 9. 43. Drucker, H.; Burges, C.J.C.; Kaufman, L.; Smola, A.; Vapnik, V. Support Vector Regression Machines. In Advances in Neural Information Processing Systems; Mozer, M.C., Jordan, M., Petsche, T., Eds.; MIT Press: Cambridge, MA, USA, 1996; p. 9. Information Processing Systems; Mozer, M.C., Jordan, M., Petsche, T., Eds.; MIT Press: Cambridge, MA, USA, 1996; p. 9. 44. Drucker, H.; Surges, C.J.C.; Kaufman, L.; Smola, A.; Vapnik, V. Support Vector Regression Machines. Adv. Neural Inf. Process. Syst. 1997, 1, 155–161. 44. Drucker, H.; Surges, C.J.C.; Kaufman, L.; Smola, A.; Vapnik, V. Support Vector Regression Machines. 1997, 1, 155–161. 45. Hasan, N.; Nath, N.C.; Rasel, R.I. A Support Vector Regression Model for Forecasting Rainfall. In Proceedings of the 2015 2nd International Conference on Electrical Information and Communication Technologies (EICT), Khulna, Bangladesh, 10–12 December 2015; pp. 554–559. [CrossRef] pp 46. Wu, J.; Liu, H.; Wei, G.; Song, T.; Zhang, C.; Zhou, H. Flash Flood Forecasting Using Support Vector Regression Model in a Small Mountainous Catchment. Water 2019, 11, 1327. [CrossRef] 47. Chen, T.; Guestrin, C. XGBoost: A Scalable Tree Boosting System. In Proceedings of the 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, San Francisco, CA, USA, 13–17 August 2016; pp. 785–794. [CrossRef] 30 of 30 Atmosphere 2023, 14, 68 48. Ibrahem Ahmed Osman, A.; Najah Ahmed, A.; Chow, M.F.; Feng Huang, Y.; El-Shaﬁe, A. Extreme Gradient Boosting (Xgboost) Model to Predict the Groundwater Levels in Selangor Malaysia. Ain. Shams Eng. J. 2021, 12, 1545–1556. References [CrossRef] g y g J [ ] 49. Dashdondov, K.; Song, M.H. Factorial Analysis for Gas Leakage Risk Predictions from a Vehicle-Based Methane Survey. Appl. Sci. 2022, 12, 115. [CrossRef] [ ] 50. Vasker Sharma Imputing Missing Data in Hydrology Using Machine Learning Models. Int. J. Eng. Res. 2021, 10, 78–82. [CrossRef] 51. Yoon, H.; Jun, S.C.; Hyun, Y.; Bae, G.O.; Lee, K.K. A Comparative Study of Artiﬁcial Neural Networks and Support Vector Machines for Predicting Groundwater Levels in a Coastal Aquifer. J. Hydrol. 2011, 396, 128–138. [CrossRef] g q J y , , [ ] 52. Che, Z.; Purushotham, S.; Cho, K.; Sontag, D.; Liu, Y. Recurrent Neural Networks for Multivariate Time Series with Missing Values. Sci. Rep. 2018, 8, 6085. [CrossRef] p [ ] 53. Sattari, M.T.; Avram, A.; Apaydin, H.; Matei, O. Soil Temperature Estimation with Meteorological Parameters by Using Tree-Based Hybrid Data Mining Models. Mathematics 2020, 8, 1407. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W2907879640	https://www.research-collection.ethz.ch/bitstream/20.500.11850/334573/3/document.pdf	English	null	Species-specific enhancement of enterohemorrhagic E. coli pathogenesis mediated by microbiome metabolites	Microbiome	2,019	cc-by	15,800	ETH Library ETH Library ( ) Tovaglieri, Alessio; Sontheimer-Phelps, Alexandra; Geirnaert, Annelies ; Prantil-Baun, Rachelle; Camacho, Diogo M.; Chou, David B.; Jalili-Firoozinezhad, Sasan; de Wouters, Tomás; Kasendra, Magdalena; Super, Michael; Cartwright, Mark J.; Richmond, Camilla A.; Breault, David T.; Lacroix, Christophe ; Ingber, Donald E. Author(s): Author(s): Tovaglieri, Alessio; Sontheimer-Phelps, Alexandra; Geirnaert, Annelies ; Prantil-Baun, Rachelle; Camacho, Diogo M.; Chou, David B.; Jalili-Firoozinezhad, Sasan; de Wouters, Tomás; Kasendra, Magdalena; Super, Michael; Cartwright, Mark J.; Richmond, Camilla A.; Breault, David T.; Lacroix, Christophe ; Ingber, Donald E. Originally published in: g y p Microbiome 7(1), https://doi.org/10.1186/s40168-019-0650-5 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Species-specific enhancement of enterohemorrhagic E. coli pathogenesis mediated Species-specific enhancement of enterohemorrhagic E. coli pathogenesis mediated by microbiome metabolites © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH Open Access Species-specific enhancement of enterohemorrhagic E. coli pathogenesis mediated by microbiome metabolites Alessio Tovaglieri1,2, Alexandra Sontheimer-Phelps1,3, Annelies Geirnaert2, Rachelle Prantil-Baun1, Diogo M. Camacho1, David B. Chou1,4, Sasan Jalili-Firoozinezhad1,5, Tomás de Wouters2, Magdalena Kasendra1,12, Michael Super1, Mark J. Cartwright1, Camilla A. Richmond6,7,8, David T. Breault7,8,9, Christophe Lacroix2 and Donald E. Ingber1,10,11* Abstract Background: Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia coli (EHEC) infection, whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities. Results: We utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites—4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid—that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity. Conclusion: Organ-on-chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection. development of novel tolerance-inducing therapeutic ap- proaches to treat pathogenic infections. For example, in the mammalian intestine, up to 100 trillion commensal bacteria influence host health, and the intestinal micro- biome differentially modulates sensitivity to infection in different species [3]. An exquisite example is the differ- ence in tolerance to enterohemorrhagic Escherichia coli (EHEC), which causes more than 100,000 infections per year in the USA [4], and can result in development of severe bloody diarrhea, hemorrhagic colitis, and Tovaglieri et al. Microbiome (2019) 7:43 https://doi.org/10.1186/s40168-019-0650-5 Tovaglieri et al. Microbiome (2019) 7:43 https://doi.org/10.1186/s40168-019-0650-5 * Correspondence: don.ingber@wyss.harvard.edu 1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA 10Vascular Biology Program and Department of Surgery, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA Full list of author information is available at the end of the article Background Host tolerance to microbial infections varies greatly be- tween different species [1, 2]. In an era of increasing bacterial antibiotic resistance, understanding of the mo- lecular basis for these differences could lead to Page 2 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Page 2 of 20 Tovaglieri et al. Microbiome (2019) 7:43 diseases in vitro [19–25]. Endoscopic analysis of human patients infected with EHEC have revealed acute inflam- mation of the colon and ex vivo infection experiments similarly demonstrated colonization as well as attaching and effacing (A/E) lesions in human colonic biopsies [26–28]. Thus, to explore whether species-specific intes- tinal microbiomes can influence host tolerance to EHEC infection, we developed a two-channel Colon Chip lined by primary human colon epithelial cells isolated from patient-derived organoids interfaced with human intes- tinal microvascular endothelial cells (HIMECs), using a recently described technique [29]. The human Colon Chips were infected with EHEC in the presence of sol- uble metabolites isolated from bioreactor cultures of complex populations of murine or human intestinal commensal microbes. These studies recapitulated the enhanced sensitivity of human colonic epithelium to EHEC in the presence of human microbiome products compared to those from mouse. Surprisingly, however, we discovered that the human microbiome metabolites increased EHEC’s ability to induce epithelial damage, ra- ther than the mouse microbiome products protecting against the damaging effects of this infectious pathogen. hemolytic uremic syndrome (HUS). The infectious dose for EHEC is 100,000-fold higher in mice compared with humans (107 versus 102 microbes), and even then mice need to be depleted of their microbiome to show symp- toms of infection [5–7]. This difference may be due in part to differential localization and expression of recep- tors for pathogen virulence factors, such as Shiga toxin produced by EHEC [8–10], though more human-relevant studies are needed because no animal model fully recapitulates human symptoms of EHEC in- fection [11]. These dissimilarities in EHEC infection limit the translational potential of murine research to human. Furthermore, studies in human patients are very limited and focused on blood and fecal samples content but do not allow for more in-depth mechanistic investi- gation of EHEC infection within the gastrointestinal tract. There is indeed a strong need for models that more closely mimic human intestinal pathophysiology in context of bacterial infection. Additional evidence suggests that products of com- mensal bacteria are important modulators of host patho- physiology. Background Metabolites generated by the host gut microbiome, such as acetate produced by Bifidobacteria, have been shown to confer protection against EHEC in- fection in mice [12]. Propionate produced by Bacteroides directly inhibits pathogen growth in vitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from Salmon- ella typhimurium [13]. Butyrate, another short-chain fatty acid produced by commensal bacteria, also modu- lates host intestinal barrier, injury response and im- munity, as well as pathogenicity of EHEC, by altering expression of genes involved in virulence and flagellar motility [14, 15]. Nevertheless, only a few tolerance- modulating microbial metabolites have been identified from fecal samples, and their effects have never been demonstrated in human intestine. In fact, most re- search on human host-microbiome-pathogen interac- tions relies on correlative genomic or meta-genomic studies, making identification of causality in humans extremely difficult [16]. Studies also have been carried out by repopulating gnotobiotic mice with complex mixtures of living human versus mouse commensal mi- crobes, but it is extremely difficult to identify soluble metabolites that mediate their effects [3, 17]. Thus, there is a great need for a human model of EHEC infection where contributions of the complex gut microbiome, and specifically soluble metabolites pro- duced by these commensal microbes, can be explored experimentally. Microbiome metabolites recapitulate species-specific tolerance in Colon Chips a A schematic representation of the experimental design illustrating how human or mouse intestinal microbiome metabolites were added to the intestinal channel (red) of optically clear, human Colon Chips that are lined by primary human colon epithelial cells (Epi) and directly opposed to a second parallel vascular microchannel (blue) in which HIMVECs (Endo.) are cultured; the two channels are separated by a thin, porous, ECM-coated membrane; F-actin filaments in epithelial and endothelial cells were stained with Phalloidin (magenta) and nuclei with DAPI (white). b–d Analysis of EHEC-induced epithelial injury on-chip. b Representative differential interference contrast (DIC) images of the colonic epithelium in the presence of Hmm or Mmm in the presence or absence of EHEC (bar, 100 μm). c Pseudo-colored images of the entire colon epithelium within the upper channel of the Colon Chip (yellow) cultured in the presence of Hmm or Mmm with or without EHEC (dark regions indicate lesion areas). d Quantification of epithelial lesion areas under the experimental conditions described in b, c. Epithelial lesion defined as regions in which cells normally contained within a continuous intact epithelium have fully detached from the ECM-coated membrane and their neighboring cells, thus, leaving exposed regions of the membrane below. e Changes in levels of various indicated cytokines released into the vascular channel of the Colon Chips by cells cultured under the conditions described in b, c. p < 0.05; p < 0.01; p < 0.001; *p < 0.0001 mouse or human microbiome samples were cultured for 2 weeks under conditions that mimic the internal milieu of the large intestine; the commensal bacterial content of the cultures was defined at the phylum and genus levels using 16S rRNA gene sequencing [33] (see “Methods” section for details) (Fig. 1a, Additional file 1: Figure S1, Additional file 2: Figure S2). On day 8 of the Colon Chip culture, the lu- minal culture medium was replaced with the same medium supplemented with human or murine microbiome metabolites (diluted 1:20 in a PBS-water based solution to 300 mOsm kg−1), while continuing to flow the same endo- thelial culture medium through the vascular channel. Per- fusion was continued for 24 h, followed by introduction of EHEC (1.7 × 105; serotype O157:H7) into the apical lumen in the same medium for 3 h under static conditions to allow for bacterial cell attachment; medium flow was then re-established and continued for 24 additional hours. Microbiome metabolites recapitulate species-specific tolerance in Colon Chips To explore how gut microbiome metabolites contribute to species-specific differences in response to infection by EHEC, we cultured healthy, primary, human colon epithe- lial cells isolated from human donor-derived organoids in close apposition to primary HIMECs within a microfluidic culture device to create a human Colon Chip. The Colon Chip contains two parallel microchannels separated by a porous (7 μm diameter) extracellular matrix (ECM)-coated membrane; the epithelial cells were cultured on the upper surface of the membrane in the top “intestinal luminal” channel with gut-specific cell culture medium in both channels, as previously described [29]. The HIMECs were seeded on the opposite side of the same membrane in the lower “vascular” channel (Fig. 1a) with gut-specific cell cul- ture medium being perfused through the intestinal luminal channel, and the same medium supplemented with endo- thelial cell supplements and growth factors through the vascular channel (see “Methods” section for details). These culture conditions induced formation of a continuous, un- dulating, colonic epithelium (Fig. 1a,b) that extended across the entire surface of the ECM-coated membrane within 1 week of culture, as visualized using immunofluor- escence microscopy (Fig. 1a, right), phase contrast (Fig. 1b), or pseudo-colored imaging (Fig. 1c). Human microbiome metabolites (Hmm) or mouse microbiome metabolites (Mmm) were collected from PolyFermS continuous intes- tinal fermentation bioreactors [30–33] in which complex In the present study, we confronted this challenge using human organ-on-a-chip (Organ Chip) microfluidic cell culture technology [18], which can be used to recap- itulate human physiology and model various human Page 3 of 20 Tovaglieri et al. Microbiome (2019) 7:43 (2019) 7:43 Tovaglieri et al. Microbiome ig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Page 4 of 20 Tovaglieri et al. Microbiome (2019) 7:43 (2019) 7:43 Tovaglieri et al. Microbiome ( g p p g ) Fig. 1 Microbiome metabolites recapitulate species-specific tolerance in Colon Chips. EHEC-induced release of cytokines in the Colon Chip is similar to in vivo As the intestinal epithelium and endothelium contribute to the host innate immune response to bacterial patho- gens by secreting inflammatory regulators [34–36], we analyzed the effects of the Hmm and Mmm on secretion of multiple pro-inflammatory cytokines and chemokines, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), in the presence or absence of EHEC infection (Fig. 1e). The pro-inflammatory chemokine interleukin-8 (IL-8) is one of the major cytokines secreted by intestinal epithelial cells [37], and it has been shown to be strongly increased during EHEC infection in humans [38–40]. Additionally, the tumor necrosis factor-α (TNF-α) pro- tein is also increased in human patients who develop EHEC-related HUS [38, 41]. We similarly observed a significant increase in abundance of these two cytokines in the human Colon Chip following EHEC infection when Hmm was present, with IL-8 increasing over five- fold compared to our control. In addition, the inflamma- tory mediator interleukin-1β (IL-1β) has been reported to enhance expression of shiga toxin receptors on endo- thelial cells, which increases EHEC-related toxicity [42], and we similarly observed increased release of IL-1β into the endothelium-lined vascular channel of these human Colon Chips exposed to Hmm. EHEC bacteria secrete molecules that modulate downstream interferon-γ (IFN-γ) signaling in epithelial cells as well [43], and remarkably, we also detected higher levels of IFN-γ following EHEC infection on chip. Moreover, EHEC in- fection increases circulating macrophage inflammatory protein-1 α (MIP-1 α) and monocyte chemoattractant protein-1 (MCP-1) levels in children [44] and the in- fected Colon Chips displayed similar increases in the levels of both of these leukocyte chemoattractants. Fi- nally, we discovered that fractalkine levels increased Using this approach, we were able to recapitulate the increased sensitivity of the human Colon Chip to EHEC infection in the presence of microbiome metabolites from human (Hmm) compared to infection when Mmm were present. This was demonstrated by a greatly in- creased loss of epithelial cells from the colonic epithe- lium in the upper channel of chips exposed to Hmm versus Mmm (Fig. 1b, c). A fivefold increase in lesion area was observed in the presence of Hmm compared to Mmm when quantified using computerized image ana- lysis (Fig. 1d), with lesions being defined as regions in which cells fully detached from the normally continuous intact epithelium and the ECM-coated membrane below. Microbiome metabolites recapitulate species-specific tolerance in Colon Chips effects seem to be mediated by direct interactions be- tween the infectious pathogens and the colon epithe- lium, and not secondary to effects on immune cells that were not present in this study. EHEC-induced release of cytokines in the Colon Chip is similar to in vivo Importantly, control studies confirmed that addition of Mmm or Hmm alone, in the absence of EHEC, did not cause any damage to the colonic epithelium (Fig. 1b–d), and EHEC did not induce epithelial lesions in Colon Chips in the absence of microbiome metabolites (Additional file 3: Figure S3). Thus, intestinal micro- biome metabolites are sufficient to recapitulate the species-specific effects on tolerance to EHEC infection observed in previous studies comparing humans and mice, even in the absence of live commensal microbes or immune cells. These findings also indicate that the Page 5 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Page 5 of 20 observed similar colonization levels in the Hmm and Mmm groups in the Colon Chip, and transcriptomics analysis confirmed that there were no significant changes in expression of LPF genes under these condi- tions (Additional file 5: Table S1). Thus, metabolites pro- duced by commensal microbes of the human and mouse gut microbiome do not appear to significantly alter EHEC colonization of the intestinal niche in the human Colon Chip. almost sixfold following EHEC infection, suggesting that this the CX3C chemokine family member also may be involved in EHEC pathogenesis. The anti-inflammatory cytokine IL-10 is of interest be- cause it has been investigated as a potential therapeutic to reduce acute inflammation in inflammatory bowel disease [45–47]. Perhaps because of the body’s attempt to counter the effects of EHEC infection, IL-10 concen- trations in blood increase with the severity of this dis- ease, and very high levels of IL-10 levels appear to correlate with HUS onset [48]. Similarly, in the infected Colon Chips, IL-10 levels increased almost tenfold and reached levels (~ 10 pg ml−1) comparable to those ob- served in human patients with severe colitis induced by EHEC [39] (Fig. 1e). Shiga toxin is believed to be one of the major EHEC virulence factors involved in pathogenesis and develop- ment of HUS; however, ex vivo experiments on human biopsies suggest that it does not play a dominant role in inducing colonization and inflammation in human colon [10, 28]. Transcriptomics analysis of Shiga toxin-related genes revealed that only stx1b was significantly upregu- lated in the presence of Mmm (Additional file 6: Figure S5A, B), but this finding cannot explain the differences in epithelial lesions as Mmm produced significantly less epithelial damage than Hmm in EHEC-infected human Colon Chips (Fig. 1c). EHEC-induced release of cytokines in the Colon Chip is similar to in vivo In addition, we could not detect any significant difference in the concentration of Shiga toxin 1 in the outflow of the vascular channels of in- fected Colon Chips cultured with Hmm versus Mmm (Additional file 6: Figure S5C). Therefore, these results indicate that Shiga toxin is likely not the cause of the differences in epithelial lesion area observed. Taken together, these results show that the human Colon Chip recapitulates the pro- and anti-inflammatory cytokine profiles induced by EHEC infection when cul- tured in the presence of metabolites secreted by the hu- man intestinal microbiome (Hmm). Importantly, when we carried out similar studies in the presence of mouse microbiome products, we found that they produced similar increases in the production of these cytokines as Hmm (Fig. 1e), suggesting that the cytokine response generated by the human intestinal cells does not appear to be responsible for the differences in epithelial injury that are induced by EHEC infection under these culture conditions. Identification of specific metabolites that mediate increased pathogenicity We next used metabolomics analysis to identify specific metabolites in Hmm that are responsible for the effects on EHEC motility we observed. To do this, we com- pared metabolite levels in the pre-fermentation medium used to culture the microbiome (and that mimics dietary food intake) with the Hmm and Mmm isolated from the final stage of PolyFermS fermentation, which emulate the contents of human proximal colon and mouse cecum colonized by their respective microbiomes. To identify relevant metabolites, we mined our metabolo- mics data set for metabolites produced by commensal bacteria (i.e., metabolites whose levels increase during the fermentation process), and within these we identified the ones enriched in either the Hmm or Mmm samples. This resulted in identification of a total of 426 metabo- lites produced by commensal bacteria that were differen- tially expressed between Hmm and Mmm (Fig. 3a, b). y p g To assess their influence on bacterial motility, we se- lected metabolites that were abundant in either Mmm or Hmm based on our metabolomics analysis. From this MSMS (tandem mass spectrometry) analysis, we in- cluded all known compounds (95% confidence), and we also included identifiers that were assigned based on the closest MSMS spectrum in the reference database to the analyte (likely a substructure of the original metabolite); however, we excluded synthetic prescription drugs and known antimicrobial compounds. We performed this se- lection in order to screen for microbial-derived com- pounds potentially affecting EHEC motility and not merely bacterial proliferation or viability (see “Methods” section for details). To further explore this potential mechanism, we eval- uated bacterial motility in the presence of Hmm or Mmm using GFP-expressing EHEC bacteria, as previ- ously described [57]. Our results showed a significantly higher fraction of highly motile bacteria in the presence of Hmm (Fig. 2e, f) that exhibited both increased vel- ocity and greater distance traveled (Fig. 2g, h). We also confirmed that expression of the fliC transcript was in- creased by Hmm (Additional file 7: Figure S6A) and that the difference in motility was not due to altered EHEC viability (Additional file 7: Figure S6B). Additionally, ΔfliC EHEC did not cause an increase in lesion area in the presence of Hmm, compared to Mmm, confirming our results and highlighting that an intact fliC gene is re- quired for expression of the different tolerance pheno- types we observed (Additional file 8: Figure S7). Species-specific injury effects are not due to changes in EHEC colonization or Shiga toxin Importantly, the tran- scriptional upregulation of these two latter genes has been directly correlated with higher bacterial motility [55, 56]. Taken together, these results suggest that Hmm preferentially stimulate the motility machinery of EHEC cells relative to Mmm, and that these changes in chemo- taxis could mediate the enhanced EHEC-induced patho- genicity we observed in the human Colon Chips. Species-specific injury effects are not due to changes in EHEC colonization or Shiga toxin We investigated genes involved in both cell and fla- gellar motility (see “Methods” section for details) due to their key role in directional cell movement and observed that multiple genes involved in these processes were up- regulated in the presence of Hmm. These include the tar and tap genes, which encode methyl-accepting chemotaxis protein (MCP) chemoreceptors; cheR and cheB that encode proteins that control MCP receptors; and cheA and cheW, which encode histidine kinase and the linker protein that associates with MCP receptors (Fig. 2c, d). In addition, the products of cheA and cheW form a complex that controls a protein encoded by cheY, which binds to the flagellar motor and promotes motility when phosphorylated (Fig. 2d). All of these genes were upregulated in the presence of Hmm compared to Mmm, and genes that encode components of the flagel- lar motor system itself, such as motA and fliC (Fig. 2d), were upregulated as well (Fig. 2c). Importantly, the tran- scriptional upregulation of these two latter genes has been directly correlated with higher bacterial motility [55, 56]. Taken together, these results suggest that Hmm preferentially stimulate the motility machinery of EHEC cells relative to Mmm, and that these changes in chemo- taxis could mediate the enhanced EHEC-induced patho- genicity we observed in the human Colon Chips. metabolites from the human gut microbiome can dir- ectly increase EHEC movement by stimulating flagellar motility. observed in the bacterial chemotaxis pathway (Fig. 2a, b). We investigated genes involved in both cell and fla- gellar motility (see “Methods” section for details) due to their key role in directional cell movement and observed that multiple genes involved in these processes were up- regulated in the presence of Hmm. These include the tar and tap genes, which encode methyl-accepting chemotaxis protein (MCP) chemoreceptors; cheR and cheB that encode proteins that control MCP receptors; and cheA and cheW, which encode histidine kinase and the linker protein that associates with MCP receptors (Fig. 2c, d). In addition, the products of cheA and cheW form a complex that controls a protein encoded by cheY, which binds to the flagellar motor and promotes motility when phosphorylated (Fig. 2d). All of these genes were upregulated in the presence of Hmm compared to Mmm, and genes that encode components of the flagel- lar motor system itself, such as motA and fliC (Fig. 2d), were upregulated as well (Fig. 2c). Species-specific injury effects are not due to changes in EHEC colonization or Shiga toxin Bacterial chemotaxis and motility are intimately linked to pathogenesis as these processes enable bacteria to de- tect and react to nutrients, sense cues from other bac- teria or the host, and reach their preferred niche [51, 52]. The majority of the intestinal pathogens possess many chemotaxis genes [53], suggesting that chemotaxis increases pathogen fitness and virulence in the gastro- intestinal tract. For this reason, there is increasing inter- est in the investigation of chemotaxis and motility as novel targets to fight infections and avoid problems re- lated to antibiotic resistance [54]. Likewise, EHEC pos- sesses chemotaxis and motility machineries that are used to sense molecules of bacterial or host origin, which can modulate bacterial movement (e.g., autoinducer-3, epi- nephrine, norepinephrine). To investigate whether en- hanced EHEC chemotaxis or motility could be responsible for the phenotype we observed, we carried out a transcriptomics analysis of EHEC bacteria present in the intestinal luminal compartment of human Colon Chips exposed to either Hmm or Mmm. Commensal bacteria of the gut microbiome have been previously shown to inhibit microbial infections by inter- fering with bacterial colonization of the intestinal epithelium via nutrient competition, secretion of antimi- crobials, or adhesion exclusion [13, 49]. For this reason, we investigated whether Hmm and Mmm may differ in their ability to prevent EHEC adhesion and colonization in the human Colon Chips. Results of fluorescence microscopic imaging and quantification of live adherent and non-adherent bacterial cells show that neither the overall distribution of the pathogenic EHEC bacteria (Additional file 4: Figure S4A) nor their adhesion to the epithelium (Additional file 4: Figure S4B) was altered by the presence of Hmm or Mmm. The numbers of non-adherent EHEC bacteria present in the medium ef- fluent collected from the epithelial lumen of the Colon Chip were also comparable in cultures containing Hmm and Mmm (Additional file 4: Figure S4C). Long polar fimbriae have been implicated in interac- tions between EHEC, Peyer’s patches, and M-cells in the terminal ileum where they may contribute to colonization, but they do not appear to be expressed in the colon [50]. Consistent with this observation, we These studies revealed that multiple EHEC genes are differentially regulated in the presence of Hmm com- pared to Mmm, with the greatest changes being Page 6 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (2019) 7:43 observed in the bacterial chemotaxis pathway (Fig. 2a, b). Identification of specific metabolites that mediate increased pathogenicity f Quantification of the fraction (%) of moving EHEC. g Mean velocity of each tracked bacterium (red and black: velocity < or > 3 μm s−1, respectively). h Distance traveled (μm) by the moving bacteria. i Fli-C-luciferase expression levels in medium supplemented with Hmm or Mmm [determined by quantifying area under the curve (AUC), and normalizing for the medium control]. p < 0.05; ***p < 0.0001 to chemotaxis and motility. Furthermore, using a meta- bolomics approach, we were able to identify four specific human gut microbiome products that were able to re- constitute the enhanced epithelial injury response ob- served with complex Hmm; addition of these four metabolites also was sufficient to convert the tolerant murine microbiome phenotype into an injury response that mimicked that produced by addition of the human microbiome products. Taken together, these data en- hance our understanding of how human commensal bacterial populations modulate intestinal pathogenesis in response to infection, and surprisingly reveal that some- times this can be a detriment to the host. analyzed the effects of each of these four compounds in- dividually over a range of concentrations (1 to 250 μM), we again observed consistent fliC-luciferase upregulation (Additional file 10: Figure S8), and all of these compounds stimulated EHEC motility when tested at 200 μM in a plate-based bacteria motility assay (Additional file 11: Figure S9). g To explore the generality of this response, we evalu- ated the effect of these four compounds in a different EHEC strain (serotype O91:H21) and found a very simi- lar increase in motility and fliC-luciferase expression to what we previously observed with the serotype O157:H7 EHEC strain, suggesting that this is a conserved mech- anism between different EHEC strains (Additional file 12: Figure S10 and Additional file 13: Figure S11). Import- antly, when all four of these compounds were combined at this same concentration and added to Colon Chips cultured in the presence of both EHEC and Mmm, we were able to reconstitute the higher levels of epithelial injury we previously observed in the presence of the complex mixture of Hmm (Fig. 4a–c). Moreover, the re- sponse we observed appears to result from a cumulative effect of the four metabolites as no single metabolite was able to increase epithelial damage when added alone (Additional file 14: Figure S12). Identification of specific metabolites that mediate increased pathogenicity From the final set of 30 compounds, we observed that 12 were present at higher levels in Mmm samples while there was a greater abundance of the remaining 18 in the Hmm set (Fig. 3c, Additional file 9: Table S2). To as- sess the impact of each one of these compounds on fla- gellar function and bacterial motility, we carried out a fliC-luciferase screening assay as previously described [56]. These studies revealed that only four of the se- lected compounds, which were all present at higher levels in the Hmm samples, significantly increased EHEC fliC-luciferase expression: 4-methyl benzoic acid, 3,4-dimethylbenzoic, hexanoic acid, and heptanoic acid (Fig. 3d). In contrast, we did not identify any metabolites in either the Hmm or Mmm samples that reduced fli- C-luciferase production, even though we could detect inhibition using the known fliC inhibitor, proanthocyani- din (PAC) [58] as a positive control (Fig. 3d). When we To corroborate these findings that Hmm stimulates flagellar motility in EHEC, we screened for metabolites that affect flagellar motility using an EHEC strain ex- pressing a fliC-luciferase reporter. Although both Hmm and Mmm increased fliC-luciferase expression when added to EHEC, its expression was significantly higher when the human metabolites were present (Fig. 2i). There were no distinguishable differences in EHEC fliC-luciferase growth under these same conditions, con- firming that the detected difference in luciferase signal is not due to differences in cell number (Additional file 7: Figure S6C). These results indicate that the presence of Page 7 of 20 (2019) 7:43 Tovaglieri et al. Microbiome Fig. 2 (See legend on next page.) Fi 2 (S l d t ) Fig. 2 (See legend on next page.) Page 8 of 20 Tovaglieri et al. Microbiome (2019) 7:43 (2019) 7:43 Tovaglieri et al. Microbiome (See figure on previous page.) Fig. 2 Human microbiome metabolites stimulate bacterial motility. a–d Changes in the EHEC transcriptome induced by exposure to human (Hmm) versus mouse (Mmm) gut microbiome metabolites. a Heatmap of differentially expressed genes (red indicates higher levels of expression). b Gene enrichment analysis. c Heatmap of chemotaxis and flagellar assembly pathways showing expression levels for relevant motility-related genes in EHEC cultured in the presence of Hmm versus Mmm. d Schematic of key genes critical in regulating chemotaxis and flagellar assembly in EHEC. e EHEC swimming motility tracking (lines: bacterial movement tracks; dots: starting points for all tracked bacteria; bar, 100 μm). Identification of specific metabolites that mediate increased pathogenicity Taken together, these re- sults show that these four metabolites produced by the human gut microbiome are responsible for increasing EHEC pathogenicity and enhancing epithelial injury in this model. Humans are susceptible to EHEC infection at a very low dose (102) [59], whereas the dose required to in- duce infections in mice is 100,000-fold higher. More- over, mice need microbiome depletion to show symptoms of infection, further suggesting a potential protective role played by commensal bacteria in mur- ine models [5–7]. Here, using microfluidic Organ Chip technology, we were able to recapitulate this species-specific difference in response to infection ob- served in humans versus mice in vivo. Similarly to what is observed in vivo, we discovered that Hmm worsens the outcome of EHEC infection, rather than Mmm inducing a tolerance response. Furthermore, by combining the Organ Chip technology with multi-omics data analysis, we were able to identify specific human microbiome metabolites that exacer- bate EHEC-induced injury of the intestinal epithelium independently of effects on bacterial growth or cyto- kine response. Specifically, we showed that exposure of EHEC to species-specific commensal metabolites alters transcription of chemotaxis-related genes and motility behavior in the bacterium, and ΔfliC EHEC lacking the fliC gene that are non-motile did not pro- duce differential epithelial lesions in the Hmm and Mmm groups. In addition, we identified four specific metabolites present in Hmm and not Mmm that upregulate a key motility-related virulence pathway involving flagellar motility. These findings are consist- ent with those of a past study which showed that flagellin is a key regulator of human intestinal inflam- mation after EHEC infection in vivo [10]. Discussion In this study, we successfully used Organ Chip technol- ogy to model injury of human colon epithelium by infection with EHEC in vitro, and to recapitulate species-specific differences in sensitivity to this pathogen by adding either human or mouse gut microbiome me- tabolites. Greater epithelial injury was observed when human metabolites were present, and interestingly, EHEC did not induce any lesion formation in the ab- sence of microbiome metabolites. This species-specific effect on epithelial damage occurred despite similar in- flammatory signatures in the Colon Chips and was, in- stead, associated with an increase in expression of EHEC genes associated with known virulence pathways related Page 9 of 20 Tovaglieri et al. Microbiome (2019) 7:43 (2019) 7:43 Tovaglieri et al. Microbiome Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) egend on next page.) Fig. 3 (See legend on next page ) Fig. 3 (See legend on next page.) Page 10 of 20 (2019) 7:43 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (See figure on previous page.) Fig. 3 Identification of specific metabolites that mediate EHEC motility. a–c Results of metabolomics analysis of human versus mouse gut microbiome metabolites. a Venn-diagram illustrating metabolomics analysis workflow and total numbers of compounds identified in the Hmm and Mmm samples compared to the pre-fermentation medium (Pre-ferm.; label p_25: human pre-fermentation medium; label p_26 murine pre- fermentation medium). b Heatmap of 426 compounds produced by commensal bacteria that were differentially abundant in human (Hmm) versus mouse (Mmm) microbiome metabolites. c Relative abundance of 30 microbiome metabolites that were tested (blue and red: higher levels in Mmm or Hmm, respectively). d Results of FliC- luciferase (FliC-lux) screening for the 30 selected metabolites (FliC-lux levels are presented based on quantification of the AUC; grape seed oligomeric proanthocyanidins (PAC) was used as a negative control; the 4 active metabolites that induced higher FliC levels are highlighted in red; all values were normalized against the DMSO control) to infection (e.g., by testing the effect of dietary nutrients on the composition and metabolic activity of cultured gut microbiota, [63]). The other two compounds we identified that increased epithelial injury are medium-chain fatty acids (heptanoic and hexanoic acid). Interestingly, fecal hexanoic acid concentrations have been found to extend over a much higher range in chil- dren than in adults [64–66], and it is known that chil- dren are more susceptible to EHEC infection. Discussion Taken together with literature showing that short-chain fatty acids influence EHEC virulence factor expression and change EHEC infection outcomes in mice [12], these data highlight a key role for microbiome-derived fatty acids in EHEC pathogenesis. Improving our understand- ing of these host-microbiome-pathogen interactions could provide insight into why certain human groups control EHEC infection while others do not. Although we used metabolic analysis to pursue the mechanism by which Hmm and Mmm produce different effects on EHEC-induced epithelial injury, we only fo- cused on known metabolites because these compounds could be obtained commercially and tested experimen- tally to validate their effects. It is possible that other un- known microbiome-derived metabolites present in the Hmm sample may have additional modulating activities, which could be explored in the future using fraction- ation of the Hmm sample and in-depth mass spectrom- etry analysis. Nevertheless, our untargeted metabolomics approach led to the identification of important EHEC injury-enhancing functions for four known molecules that were found at significantly higher levels within Hmm versus Mmm. Importantly, a similar experimental approach could be used to identify microbiome-derived modulators of other enteropathogens that exhibit species-specific differences in pathogenicity in the future. This general strategy of comparing how species-specific microbiome metabolites influence host- pathogen interactions in controlled experimental settings could prove to be effective in uncovering new functions for other microbiome metabolites. It also might offer new mechanistic insights into why certain species are more tolerant to specific infectious pathogens than others in the future. This work with Organ Chip technology represents an initial step toward developing advanced in vitro human disease models that can enable direct investigation into the cellular and molecular basis of human host-micro- biome-pathogen relationships. The human Colon Chip model of EHEC infection described here only included living epithelium and endothelium with soluble microbiome-derived metabolites, and thus, we were not able to examine mechanisms related to direct interac- tions between living commensal microbes and bacterial pathogens, between the microbes and the host cells, or to address questions centered on contributions of im- mune cells. However, these are feasible future directions for study as Organ Chip technology can incorporate multiple layers of complexity at the cell, tissue, and organ levels [18], including co-culture of human intes- tinal epithelium with complex living gut microbiome [67]. Discussion p < 0.05; *p < 0.01 from four healthy wild-type C57BL/6 mice cohoused in the same cage. These commensal bacteria were immobi- lized as described [33] and used to inoculate the Poly- FermS platform. In humans, the proximal colon is the main site of carbohydrate fermentation [70], whereas in mice, this mainly takes place in the cecum [71, 72]. There- fore, we isolated Hmm and Mmm from the final stage of PolyFermS fermentation reactors that respectively emulate the contents of human proximal colon and mouse cecum colonized by their species-specific microbiomes. The Poly- FermS pre-fermentation medium for the proximal colon was based on the composition described by Macfarlane et al. 1998 for simulation of the intestinal content [73]. It in- cludes (g l−1 of distilled water) pectin (citrus) (2), mucine (4), L-cysteine HCl (0.8), bile salts (0.4), KH2PO4 (0.5), NaHCO3 (1.5), NaCl (4.5), KCl (4.5), MgSO4 anhydrated (0.61), CaCl22 H2O (0.1), MnCl2* 4 H2O (0.2), FeSO4* 7H20 (0.005), hemin (0.05), and Tween 80 (1 ml). One milliliter of a filter-sterilized (0.2 μm pore-size) vitamin so- lution was added to the sterilized (20 min, 120 °C) and cooled down medium. The final concentration of vitamins in PolyFermS medium (μg l−1) are pyridoxine-HCl (Vit. B6) (100); 4-aminobenzoic acid (PABA) (50); nicotinic acid (Vit. B3) (50); biotine (20); folic acid (20); cyanocobalamin (5); thiamine (50); riboflavin (50); phylloquinone (0,075); menadione (10); and pantothenate (100). Human pre-fermentation medium included (g l−1 of distilled water) xylan (oat spelts) (2), arabinogalactan (larch) (2), guar gum (1), inulin (1), soluble potato starch (5), casein acid hydrol- ysate (3), peptone water (5), Bacto tryptone (5), and yeast extract (4.5). The PolyFermS pre-fermentation medium for the mouse cecum model was also based on the compos- ition described by Macfarlane et al. (1998), modified for its carbohydrate and protein concentrations for mimicking the chyme of a mouse, accounting for the mouse chow and different digestion physiology [33]. Murine pre-fer- mentation medium included (g l−1 of distilled water) xylan (oat spelts) (4.8), arabinogalactan (larch) (4.8), soluble corn starch (4), mucine (4), casein acid hydrolysate (3.6), pep- tone water (6), tryptone (6), yeast extract (5.4), and guar gum was omitted (see Additional file 15: Table S3 for ma- terial details). Human PolyFermS reactors were operated at proximal from the same patient, and thus, significantly advances personalized medicine in the future. Discussion Given that the Colon Chip has easily accessible in- testinal and vascular channels that can be sampled indi- vidually, it also could be used to investigate mucus physiology, effects of varying oxygen gradients and con- centrations, and other critical components of EHEC in- fection and pathogenesis [28, 68, 69]. Because Organ Chips can be made using epithelial cells isolated from patient-derived organoids as we did here, this approach also can be used to explore interactions between the host epithelium and commensal microbiome isolated Microbiome metabolites are products of the break- down of dietary foods and nutrients derived from deg- radation of host molecules (e.g., mucin) by commensal microbes. Two of the compounds identified (4-methyl- benzoic acid, 3,4-dimethylbenzoic acid) are structurally simple phenolic compounds, which can be generated by the degradation of more complex phenolic compounds, such as caffeic acid, ferulic acid, and tannins contained in coffee, cereal, and grapes [60–62]. Interestingly, these molecules are often promoted as having health benefits based on their anti-oxidant effects; however, our results raise the possibility that they could have negative effects on health if they are metabolized into products that en- hance the pathogenicity of EHEC. Taken together, these findings emphasize how further investigation of dietary compounds altering microbiome metabolite production could improve our understanding of how diet alters the intestinal environment and influences patient responses Page 11 of 20 Tovaglieri et al. Microbiome (2019) 7:43 (2019) 7:43 Tovaglieri et al. Microbiome Fig. 4 (See legend on next page.) Fig. 4 (See legend on next page.) Page 12 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (See figure on previous page.) Fig. 4 The identified active metabolites mediate increased pathogenicity. a–c Effect of 3,4-dimethylbenzoic acid, 4-methylbenzoic acid, hexanoic acid, and heptanoic acid (4 metab.) on epithelial injury in the Colon Chip in the presence or absence of EHEC, with or without Mmm, compared to the effects of Hmm with EHEC. a Representative DIC images of the colon epithelium under the various experimental conditions (bar, 100 μm). b Pseudocolored view of the entire epithelial layer in the Colon Chip (yellow) under the same conditions. c Quantification of epithelial lesion area size under conditions shown in b. Epithelial lesion defined as regions in which cells normally contained within a continuous intact epithelium have fully detached from the ECM-coated membrane and their neighboring cells, thus, leaving exposed regions of the membrane below. Discussion In addition, this in vitro human infection model could be used to develop new biomarkers for susceptibility to infection as well as therapeutics or prophylactics that target microbiome-pathogen interactions, and thereby protect against enteric bacterial infections in an antibiotic-independent manner. Conclusion Application of Organ Chip technology to create a hu- man Colon Chip, combined with microbial metabolites isolated from microbial fermentation reactors (Poly- FermS), allowed analysis of pathogen-microbiome-hu- man host interactions under in vitro conditions that emulate human intestinal physiology in vivo. The gut microbiome products identified as active were sufficient to recapitulate species-specific differences in response to EHEC infection, with greater injury being observed when human metabolites were present compared to murine metabolites. The four human gut microbiome products we identified—4-methyl benzoic acid, 3,4-dimethylbenzoic, hexanoic acid, and heptanoic acid—were sufficient to convert the tolerant murine microbiome phenotype into a higher injury response comparable to the one observed in the presence of hu- man microbiome products. This study offers new mech- anistic insights in EHEC pathogenesis mediated by human microbiome metabolites, which also could po- tentially explain different susceptibilities within different human populations, such as the greater sensitivity ob- served in children. These findings also provide a basis to explore therapeutic and prophylactic modulation of hu- man intestinal contents in order to protect from infec- tion by this potentially life-threatening pathogen. Organoid cultures Human intestinal epithelium was isolated from resec- tions or endoscopic tissue biopsies from two females and one male. Resections, consisting of full thickness pieces of human colon, were obtained anonymously from healthy regions of colonic resection specimens processed in the Department of Pathology at Massachu- setts General Hospital under an existing Institutional Review Board approved protocol (#2015P001859). Speci- mens were restricted to healthy (non-neoplastic) disease samples, and tissue was taken from healthy normal re- gions as determined by careful gross examination. Endo- scopic biopsies were collected from macroscopically normal (grossly unaffected) areas of the colon of de-identified patients undergoing endoscopy for abdom- inal complaints. Informed consent and developmentally appropriate assent were obtained at Boston Children’s Hospital from the donors’ guardian and the donor, re- spectively. All methods were carried out in accordance with the Institutional Review Board of Boston Children’s Hospital (Protocol number IRB-P00000529) approval. p p ( ) After 7 days, HIMEC were seeded in the vascular side of the Colon Chip (250,000 cells/chip) in expansion medium supplemented with human epidermal growth factor, vas- cular endothelial growth factor, human fibroblastic growth factor-B, R3-Insulin-like growth factor-1, ascorbic acid, and no antibiotics. To allow endothelial cells to adhere to the membrane, chips were inverted under static condi- tions for 1 h, and then the chips were placed upright and perfusion was restored. After 24 h, the vascular channel medium was switched to recombinant organoid expansion medium. Meanwhile, the intestinal luminal channel medium was switched to 5% (vol vol−1) human (Hmm) or mouse (Mmm) gut microbiome metabolites isolated from PolyFermS bioreactors, diluted in phosphate-buffered sa- line (PBS) containing calcium and magnesium (final osmolarity = 300 mOsm kg−1), filtered through a 0.2 μm filter (Corning), and stored at −80 °C. Recombinant orga- noid expansion medium consists of organoid expansion medium except that of L-WRN conditioned medium was substituted with recombinant Wnt-3a (100 ng ml−1), mur- ine noggin (100 ng ml−1), murine R-spondin-1 (1 μg ml−1), Tissues from the resections were dissected to detach the epithelium, and the epithelial layer or the entire biopsy was digested with 2 mg ml−1 collagenase I for 40 min at 37 °C followed by mechanical dissociation, as previously described [29]. Organoids were grown em- bedded in expansion medium in growth factor-reduced Matrigel [29, 74]. Experimental model and subject details PolyFermS The supernatant was filter sterilized using a 0.2 μm fil- ter (Minisart-plus 0.2 μm filter, Satorius Stedim Biotech GmbH, Goettingen, Germany) and stored in 1 ml ali- quots at −80 °C until usage. Colon Chip cultures p Organ Chips composed of poly-dimethylsiloxane (PDMS) were obtained from Emulate Inc. (Boston, MA). The Organ Chips consist of two parallel microchannels (1000 × 1000 μm and 1000 × 200 μm; width × height) separated by a thin (50 μm) porous membrane (7 μm pore diameter, 40 μm spacing). Organ Chips were ac- quired from Emulate Inc. (Boston, MA; Cat#10231-2) and activated using ER-1 and ER-2 solutions provided by the company before being coated with type I collagen (200 μg ml−1) and Matrigel (1% in PBS), as described [29]. Colonic organoids were fragmented by incubating for 2 min at 37 °C in Triple E express diluted in PBS 1:1 (vol:vol) supplemented with 10 μM Y-27632, and seeded on the ECM-coated membrane in the intestinal luminal channel of the Colon Chip (210,000 cells/chip) in expansion medium supplemented with 10 μM Y-27632. Chips were incubated overnight at 37 °C in expansion medium. The following day expansion medium was perfused with cell culture medium at 60 μl h−1 through top and bottom channels. Medium flow was driven at a constant flow rate using a peri- staltic pump (Ismatec; Cat#ISM938D). Experimental model and subject details PolyFermS The gut microbial metabolite suspensions used in this study were generated by the PolyFermS platform, a vali- dated continuous in vitro intestinal fermentation model [30](ETH Zürich). Two distinct sources of human and mouse gut microbiome were utilized: feces from a single healthy human adult and murine cecal content pooled Human PolyFermS reactors were operated at proximal colon conditions (pH 5.8, 37 °C) and a hydraulic retention time of 8 h [33]. Murine PolyFermS reactors were Page 13 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (2019) 7:43 A83-01, and primocin (100 μg ml−1) [74, 75] (see Additional file 15: Table S3 for material details). A83-01, and primocin (100 μg ml−1) [74, 75] (see Additional file 15: Table S3 for material details). operated at murine cecal conditions (pH 6.5, 37 °C) and hydraulic retention time of 12 h [33]. In both models, re- actor headspace was continuously flushed with sterile CO2 to control for anaerobiosis. After operating in con- tinuous mode for at least 10 days, microbial composition in both systems remained stable for collection of effluents for the Colon Chip experiments, as indicated by stable base consumption, fermentation metabolite production, and bacterial population composition [33]. Therefore, the PolyFermS effluents used for the Colon Chip experi- ments were derived after this 10-day stabilization period. Fresh effluents were collected from PolyFermS reactor, centrifuged at 16,000×g for 10 min in a pre-cooled centrifuge (Heraeus Biofuge Primo 230, MultiTemp Scientific AG, Kloten, Switzerland) at 4 °C. The supernatant was filter sterilized using a 0.2 μm fil- ter (Minisart-plus 0.2 μm filter, Satorius Stedim Biotech GmbH, Goettingen, Germany) and stored in 1 ml ali- quots at −80 °C until usage. operated at murine cecal conditions (pH 6.5, 37 °C) and hydraulic retention time of 12 h [33]. In both models, re- actor headspace was continuously flushed with sterile CO2 to control for anaerobiosis. After operating in con- tinuous mode for at least 10 days, microbial composition in both systems remained stable for collection of effluents for the Colon Chip experiments, as indicated by stable base consumption, fermentation metabolite production, and bacterial population composition [33]. Therefore, the PolyFermS effluents used for the Colon Chip experi- ments were derived after this 10-day stabilization period. Fresh effluents were collected from PolyFermS reactor, centrifuged at 16,000×g for 10 min in a pre-cooled centrifuge (Heraeus Biofuge Primo 230, MultiTemp Scientific AG, Kloten, Switzerland) at 4 °C. Endothelial cell culture Human intestinal microvascular endothelial cells (HIMECs) were obtained from ScienCell (Cat#2900) ex- panded in Microvascular Endothelial Cell Growth Medium-2 BulletKit (EGM-2MV). Endothelial cells were subcultured less than five times before use. Methods details Colon Chip infection Colon Chips were cultured in the intestinal lumen chan- nel of the chip in 5% (vol vol−1) human or mouse gut microbiome metabolites isolated from PolyFermS biore- actors, diluted in phosphate-buffered saline (PBS; final osmolarity = 300 mOsm kg−1) for 24 h. The following day, the intestinal channel was infected with 1.7 × 105 EHEC-GFP or EHEC ΔfliC (both generated from NR-3 E. coli / EDL931; serotype O157:H7), by adding the bac- teria to into the channel lumen in medium again with or without Hmm or Mmm. Chips were maintained under static conditions for 3 h to promote EHEC colonization, and then perfused at 60 μl h−1. Bacterial growth conditions EHEC-GFP was generated from NR-3 Escherichia coli, EDL931, serotype O157:H7, transformed with pGEN-GFP(LVA) CbR plasmid. The EHEC fliC-lucifer- ase strain was generated by transforming EHEC EDL931 (serotype O157:H7) and EHEC B2F1 (serotype O91:H21) with fliC-luciferase plasmid kindly provided by H.L. Mobley, as described [56]. EHEC ΔfliC was generated from NR-3 Escherichia coli, EDL931, serotype O157:H7, transformed with a DNA fragment from K12 Keio col- lection fliC single-gene knockout [76], and the correct insertion was confirmed by PCR and sequencing (see Additional file 15: Table S3 for details). EHEC, EHEC-GFP, and EHEC ΔfliC were grown to 0.5 McFar- land (1.5 × 108 CFUs) in RPMI medium supplemented with 10% glucose, centrifuged, and stored at −80 °C in saline 10% glycerol, while EHEC fliC-luciferase bacteria were grown in Luria Broth (LB) containing (g l−1 of dis- tilled water) Bacto Tryptone (10), Bacto Yeast Extract (5) and sodium chloride (10), and the appropriate antibiotic; the following day, the bacteria were diluted or resuspended in medium and at the dose indicated, ac- cording to the experimental need (see Additional file 15: Table S3 for material details). All experiments were car- ried with NR-3 E. coli/EDL931 (serotype O157:H7) if not otherwise indicated. 16S rRNA gene sequencing Genomic DNA of PolyFermS microbiota was extracted with the FastDNA® SPIN Kit for soil (MP Biomedicals Europe, Illkirch, France) following the protocol of the supplier. Pellets of 2 ml PolyFermS effluent were resus- pended in MT lysis buffer and disrupted in Lysing Matrix E tubes with the Omni Bead Ruptor 24 (OMNI International, Kennesaw, United States) at 16 m s−1 for 40 s. Quality of genomic DNA was assessed by 1.5% (m/ v) agarose electrophoresis and concentration was deter- mined by Nanodrop ND-100 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). DNA samples were diluted in nuclease-free water to 20 ng μl−1 and stored at 4 °C until further processing. The V4 re- gion of the 16S rRNA gene was amplified by PCR with forward 515F (GTG CCA GCM GCC GCG GTA A) and reverse 806R (GGACT ACH VGG GTW TCT AAT) pri- mer (Caporaso et al. 2011). Negative controls with PCR water as template were included. Library preparation and sequencing was conducted at the Genetic Diversity Center (GDC, ETH Zurich, Switzerland) following a pre- viously described protocol for library preparation [78]. Sequencing was performed using an Illumina MiSeq flow cell with V2 2 × 250 bp paired end chemistry sup- plemented with 20% of PhiX. Shiga toxin quantification A Shiga toxin ELISA was performed on several dilutions of outflow from the vascular channel of Colon Chips at 6 h following EHEC infection. The assay was performed following the protocol of the supplier (Abraxis). Organoid cultures Expansion medium consists of ad- vanced DMEM F12 supplemented with L-WRN (Wnt3a, R-spondin, noggin) conditioned medium (65% vol vol−1), glutamax, 10 mM HEPES, murine epidermal growth fac- tor (50 ng ml−1), N2 supplement, B27 supplement, 10 nM human [Leu15]-gastrin I, 1 mM n-acetyl cysteine, 10 mM nicotinamide, 10 μM SB202190, 500 nM Tovaglieri et al. Microbiome (2019) 7:43 Page 14 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome cells and the total area of the chip were measured using Fiji software [77]. and supplemented with human epidermal growth factor, vascular endothelial growth factor, human fibroblastic growth factor-B, R3-insulin-like growth factor-1, ascorbic acid, and no antibiotics (see Additional file 15: Table S3 for material details). fliC-luciferase reporter assay The EHEC bacteria were grown overnight in LB medium diluted 1:1000 and the fliC-luciferase assay was carried as described [58]. Compound screening was carried at a concentration of 10 μM of the compound diluted in dimethyl sulfoxide (DMSO; final concentration 0.1% vol vol−1) or an equivalent volume of DMSO as a vehicle control. Grape seed oligomeric proanthocyanidins (PAC) was used as negative control at a dose of 100 μg ml−1 [58]. All compounds used for motility screening were purchased from MedChemExpress except DiHOME (Cayman Chemicals) and PAC (Sigma). Compounds were dosed from 0.97 to 250 μM, followed by sealing the plate with a gas-permeable membrane (EK Scientific) and incubating at 37 °C in a Synergy HT Microplate Reader. Luciferase luminescence and optical density at 600 nm wavelength (OD600) were measured at 20-min interval for 11 h. Epithelial lesion analysis One day post infection, Colon Chips were washed with PBS and fixed with 4% paraformaldehyde in PBS for 2 h. The Chips were imaged using a Leica DM IL LED microscope and images were stitched together with Basler Phylon Software. The area occupied by Page 15 of 20 Page 15 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome Bacterial motility tracking y g EHEC-GFP bacteria were grown 6 h at 37 °C in Hmm or Mmm, then transferred to plasma-treated cover slips, and imaged using a Zeiss Axio Observer Z1 microscope for 3 min, as described [57]. The videos were then proc- essed using Fiji, an image processing package of ImageJ, StackReg to stabilize the video, cropped to remove video edges, and particles were tracked using TrackMate plu- gin [77, 79, 80]. Particles tracked for less than 1 s were removed from the analysis. Bacteria with a speed higher than 3 μm s−1 were considered motile. We tracked a total of 1255 (61 motile) and 519 (157 motile) bacteria in the Mmm and Hmm groups, respectively. TrackMate mean velocity was calculated as the mean of the instant- aneous velocity and distance traveled shown over the total video time (3 min). For better visualization of the full particle tracks, we changed the color Look-Up Table to have a white background using Fiji and applied a minimum filter in Adobe Photoshop to widen the tracks path and make it visible once the image size was re- duced for publication purposes. Metabolomics sequence to allow for multiplexing (custom oligo order from Integrated DNA Technologies). Excess PCR re- agents were removed using magnetic bead-based cleanup on an Apollo324 automated workstation (PCR Clean DX beads, Aline Biosciences). Resulting libraries were assessed using a 2200 TapeStation (Agilent Tech- nologies) and quantified by qPCR (Kapa Biosystems). Li- braries were pooled and sequenced on four lanes of a HiSeq 2500 v4 high output flow cell using single end, 50 bp reads (Illumina, Inc.). Samples were centrifuged at 10,000×g for 5 min followed by biphasic chloroform-methanol extraction. All samples were run for untargeted mass spectrometry on a Ther- moFisher Q-exactive mass spectrometer (Small Mol- ecule Mass Spectrometry Facility, FAS Division of Science Operations Harvard University). Compound Discovery Software was utilized to assign compound names (95% confidence). If the parent ion was not found, the compound with the closest spectrum was used as an identifier, thus indicating a potential sub- structure of the original metabolite. In the case of mul- tiple metabolites matching to the same identifier, priority was given to the metabolite identified with the highest average area value. From our analysis, we identi- fied 426 metabolites enriched in either Hmm or Mmm, and selected all the metabolites with an assigned compound name. Within these metabolites, we se- lected all 30 commercially available compounds, while excluding known synthetic prescription drugs, anti- microbial agents, or potential chemical contaminants (Additional file 9: Table S2) and screened them for their effect on EHEC flagellar motility. Cytokines/chemokines analysis Levels of cytokines and chemokines within medium col- lected from the effluent of the vascular channel were measured using MSD U-plex Assay (Meso Scale Diag- nostic). Medium samples were collected 6 h post EHEC infection (3 h after restoring the flow to the Colon Chip). RNA isolation and gene expression Endothelial cells were first removed using Trypsin- EDTA (0.25%) from the vascular channel of the Colon Chip. Epithelial cells were then isolated from the intes- tinal luminal compartment and RNA was isolated using an RNAeasy Mini Kit. For qPCR measurement, cDNA was synthetized using SuperScript IV VILO Master Mix (Thermo Fisher Scientific) and primers (Additional file 15: Table S3) and Powerup SYBR Green Master Mix (Thermo Fisher Scientific) were utilized for amplifica- tion. For RNA seq analysis, the RNA concentration was measured using a Qubit instrument and Quant-it re- agents (Thermo Fisher Scientific). RNA purity was assessed by measuring the ratio of absorbance at 260/ 230 nm, and 260/280 nm on a Nanodrop Instrument (Thermo Fisher Scientific). RNA integrity was measured using TapeStation 2200 (Agilent Technologies). Bacterial and human ribosomal RNA was depleted from total RNA samples using an Epidemiology Ribo-Zero Gold rRNA Removal kit (Illumina, Inc.) on an Apollo324 au- tomated workstation (Takara Bio USA). The resulting ribosomal-RNA-depleted RNA samples were immedi- ately converted into stranded Illumina sequencing librar- ies using 200 bp fragmentation and sequential adapter addition on an Apollo324 automated workstation follow- ing manufacturer’s specifications (PrepX RNA-seq for Illumina Library kit, Takara Bio, USA). Libraries were enriched and indexed using 15 cycles of amplification (LongAmp Taq 2x MasterMix, New England BioLabs Inc.) with PCR primers which include a 6 bp index Quantification of bacterial numbers Q To quantify adherent bacteria, epithelial cells were washed with PBS and isolated with Trypsin-EDTA (0.25%)–Type IV Collagenase (1 mg ml−1) 20 min at 37 °C. Adherent bacteria and bacteria contained within samples of medium effluent, collected 6 h post infection, were diluted and plated on soy agar plates with sheep blood with an Eddy Jet 2 automated spiral plater (UL Instruments). Plates were incubated overnight at 37 ° C and colony-forming units (CFUs) were quantified using a Flash & Go automatic colony counter (UL Instruments). Page 16 of 20 Page 16 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome Analysis of metabolomics Raw data were normalized using R metabolomics pack- age [89] and groups compared using Linear Models for Microarray Data (limma) package [90]. We applied a cut off of 0.05 on adjusted p value and fold change greater than 1.5. Colon Chip epithelium and bacteria imaging The data was processed using bcbio-nextgen. We used the STAR alignments, FASTWQ files, and Salmon quan- tification to generate quality control metrics of the sam- ples [91, 92]. Differential expression was computed using DESeq2 package in R [93]. Genes were considered differentially expressed based on both an absolute fold-change larger than 1.5 and an FDR-corrected p value cutoff less than 0.05. We performed pathway en- richment on this gene signature using the enrichKEGG function in the clusterProfiler package in R [94]. To compare gene clusters associated with a KEGG pathway, we used the compareCluster function in the clusterProfi- ler package with a p value cutoff less than 0.001. Repre- sented pathways are based on the following KEGG IDs from the E. coli O157:H7 EDL933 annotations: Bacterial chemotaxis pathway (KEGG ID: ece02030), Cellular mo- tility (KEGG ID: 09142: chemotaxis + flagellar assembly), Pathogenic E. coli infection genes (KEGG ID: ece05130), Arginine and proline metabolism (KEGG ID: ece00330), Galactose metabolism (KEGG ID: ece00052), and Sulfur metabolism (KEGG ID: ece00920). Colon Chips infected with EHEC-GFP and uninfected controls were washed with PBS and fixed with 4% para- formaldehyde for 2 h. Following fixation, epithelial cells and bacteria were labeled with Alexa Fluor 647 Phal- loidin, 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), and anti-green fluorescent protein-Alexa Fluor 488 conjugate. Images were acquired with an inverted laser-scanning confocal microscope (Leica SP5 X MP DMI-6000) and processed using IMARIS. Quantification and statistical analysis Analysis of fliC-luciferase reporter assay data y y Raw luciferase signal at each time point was divided by OD600 to control for bacterial growth. The area under the curve (AUC) was calculated and all the statistics were performed using R language and environment for statistical computing [83]. Each compound was run in quadruplicate and the screening repeated three times. A Mann–Whitney–Wilcoxon test was performed followed by Bonferroni correction for multiple comparisons [84– 86]. Significant differences were selected with fold change higher than 20% and adjusted p value < 0.0001. We then generated dose curves for the newly identified compounds modulating fliC-luciferase serially diluting 1:2 from 250 to 0.97 μM. For fliC-luciferase compound dose curves, each dot indicates the mean and the stand- ard error of the mean (SEM). Bacteria viability corresponding quality scores were merged and trimmed using fastq_mergepairs and fastq_filter scripts imple- mented in the UPARSE pipeline [87]. The minimum overlap length of trimmed reads (150 bp) was set to 50 bp. The minimum length of merged reads was 150 bp. The max expected error E = 2.0, and first truncating position with quality score N ≤4. We used the DADA2 pipeline in R to process the data and ob- tain the operational taxonomic units (OTU) table (Additional file 16: Table S4), following the described protocol [88]. The resulting data was analyzed using custom scripts in R. EHEC-GFP bacteria were grown 6 h at 37 °C in medium containing Hmm or Mmm, then propidium iodide solu- tion was added at a final concentration of 10 mg ml−1 for 5 min at room temperature as reported [81]. Bacter- ial GFP and propidium iodide were imaged using a Zeiss Axio Observer Z1 microscope; the dead bacteria fraction was calculated as PI positive bacteria divided by the total number of bacteria. Bacteria swimming plate assay Swimming motility was assessed using 0.25% agar LB plates. Overnight cultures of EHEC or EHEC-GFP bac- teria were standardized at 1 OD600 and 1.5 μl of the culture medium was added to the center of the agar plate with a sterile pipette tip as described [82]. Bacterial swimming was quantified at 12 h, imaging the plates using a FluorChem M imaging system (ProteinSimple). The area occupied by bacteria was then measured using Fiji [77]. Statistics All statistical analyses were carried out in R using cus- tom scripts (see Additional file 15: Table S3 for software and algorithms details). The Mann–Whitney test was used to compare lesion area of Colon Chips, cytokines expression, percentage of moving bacteria, their velocity, distance traveled, qPCR, and bacterial motility plate assay. For the fliC-luciferase screening, significance was calculated using a Mann–Whitney test, with p values ad- justed for multiple comparisons using the Bonferroni method. All boxplots represent median, first, and third Analysis of 16S rRNA gene sequencing The raw data set containing pair-ended reads with corre- sponding quality scores were merged using settings as previously described [33]. Briefly, pair-end reads with Page 17 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome quartile of the data distribution, with whiskers extending to the largest value no further than 1.5 times the inter-quartile range. Bar plots represent mean value of the data with SEM; dots in bar- or box-plots indicate the sample size N for each experiment. For Colon Chips ex- periments, N is equal to the number of chips used; for the fliC-luciferase experiment, N corresponds to a single well; for the quantification of the fraction of moving bac- teria, N indicates the number of videos analyzed; for all the other bacterial tracking experiments, N indicates a single bacterium tracked; for plate-based swimming as- says, N indicates an individual plate. levels (determined by quantifying the AUC and normalizing for the DMSO control) of 4-methylbenzoic acid, 3,4 dimethylbenzoic acid, hexanoic acid, and heptanoic acid metabolites measured at indicated concentrations. (TIF 1914 kb) levels (determined by quantifying the AUC and normalizing for the DMSO control) of 4-methylbenzoic acid, 3,4 dimethylbenzoic acid, hexanoic acid, and heptanoic acid metabolites measured at indicated concentrations. (TIF 1914 kb) Additional file 11: Figure S9. The 4 identified active metabolites increase EHEC motility in a plate-based swimming assay. (A, B). Effects of 3,4-dimethylbenzoic acid, 4-methylbenzoic acid, hexanoic acid, and heptanoic acid (all at 200 μM) individually on EHEC-GFP swimming motility. (A) Photographic image of the plate containing EHEC-GFP bacteria (white) cultured with each of the 4 metabolites (black: plate background; blue arrows indicate the edge of the area occupied by bacteria). (B) Quantification of the area occupied by EHEC-GFP in A. p < 0.01. (TIF 2040 kb) Additional file 11: Figure S9. The 4 identified active metabolites increase EHEC motility in a plate-based swimming assay. (A, B). Effects of 3,4-dimethylbenzoic acid, 4-methylbenzoic acid, hexanoic acid, and heptanoic acid (all at 200 μM) individually on EHEC-GFP swimming motility. (A) Photographic image of the plate containing EHEC-GFP bacteria (white) cultured with each of the 4 metabolites (black: plate background; blue arrows indicate the edge of the area occupied by bacteria). (B) Quantification of the area occupied by EHEC-GFP in A. p < 0.01. (TIF 2040 kb) Additional file 12: Figure S10. Each of the 4 identified metabolites increases fliC expression in EHEC serotype O91:H21. Analysis of 16S rRNA gene sequencing FliC-luciferase levels (determined by quantifying the AUC and normalizing for the DMSO control) of 4-methylbenzoic acid, 3,4 dimethylbenzoic acid, hexanoic acid, and heptanoic acid metabolites at a concentration of 200 μM. *p < 0.001 (TIF 1525 kb) Availability of data and materials RNA-seq data have been deposited to the Sequence Read Archive (accession: PRJNA497914). 16S rRNA gene data are available in Additional file 16: Table S4. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Donald E. Ingber (don.ingber@wyss.harvard.edu). Additional file 9: Table S2. List of 30 known metabolites enriched in Hmm compared to Mmm that were selected for fliC-luciferase screening (CAS n: Chemical Abstracts Service number; “name”: metabolites with a known name; “similarity”: closest MSMS spectrum in the reference database to the analyte, with a 95% confidence in identification). (XLSX 10 kb) Additional files Additional file 1: Figure S1. Analysis of relative abundance of PolyFermS commensal bacteria phyla. Relative abundance of phyla measured in the last stage of human and murine microbial fermentation in PolyFermS. (TIF 2104 kb) Additional file 13: Figure S11. The 4 identified active metabolites increase motility in a plate-based swimming assay in EHEC serotype O91:H21. (A, B) Effects of 3,4-dimethylbenzoic acid, 4-methylbenzoic acid, hexanoic acid, and heptanoic acid (all at 200 μM) individually on EHEC (serotype O91:H21) swimming motility. (A) Bright field photographic image of the plate containing EHEC bacteria (white) cultured with each of the 4 metabolites (black: plate background; blue arrows indicate the edge of the area occupied by bacteria). (B) Quantification of the area occupied by EHEC in A. p < 0.01. (TIF 1998 kb) Additional file 2: Figure S2. Analysis of relative abundance of PolyFermS commensal bacteria genera. Relative abundance of genera measured in the last stage of human and murine microbial fermentation in PolyFermS. (TIF 3460 kb) Additional file 3: Figure S3. EHEC infection of Colon Chips in the absence of microbial metabolites. Analysis of EHEC-induced epithelial injury on-chip. (TIF 1389 kb) Additional file 14: Figure S12. The compound effect of the identified metabolites mediates increased pathogenicity. Effect of 3,4- dimethylbenzoic acid, 4-methylbenzoic acid, hexanoic acid, heptanoic acid individually and together (4 metab.) on epithelial injury in the Colon Chip with Mmm. Quantification of epithelial lesion area size. Data compounded from 3 experiments. *p < 0.05. (TIF 1541 kb) Additional file 4: Figure S4. Species-specific injury effects are not due to changes in EHEC colonization. (A-C) EHEC colonization of the human Colon Chip. (A) Representative fluorescence images showing the epithelial layer of infected and control Colon Chips in the presence or absence of Hmm or Mmm, with or without EHEC present (red: F-actin, green: GFP-EHEC, white: nuclei; bar, 100 μm). (B) Quantification of EHEC bacteria adherent to the intestinal epithelium. (C) Quantification of non- adherent EHEC quantification floating in the culture medium (TIF 5673 kb) Additional file 15: Table S3. Reagents and resources. (XLSX 16 kb) Additional file 15: Table S3. Reagents and resources. (XLSX 16 kb) Additional file 16: Table S4. 16 s rRNA gene data. (CSV 291 kb) Additional file 16: Table S4. 16 s rRNA gene data. (CSV 291 kb) Funding h k This work was funded by Defense Advanced Research Projects Agency under Cooperative Agreement Number W911NF-12-2-0036 (to D.E.I.) and the Wyss Institute for Biologically Inspired Engineering at Harvard University. The commensal bacteria fermentation was supported Sinergia grant 35150 of the Swiss National Science Foundation (to C.L., A.G and T.d-W). The production of human organoids was supported by NIH grants R01DK084056 and P30DK034854 (to D.T.B.) and by NIH grant 5T32CA009216–37 (to D.B.C.). Additional file 8: Figure S7. ΔfliC EHEC does not produce differential epithelial lesions in the Hmm and Mmm groups. Analysis of ΔfliC EHEC induced epithelial injury on-chip. (TIF 1400 kb) Acknowledgments h k bl Additional file 5: Table S1. Transcriptomics analysis of long polar fimbriae genes.. (XLSX 8 kb) We thank H.L. Mobley for his generous gift of fliC-lux plasmid; Marika Ziesack and Jeffrey Way for providing us E. coli K12 Keio collection fliC single-gene knockout and for their support and advice; A. Sanz Garcia and S. Leng for their help with R coding; the Bauer Core at Harvard University, Harvard Chan Bioinformatic Core, C. Vidoudez and S.A. Trauger at the Harvard Small Molecule Mass Spectrometry Core for their support in the data collection; A. Monreal, O. Levy, and A. Chalkiadaki for their expert advice; and T. Ferrante for assistance with imaging. Additional file 6: Figure S5. Species-specific injury effects are not due to Shiga toxin. (A) Table with transcriptomics comparison of EHEC shiga toxin genes from Hmm and Mmm Colon Chips (FC: fold change). (B) Heatmap of the differentially expressed gene stx1b. (C) Quantification of shiga toxin one released in the vascular channel of Colon Chips. (TIF 1494 kb) Additional file 7: Figure S6. FliC gene transcript is upregulated by Hmm, the species-specific motility effect is not due to changes bacteria viability, and the fliC-luciferase increase in the presence of Hmm is not due to altered bacterial growth. (A) FliC mRNA levels in EHEC cultured with Hmm or Mmm (shown as linearized, normalized fold change). (B) Fluorescence microscopic image of GFP-EHEC (green) and quantification of EHEC viability by staining with propidium iodide (red; bar, 100 μm). (C) Bacterial concentration determined as optical density measured at 600 nm (OD600) of EHEC fliC-luciferase in the presence of Mmm or Mmm. (TIF 1939 kb) References 1. Cheng Y-L, Song L-Q, Huang Y-M, Xiong Y-W, Zhang X-A, Sun H, et al. Effect of enterohaemorrhagic Escherichia coli O157:H7-specific enterohaemolysin on interleukin-1β production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation. Immunology. 2015;145:258–67. https://doi.org/10.1111/imm.12442. 1. Cheng Y-L, Song L-Q, Huang Y-M, Xiong Y-W, Zhang X-A, Sun H, et al. Effect of enterohaemorrhagic Escherichia coli O157:H7-specific enterohaemolysin on interleukin-1β production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation. Immunology. 2015;145:258–67. https://doi.org/10.1111/imm.12442. 1. Cheng Y-L, Song L-Q, Huang Y-M, Xiong Y-W, Zhang X-A, Sun H, et al. Effect of enterohaemorrhagic Escherichia coli O157:H7-specific enterohaemolysin on interleukin-1β production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation. Immunology. 2015;145:258–67. https://doi.org/10.1111/imm.12442. 22. Barrile R, van der Meer AD, Park H, Fraser JP, Simic D, Teng F, et al. Organ- on-Chip recapitulates thrombosis induced by an anti-CD154 monoclonal antibody: translational potential of advanced microengineered systems. Clin Pharmacol Ther. 2018; https://doi.org/10.1002/cpt.1054. 2. Perlman RL. Mouse models of human disease: an evolutionary perspective. Evol Med public Heal. 2016;2016:170–6. https://doi.org/10.1093/emph/ eow014. 2. Perlman RL. Mouse models of human disease: an evolutionary perspective. Evol Med public Heal. 2016;2016:170–6. https://doi.org/10.1093/emph/ eow014. 23. Kim HJ, Li H, Collins JJ, Ingber DE. Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip. Proc Natl Acad Sci U S A. 2016;113: E7–15. https://doi.org/10.1073/pnas.1522193112. 3. Chung H, Pamp SJSJ, Hill JA, Surana NK, Edelman SM, Troy EB, et al. Gut immune maturation depends on colonization with a host-specific microbiota. Cell. 2012;149:1578–93. https://doi.org/10.1016/j.cell.2012.04. 037. 3. Chung H, Pamp SJSJ, Hill JA, Surana NK, Edelman SM, Troy EB, et al. Gut immune maturation depends on colonization with a host-specific microbiota. Cell. 2012;149:1578–93. https://doi.org/10.1016/j.cell.2012.04. 037. 24. Wang G, McCain ML, Yang L, He A, Pasqualini FS, Agarwal A, et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat Med. 2014;20: 616–23. https://doi.org/10.1038/nm.3545. 4. Eppinger M, Cebula TA. Future perspectives, applications and challenges of genomic epidemiology studies for food-borne pathogens: a case study of Enterohemorrhagic Escherichia coli (EHEC) of the O157:H7 serotype. Gut Microbes. 2015;6:194–201. https://doi.org/10.4161/ 19490976.2014.969979. 4. Eppinger M, Cebula TA. Future perspectives, applications and challenges of genomic epidemiology studies for food-borne pathogens: a case study of Enterohemorrhagic Escherichia coli (EHEC) of the O157:H7 serotype. Gut Microbes. 2015;6:194–201. https://doi.org/10.4161/ 19490976.2014.969979. 25. Authors’ contributions Conceptualization, AT, RP-B, MK, CL, and DEI; Methodology, AT, AS-P, MK, DBC, AG, Td-W, DTB, CAR, CL, and DEI; Software, Data Curation, and Formal Analysis, AT and DMC; Investigation: AT, AS-P, SJ-F, AG, and Td-W; Resources, DTB, CAR, DBC, MS, and MJC; Writing—Original Draft, AT and DEI; Conceptualization, AT, RP-B, MK, CL, and DEI; Methodology, AT, AS-P, MK, DBC, AG, Td-W, DTB, CAR, CL, and DEI; Software, Data Curation, and Formal Analysis, AT and DMC; Investigation: AT, AS-P, SJ-F, AG, and Td-W; Resources, DTB, CAR, DBC, MS, and MJC; Writing—Original Draft, AT and DEI; Additional file 10: Figure S8. Each of the 4 identified metabolites increases fliC expression in a dose-dependent manner. FliC-luciferase Additional file 10: Figure S8. Each of the 4 identified metabolites increases fliC expression in a dose-dependent manner. FliC-luciferase Page 18 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (2019) 7:43 (2019) 7:43 Tovaglieri et al. Microbiome Writing—Review & Editing, AT, DBC, DTB, CL, AG, MK, RP-B, AS-P, DMC, SJ-F, and DEI; Visualization, AT, DBC, and DEI, Supervision, RP-B, CL, and DEI, Writing—Review & Editing, AT, DBC, DTB, CL, AG, MK, RP-B, AS-P, DMC, SJ-F, and DEI; Visualization, AT, DBC, and DEI, Supervision, RP-B, CL, and DEI, Funding Acquisition, DBC, DTB, CAR, MS, CL, and DEI. All authors read and approved the final manuscript. Writing—Review & Editing, AT, DBC, DTB, CL, AG, MK, RP-B, AS-P, DMC, SJ-F, and DEI; Visualization, AT, DBC, and DEI, Supervision, RP-B, CL, and DEI, Funding Acquisition, DBC, DTB, CAR, MS, CL, and DEI. All authors read and approved the final manuscript. Writing—Review & Editing, AT, DBC, DTB, CL, AG, MK, RP-B, AS-P, DMC, SJ-F, and DEI; Visualization, AT, DBC, and DEI, Supervision, RP-B, CL, and DEI, response of human disease. J Am Soc Nephrol. 2006;17:3404–14. https://doi. org/10.1681/ASN.2006050419. Writing—Review & Editing, AT, DBC, DTB, CL, AG, MK, RP-B, AS-P, DMC, SJ-F, and DEI; Visualization, AT, DBC, and DEI, Supervision, RP-B, CL, and DEI, Funding Acquisition, DBC, DTB, CAR, MS, CL, and DEI. All authors read and approved the final manuscript. response of human disease. J Am Soc Nephrol. 2006;17:3404–14. https://doi. org/10.1681/ASN.2006050419. and DEI; Visualization, AT, DBC, and DEI, Supervision, RP-B, CL, and DEI, Funding Acquisition, DBC, DTB, CAR, MS, CL, and DEI. All authors read and approved the final manuscript. 7. Ikeda M, Ito S, Honda M. Hemolytic uremic syndrome induced by lipopolysaccharide and Shiga-like toxin. Pediatr Nephrol. 2004;19:485–9. Author details 1 1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. 2Department of Health Sciences and Technology, ETH Zurich, 8092 Zurich, Switzerland. 3Department of Biology, University of Freiburg, 79085 Freiburg, Germany. 4Department of Pathology, Massachusetts General Hospital, Boston, MA 02115, USA. 5Department of Bioengineering and iBB, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1649-004 Lisbon, Portugal. 6Division of Gastroenterology, Boston Children’s Hospital, Boston, MA 02115, USA. 7Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. 8Harvard Stem Cell Institute, Harvard University, Boston, MA 02139, USA. 9Division of Endocrinology, Boston Children’s Hospital, Boston, MA 02115, USA. 10Vascular Biology Program and Department of Surgery, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA. 11Harvard John A. Paulson School of Engineering and Applied Sciences, Cambridge, MA 02139, USA. 12Present Address: Emulate Inc., 27 Drydock Avenue Boston MA 02210 USA 15. Tobe T, Nakanishi N, Sugimoto N. Activation of motility by sensing short- chain fatty acids via two steps in a flagellar gene regulatory cascade in enterohemorrhagic Escherichia coli. Infect Immun. 2011;79:1016–24. https:// doi.org/10.1128/IAI.00927-10. 16. Surana NK, Kasper DL. Moving beyond microbiome-wide associations to causal microbe identification. Nature. 2017;552:244–7. 17. Sridharan GV, Choi K, Klemashevich C, Wu C, Prabakaran D, Bin PL, et al. Prediction and quantification of bioactive microbiota metabolites in the mouse gut. Nat Commun. 2014;5:1–13. https://doi.org/10.1038/ncomms6492. 18. Ingber DE. Reverse engineering human pathophysiology with organs-on- chips. Cell. 2016;164:1105–9. https://doi.org/10.1016/j.cell.2016.02.049. 19. Jalili-Firoozinezhad S, Prantil-Baun R, Jiang A, Potla R, Mammoto T, Weaver JC, et al. Modeling radiation injury-induced cell death and countermeasure drug responses in a human gut-on-a-chip article. Cell Death Dis. 2018;9 https://doi.org/10.1038/s41419-018-0304-8. Cambridge, MA 02139, USA. 12Present Address: Emulate Inc., 27 Drydock Avenue, Boston, MA 02210, USA. 20. Huh D, Leslie DC, Matthews BD, Fraser JP, Jurek S, Hamilton GA, et al. A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Sci Transl Med. 2012;4:159ra147. https://doi.org/ 10.1126/scitranslmed.3004249. Competing interests D.E.I. holds equity in Emulate, Inc., consults to the company, and chairs its scientific advisory board; he also is an inventor on relevant patents. 13. Jacobson A, Lam L, Rajendram M, Tamburini F, Honeycutt J, Pham T, et al. A gut commensal-produced metabolite mediates colonization resistance to salmonella infection. Cell Host Microbe. 2018;24:296–307.e7. https://doi.org/ 10.1016/j.chom.2018.07.002. Received: 25 November 2018 Accepted: 21 February 2019 21. Benam KH, Novak R, Nawroth J, Hirano-Kobayashi M, Ferrante TC, Choe Y, et al. Matched-comparative modeling of normal and diseased human airway responses using a microengineered breathing lung chip. Cell Syst. 2016;3: 456–466.e4. https://doi.org/10.1016/j.cels.2016.10.003. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 14. Nakanishi N, Tashiro K, Kuhara S, Hayashi T, Sugimoto N, Tobe T. Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli. Microbiology. 2009;155(Pt 2):521–30. https://doi.org/10.1099/mic.0.023499-0. Consent for publication Not applicable. 12. Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y, Yoshimura K, et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature. 2011;469:543–7. https://doi.org/10.1038/ nature09646. 12. Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y, Yoshimura K, et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature. 2011;469:543–7. https://doi.org/10.1038/ nature09646. Authors’ contributions https://doi.org/10.1007/s00467-003-1395-7. Funding Acquisition, DBC, DTB, CAR, MS, CL, and DEI. All authors read and approved the final manuscript. Ethics approval and consent to participate 8. Schüller S. Shiga toxin interaction with human intestinal epithelium. Toxins (Basel). 2011;3:626–39. https://doi.org/10.3390/toxins3060626. Human colonic resections were obtained anonymously from the Department of Pathology at Massachusetts General Hospital under an existing Institutional Review Board approved protocol (#2015P001859). Endoscopic biopsies were collected from de-identified patients from Boston Children’s Hospital. Informed consent and developmentally appropriate assent were obtained at Boston Children’s Hospital from the donors’ guardian and the donor, respectively. All methods were carried out in accordance with the Institutional Review Board of Boston Children’s Hospital (Protocol number IRB-P00000529) approval. 9. Schüller S, Frankel G, Phillips AD. Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture. Cell Microbiol. 2004;6:289–301. https:// doi.org/10.1111/j.1462-5822.2004.00370.x. 10. Miyamoto Y, Iimura M, Kaper JB, Torres AG, Kagnoff MF. Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo. Cell Microbiol. 2006;8:869–79. https://doi. org/10.1111/j.1462-5822.2005.00673.x. 11. Ritchie JM. Animal models of Enterohemorrhagic Escherichia coli infection. Microbiol Spectr. 2014;2:1–13. https://doi.org/10.1128/microbiolspec.EHEC- 0022-2013.Correspondence. References Novel Polyfermentor intestinal model (PolyFermS) for controlled ecological studies: validation and effect of pH. PLoS One. 2013;8: e77772. https://doi.org/10.1371/journal.pone.0077772. 49. Sassone-Corsi M, Raffatellu M. No vacancy: how beneficial microbes cooperate with immunity to provide colonization resistance to pathogens. J Immunol. 2015;194:4081–7. https://doi.org/10.4049/jimmunol.1403169. 31. Tanner SA, Zihler Berner A, Rigozzi E, Grattepanche F, Chassard C, Lacroix C. In vitro continuous fermentation model (PolyFermS) of the swine proximal colon for simultaneous testing on the same gut microbiota. PLoS One. 2014;9:e94123. https://doi.org/10.1371/journal.pone.0094123. 50. Cordonnier C, Etienne-Mesmin L, Thévenot J, Rougeron A, Rénier S, Chassaing B, et al. Enterohemorrhagic Escherichia coli pathogenesis: role of long polar fimbriae in Peyer’s patches interactions. Sci Rep. 2017;7:44655. https://doi.org/10.1038/srep44655 32. Fehlbaum S, Chassard C, Haug MC, Fourmestraux C, Derrien M, Lacroix C. Design and investigation of PolyFermS in vitro continuous fermentation models inoculated with immobilized fecal microbiota mimicking the elderly Colon. PLoS One. 2015;10:e0142793. https://doi.org/10.1371/journal.pone. 0142793. 51. Wadhams GH, Armitage JP. Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol. 2004;5:1024–37. 52. Lopes JG, Sourjik V. Chemotaxis of Escherichia coli to major hormones and polyamines present in human gut. ISME J. 2018;96:1272–82. https://doi.org/ 10.1038/s41396-018-0227-5. 33. Poeker SA, Geirnaert A, Berchtold L, Greppi A, Krych L, Steinert RE, et al. Understanding the prebiotic potential of different dietary fibers using an in vitro continuous adult fermentation model (PolyFermS). Sci Rep. 2018;8: 4318. https://doi.org/10.1038/s41598-018-22438-y. 53. Matilla MA, Krell T. The effect of bacterial chemotaxis on host infection and pathogenicity. FEMS Microbiol Rev. 2018;42:40–67. https://doi.org/10.1093/ femsre/fux052. 34. Andrews C, McLean MH, Durum SK. Cytokine tuning of intestinal epithelial function. Front Immunol. 2018;9:1270. https://doi.org/10.3389/fimmu.2018. 01270. 54. Erhardt M. Strategies to block bacterial pathogenesis by interference with motility and chemotaxis. In: Stadler M, Dersch P, editors. How to overcome the antibiotic crisis : facts, challenges, technologies and future perspectives. Cham: Springer International Publishing; 2016. p. 185–205. https://doi.org/ 10.1007/82_2016_493. 35. Kagnoff MF. The intestinal epithelium is an integral component of a communications network. J Clin Invest. 2014;124:2841–3. https://doi.org/10. 1172/JCI75225. 36. Maaser C, Heidemann J, von Eiff C, Lugering A, Spahn TW, Binion DG, et al. Human intestinal microvascular endothelial cells express toll-like receptor 5: a binding partner for bacterial flagellin. J Immunol. 2004;172:5056–62. https://doi.org/10.4049/jimmunol.172.8.5056. 55. Ravichandar JD, Bower AG, Julius AA, Collins CH. Transcriptional control of motility enables directional movement of Escherichia coli in a signal gradient. Sci Rep. 2017;7:1–14. https://doi.org/10.1038/s41598-017-08870-6. 56. Lane MC, Alteri CJ, Smith SN, Mobley HLT. References Dawson A, Dyer C, Macfie J, Davies J, Karsai L, Greenman J, et al. A microfluidic chip based model for the study of full thickness human intestinal tissue using dual flow. Biomicrofluidics. 2016;10:064101. https://doi. org/10.1063/1.4964813. 5. Mohawk KL, O’Brien AD. Mouse models of Escherichia coli O157:H7 infection and Shiga toxin injection. J Biomed Biotechnol. 2011;2011:258185. https://doi.org/10.1155/2011/258185. 5. Mohawk KL, O’Brien AD. Mouse models of Escherichia coli O157:H7 infection and Shiga toxin injection. J Biomed Biotechnol. 2011;2011:258185. https://doi.org/10.1155/2011/258185. 26. Griffin PM, Olmstead LC, Petras RE. Escherichia coli O157:H7-associated colitis. A clinical and histological study of 11 cases. Gastroenterology. 1990; 99:142–9. https://doi.org/10.1016/0016-5085(90)91241-W. 6. Keepers TR, Psotka MA, Gross LK, Obrig TG. A murine model of HUS: Shiga toxin with lipopolysaccharide mimics the renal damage and physiologic Page 19 of 20 Page 19 of 20 Page 19 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (2019) 7:43 27. Shigeno T, Akamatsu T, Fujimori K, Nakatsuji Y, Nagata A. The clinical significance of colonoscopy in hemorrhagic colitis due to enterohemorrhagic Escherichia coli O157:H7 infection. Endoscopy. 2002;34: 311–4. https://doi.org/10.1055/s-2002-23644. 45. Franke A, Balschun T, Karlsen TH, Sventoraityte J, Nikolaus S, Mayr G, et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet. 2008;40:1319–23. https://doi.org/ 10.1038/ng.221. 28. Lewis SB, Cook V, Tighe R, Schüller S. Enterohemorrhagic Escherichia coli colonization of human colonic epithelium in vitro and ex vivo. Infect Immun. 2015;83:942–9. 28. Lewis SB, Cook V, Tighe R, Schüller S. Enterohemorrhagic Escherichia coli colonization of human colonic epithelium in vitro and ex vivo. Infect Immun. 2015;83:942–9. 46. Marlow GJ, van Gent D, Ferguson LR. Why interleukin-10 supplementation does not work in Crohn’s disease patients. World J Gastroenterol. 2013;19:3931–41. 47. Fioranelli M, Roccia MG. Twenty-five years of studies and trials for the therapeutic application of IL-10 immunomodulating properties. From high doses administration to low dose medicine new paradigm. J Integr Cardiol. 2014;1:2–6. 29. Kasendra M, Tovaglieri A, Sontheimer-Phelps A, Jalili-Firoozinezhad S, Bein A, Chalkiadaki A, et al. Development of a primary human small intestine-on-a- chip using biopsy-derived organoids. Sci Rep. 2018;8:2871. https://doi.org/ 10.1038/s41598-018-21201-7. 48. Yamamoto T, Nagayama K, Satomura K, Honda T, Okada S. Increased serum IL-10 and endothelin levels in hemolytic uremic syndrome caused by Escherichia coli O157. Nephron. 2000;84:326–32. https://doi.org/10.1159/ 000045607. 30. Zihler Berner A, Fuentes S, Dostal A, Payne AN, Vazquez Gutierrez P, Chassard C, et al. References Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract. Proc Natl Acad Sci. 2007;104:16669–74. https://doi.org/10.1073/ pnas.0607898104. 37. Eckmann L, Kagnoff MF, Fierer J. Epithelial cells secrete the chemokine interleukin-8 in response to bacterial entry. Infect Immun. 1993;61:4569–74. doi:<p></p> 38. Shimizu M, Kuroda M, Sakashita N, Konishi M, Kaneda H, Igarashi N, et al. Cytokine profiles of patients with enterohemorrhagic Escherichia coli O111- induced hemolytic-uremic syndrome. Cytokine. 2012;60:694–700. https://doi. org/10.1016/j.cyto.2012.07.038. 57. Valeriani C, Li M, Novosel J, Arlt J, Marenduzzo D. Colloids in a bacterial bath: simulations and experiments. Soft Matter. 2011;7:5228. https://doi.org/ 10.1039/c1sm05260h. 58. Hidalgo G, Chan M, Tufenkji N. Inhibition of Escherichia coli CFT073 flic expression and motility by cranberry materials. Appl Environ Microbiol. 2011;77:6852–7. 39. Murata A, Shimazu T, Yamamoto T, Taenaka N, Kichi N, Honda T, et al. Profiles of circulating inflammatory- and anti-inflammatory cytokines in patients with hemolytic uremic syndrome due to E. coli O157 infection. Cytokine. 1998;10:544–8. 59. Tuttle J, Gomez T, Doyle MP, Wells JG, Zhao T, Tauxe RV, et al. Lessons from a large outbreak of Escherichia coli O157:H7 infections: insights into the infectious dose and method of widespread contamination of hamburger patties. Epidemiol Infect. 1999;122:185–92. https://doi.org/10.1017/ S0950268898001976. 40. Fitzpatrick MM, Shah V, Trompeter RS, Dillon MJ, Barratt TM. Interleukin-8 and polymorphoneutrophil leucocyte activation in hemolytic uremic syndrome of childhood. Kidney Int. 1992;42:951–6. https://doi.org/10.1038/ki. 1992.372. 60. Gonthier M-P, Remesy C, Scalbert A, Cheynier V, Souquet J-M, Poutanen K, et al. Microbial metabolism of caffeic acid and its esters chlorogenic and caftaric acids by human faecal microbiota in vitro. Biomed Pharmacother. 2006;60:536–40. https://doi.org/10.1016/j.biopha.2006.07.084. 41. López EL, Contrini MM, Devoto S, de Rosa MF, Graña MG, Genero MH, et al. Tumor necrosis factor concentrations in hemolytic uremic syndrome patients and children with bloody diarrhea in Argentina. Pediatr Infect Dis J. 1995;14:594–8. http://www.ncbi.nlm.nih.gov/pubmed/7567288 61. Andreasen MF, Christensen LP, Meyer AS, Hansen Å. Content of phenolic acids and ferulic acid dehydrodimers in 17 rye (Secale cereale L.) varieties. J Agric Food Chem. 2000;48:2837–42. 42. van de Kar NC, Monnens LA, Karmali MA, van Hinsbergh VW. Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome. Blood. 1992;80:2755– 64. http://www.ncbi.nlm.nih.gov/pubmed/1333300 62. Ozdal T, Sela DA, Xiao J, Boyacioglu D, Chen F, Capanoglu E. The reciprocal interactions between polyphenols and gut microbiota and effects on bioaccessibility. Nutrients. 2016;8:1–36. References 43. Ceponis PJM, McKay DM, Ching JCY, Pereira P, Sherman PM. Enterohemorrhagic Escherichia coli O157:H7 disrupts Stat1-mediated gamma interferon signal transduction in epithelial cells. Infect Immun. 2003; 71:1396–404. https://doi.org/10.1128/IAI.71.3.1396. 63. Lacroix C, De Wouters T, Chassard C. ScienceDirect integrated multi-scale strategies to investigate nutritional compounds and their effect on the gut microbiota. Curr Opin Biotechnol. 2015;32:149–55. https://doi.org/10.1016/j. copbio.2014.12.009. copbio.2014.12.009. 44. Proulx F, Toledano B, Phan V, Clermont M-J, Mariscalco MM, Seidman EG. Circulating granulocyte colony-stimulating factor, C-X-C, and C-C chemokines in children with Escherichia coli O157:H7 associated hemolytic uremic syndrome. Pediatr Res. 2002;52:928–34. https://doi.org/10.1203/ 00006450-200212000-00019. 64. Zheng X, Qiu Y, Zhong W, Baxter S, Su M, Li Q, et al. A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids. Metabolomics. 2013;9:818–27. https://doi.org/10.1007/s11306-013-0500-6. 65. Han J, Lin K, Sequeira C, Borchers CH. An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in 65. Han J, Lin K, Sequeira C, Borchers CH. An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in Page 20 of 20 Page 20 of 20 Tovaglieri et al. Microbiome (2019) 7:43 Tovaglieri et al. Microbiome (2019) 7:43 human feces by liquid chromatography–tandem mass spectrometry. Anal Chim Acta. 2015;854:86–94. https://doi.org/10.1016/j.aca.2014.11.015. human feces by liquid chromatography–tandem mass spectrometry. Anal Chim Acta. 2015;854:86–94. https://doi.org/10.1016/j.aca.2014.11.015. 87. Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10:996–8. https://doi.org/10.1038/nmeth.2604. human feces by liquid chromatography–tandem mass spectrometry. Anal Chim Acta. 2015;854:86–94. https://doi.org/10.1016/j.aca.2014.11.015. 66. Di Cagno R, De Angelis M, De Pasquale I, Ndagijimana M, Vernocchi P, Ricciuti P, et al. Duodenal and faecal microbiota of celiac children: molecular, phenotype and metabolome characterization. BMC Microbiol. 2011;11:219. https://doi.org/10.1186/1471-2180-11-219. 88. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–3. https://doi.org/10.1038/nmeth.3869. 89. Bowne JB DLA. Metabolomics: analysis of metabolomics data. 2014. https:// cran.r-project.org/package=metabolomics. 67. Jalili-Firoozinezhad S, Gazzaniga FS, Calamari EL, Camacho DM, Fadel CW, Nestor B, et al. Complex human gut microbiome cultured in anaerobic human intestine chips. bioRxiv. 2018. http://biorxiv.org/content/early/2018/ 09/20/421404.abstract. 90. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47. https://doi.org/10.1093/nar/gkv007. 91. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017; 14:417–9. https://doi.org/10.1038/nmeth.4197. 68. Carlson-Banning KM, Sperandio V. Catabolite and oxygen regulation of Enterohemorrhagic Escherichia coli virulence. MBio. 2016;7 https://doi.org/ 10.1128/mBio.01852-16. 92. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. https://doi. org/10.1093/bioinformatics/bts635. 69. Grys TE, Siegel MB, Lathem WW, Welch RA. copbio.2014.12.009. The StcE protease contributes to intimate adherence of enterohemorrhagic Escherichia coli O157 : H7 to host cells. 2005;73:1295–303. 93. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. https://doi.org/10.1186/s13059-014-0550-8. 70. Macfarlane S, Macfarlane GT. Regulation of short-chain fatty acid production. Proc Nutr Soc. 2003;62:67–72. https://doi.org/10.1079/ PNS2002207. 94. Yu G, Wang L-G, Han Y, He Q-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16:284–7. https://doi. org/10.1089/omi.2011.0118. 71. Brown K, Abbott DW, Uwiera RRE, Inglis GD. Removal of the cecum affects intestinal fermentation, enteric bacterial community structure, and acute colitis in mice. Gut Microbes. 2018;9:218–35. https://doi.org/10.1080/ 19490976.2017.1408763. 72. Nguyen TLA, Vieira-Silva S, Liston A, Raes J. How informative is the mouse for human gut microbiota research? Dis Model Mech. 2015;8:1–16. https:// doi.org/10.1242/dmm.017400. 73. Macfarlane GT, Macfarlane S, Gibson GR. Validation of a three-stage compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon. Microb Ecol. 1998; 35: 180–187. 74. VanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon C, Tarr PI, et al. Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays. Gut. 2015;64:911–20. https://doi. org/10.1136/gutjnl-2013-306651. 75. Sato T, Clevers H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science. 2013;340:1190–4. https:// doi.org/10.1126/science.1234852. 76. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2:2006.0008. https://doi.org/10.1038/ msb4100050. 77. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. https://doi.org/10.1038/nmeth.2019. 78. Krych Ł, Kot W, Bendtsen KMB, Hansen AK, Vogensen FK, Nielsen DS. Have you tried spermine? A rapid and cost-effective method to eliminate dextran sodium sulfate inhibition of PCR and RT-PCR. J Microbiol Methods. 2018;144: 1–7. https://doi.org/10.1016/j.mimet.2017.10.015. 79. Tinevez J-Y, Perry N, Schindelin J, Hoopes GM, Reynolds GD, Laplantine E, et al. TrackMate: an open and extensible platform for single-particle tracking. Methods. 2017;115:80–90. https://doi.org/10.1016/j.ymeth.2016.09.016. 80. Thévenaz P, Ruttimann UE, Unser M. A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process. 1998;7:27–41. https://doi.org/10.1109/83.650848. 81. Yamaguchi N, Nasu M. Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment. J Appl Microbiol. 1997;83:43–52. https://doi.org/10.1046/j.1365-2672.1997.00165.x. 82. copbio.2014.12.009. Allison SE, Silphaduang U, Mascarenhas M, Konczy P, Quan Q, Karmali M, et al. Novel repressor of Escherichia coli O157:H7 motility encoded in the putative fimbrial cluster OI-1. J Bacteriol. 2012;194:5343–52. p y putative fimbrial cluster OI-1. J Bacteriol. 2012;194:5343–52. 83. Team R. R: a language and environment for statistical computing. R Foundation for Statistical Computing; 2016. 84 Wilcoxon F Individual comparisons of grouped data by ranking methods J 83. Team R. R: a language and environment for statistical computing. R Foundation for Statistical Computing; 2016. 83. Team R. R: a language and environment for statistical computing. R Foundation for Statistical Computing; 2016. 84. Wilcoxon F. Individual comparisons of grouped data by ranking methods. J Econ Entomol. 1946;39:269. https://doi.org/10.1093/jee/39.2.269. 84. Wilcoxon F. Individual comparisons of grouped data by ranking methods. J Econ Entomol. 1946;39:269. https://doi.org/10.1093/jee/39.2.269. 85. Bonferroni CE. Teoria statistica delle classi e calcolo delle probabilità. Pubblicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze. 1936;8:3–62. 86. Mann HB, Whitney DR. On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat. 1947;18:50–60. https:// doi.org/10.1214/aoms/1177730491. 86. Mann HB, Whitney DR. On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat. 1947;18:50–60. https:// doi.org/10.1214/aoms/1177730491.
https://openalex.org/W4286970748	https://zenodo.org/records/5517784/files/BIOTECHNOLOGY.pdf	English	null	BIO-TECHNOLOGY: Newer Biology -An Impression	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	255	BIO-TECHNOLOGY: Newer Biology -An Impression ( Jaydip Datta et al ) In this Impression a modern scope of bio-technology is discussed. The word Bio-Technology literally means a combinatorial hybrid of classical Biological - Science with some Chemical Industrial aspects. The micro biomes – the origin of this newer biology ie MICROBIOLOGY , MOLECULAR BIOLOGY ,rDNA science , Virology ( Most important biological entity – Corona Virus –Latest Global Issue !!! ) . The recombination power of Microbial Kinetics including bacterial as well as Viral cloning to more powerful as well as Virulent type – This type of cloning is irreproducible very often as per Statistical Relevance – a Limitation of Genetic Engineering. Keywords : Newer biology , rDNA , Pharmaceutical biotechnology , SARS-COV-2 Structural dynamics . Discussion Reference : 1.Jaydip Datta , INVISIBLE UNIVERSE OF MICROBIOME , HUMAN HEALTH & FUTURE PERSPECTIVE : A SEMINAR PARTICIPATION , February 2016 ,DOI: 10.13140/RG.2.2.30623.51367 . (https://www.researchgate.net/publication/335868820_INVISIBLE_UNIVERSE_OF_MICROBIOME_H UMAN_HEALTH_FUTURE_PERSPECTIVE_A_SEMINAR_PARTICIPATION?_sg=VpcuLhsmbw8Cw feIONqzvOMCKIibiV__H437L-uG_w0Mmk0-ASIEvnKDzofwh7- Ff4xk2_uwB2UXV9K5rkcWAqDYZ6zoiUecuXcvZI2d.tLz39x_LkDr2f6bLXRVsF8sJgnx4BFTd3uwZWF R270OzL4vM47vgesTUwJqsHosC8z-NZHO0E8nDGbg2fKWikw ) ( MICROBIOME & HEALTH PROBIOTICS ) 2. Jaydip Datta , MICROBIAL GROWTH KINETICS : A MATHEMATICAL REVIEW ( SPECIAL ADDENDUM - COVID19 ) ,May 2020 ,DOI: 10.6084/m9.figshare.12280790.v2 ,License CC BY-ND 4.0 (https://www.researchgate.net/publication/334965185_MICROBIAL_GROWTH_KINETICS_A_MATH EMATICAL_REVIEW_SPECIAL_ADDENDUM_- _COVID19?_sg=VpcuLhsmbw8CwfeIONqzvOMCKIibiV__H437L-uG_w0Mmk0-ASIEvnKDzofwh7- Ff4xk2_uwB2UXV9K5rkcWAqDYZ6zoiUecuXcvZI2d.tLz39x_LkDr2f6bLXRVsF8sJgnx4BFTd3uwZWF R270OzL4vM47vgesTUwJqsHosC8z-NZHO0E8nDGbg2fKWikw) INDUSTRIAL MICROBIOLOGY : FERMENTATION BIO-TECH . 3. Jaydip Datta , PHARMACEUTICAL & HOUSE HOLD PRODUCTS MANUFACTURING : IMPORTANTANCE OF INDUSTRIAL TRAINING , October 2019 ,DOI: 10.13140/RG.2.2.10980.32643/3 . (https://www.researchgate.net/publication/336304835_PHARMACEUTICAL_HOUSE_HOLD_PRODU CTS_MANUFACTURING_IMPORTANTANCE_OF_INDUSTRIAL_TRAINING?_sg=XDMhms1DSeA4 ALwV4ELDMZ_GF3Ap2uk5OmuapQmq_k5ytJ58W7IS-- v5RiaigQFVCnkcxL3tUEabG_TOxEweGprQ__KefZE9SMdeQohU.6hUikh1ncpMQUglYMRppap0hfvV OfkQrYGUX9BU3-xyX6Nj_nzv4G7KWFtG9B6Oo2mnUmC5BFoFZzPgf83vnzw ) PHARMACEUTOCAL BIOTECHNOGY : MICROBIAL QC , VACCINE PROCESSING UNIT . (https://www.researchgate.net/publication/335868820_INVISIBLE_UNIVERSE_OF_MICROBIOME_H UMAN_HEALTH_FUTURE_PERSPECTIVE_A_SEMINAR_PARTICIPATION?_sg=VpcuLhsmbw8Cw feIONqzvOMCKIibiV__H437L-uG_w0Mmk0-ASIEvnKDzofwh7- Ff4xk2_uwB2UXV9K5rkcWAqDYZ6zoiUecuXcvZI2d.tLz39x_LkDr2f6bLXRVsF8sJgnx4BFTd3uwZWF R270OzL4vM47vgesTUwJqsHosC8z-NZHO0E8nDGbg2fKWikw ) ( MICROBIOME & HEALTH PROBIOTICS ) INDUSTRIAL MICROBIOLOGY : FERMENTATION BIO-TECH .
https://openalex.org/W2942396464	https://sciencepubco.com/index.php/ijet/article/download/24334/12281	English	null	Modelling Economic Growth Based on Factors of Innovation Production Potential Development	International journal of engineering & technology	2,018	cc-by	3,006	1. Introduction One of the fundamental factors of the economic growth and the materials sector, in particular, is the promotion of innovative and high-tech products on the domestic and world markets. The current actual task of the economy is to solve the problem on assessing the innovation activity of Russian regions. Probably, one of the main reasons of difficulties related to this is the reporting system where enterprises are not interested in displaying their innovative activity. In accordance with the recommended methodology for collecting statistical information when classifying products and referring to them as high-tech products, it is necessary to collect a lot of information, which is quite difficult. It goes not only about expenditures for high-tech products, the number of employees involved in production, but also about the correlation of innovations in terms of technological, marketing or organizational categories with the relevant amount of information. It is easier for enterprises to not display this information at all or to display its minimum. However, state authorities need information about the performed work and the tempo and volume of innovation activity in general [5], because, as a rule, high-tech products are developed by using the budget. There are a number of reasons that restrain the economic growth. They include [1] the arrears of financial and organizational infrastructures, the lack of developed systems of export support by the state, difficulties in adapting competitive technologies to mass production, insufficient level of the specialized engineering development, low efficiency of production, and rather considerable material consumption of agricultural and industrial products. The instability of the global economy and the vagueness of its formation prospects, subject to reducing resources, decrease profitability in processing sectors, and complicate the development of the industrial sector of economy. Today promising innovative technologies that enable developing of new large oil production centers [2] and food production in large companies have become the main areas of the materials sector development. In order to process the information, it is necessary to have methodological instruments that would display and assess the innovation activity of all organizations supported by state regulation. Not only national but also foreign researchers have been interested in this task for a long time. Thus, production functions’ models were offered as the first methodological instruments. When improving them, the economic growth models were developed. Abstract It goes not only about expenditures for high-tech products, the number of employees involved in production, but also about the correlation of innovations in terms of technological, marketing or organizational categories with the relevant amount of information. It is easier for enterprises to not display this information at all or to display its minimum. However, state authorities need information about the performed work and the tempo and volume of innovation activity in general [5], because, as a rule, high-tech products are developed by using the budget. In order to process the information, it is necessary to have methodological instruments that would display and assess the economy, it is necessary to modify the attitude towards the resource potential of Russia, to stimulate investments in processing sectors and high-tech industries, and to reduce the flow of capital from the country [3]. To increase export on a qualitative level, it is necessary to increase the share of finished goods [4]. International Journal of Engineering & Technology, 7 (4.38) (2018) 112-114 Abstract The Russian economic growth is associated with the increase in the competitive advantages of the domestic industry and the material sector of economy, including agriculture. The article studies the factors that have positive impact on the economic development and growth, as well as those that restrain these processes. The problem of assessing the need in innovative activity of economic sectors is actualized taking into account the differences of Russian regions in terms of agricultural productivity. The use of the imitations and innovations’ theory and methodological instruments of production functions’ models is substantiated. Thus, the Solow model is studied, and the improvement of the model, including the funds’ introduction delay when the optimal accumulation rates are equal to the elasticity coefficients by funds, is shown. The factors that include various economic growth models: volumes of capital, physical and intellectual labor, labor productivity, the production returns rate, the scientific and technological progress rate, the population growth rate, the consumer demand, production structure, institutional factors, and saving rates, are studied. It is proved that the study of neoclassical models of the economic growth shows their incomplete adequacy taking into account all differentiating factors, competitive processes and differentiating branch structures of economy. In the work, the hypothesis of the interrelationship of the saving rate, labor productivity and the volume of investments in Russia is mathematically proven taking into account the differentiation of Russian regions and peculiarities of statistical accounting. Economic Growth, Solow Model, Regional Differentiation, Industrial and Agricultural Production, Saving Rate, Labor V l Keywords: Innovations, Economic Growth, Solow Model, Regional Differentiation, Industrial and Agricultural Productio Productivity, Investment Volume. economy, it is necessary to modify the attitude towards the resource potential of Russia, to stimulate investments in processing sectors and high-tech industries, and to reduce the flow of capital from the country [3]. To increase export on a qualitative level, it is necessary to increase the share of finished goods [4]. The current actual task of the economy is to solve the problem on assessing the innovation activity of Russian regions. Probably, one of the main reasons of difficulties related to this is the reporting system where enterprises are not interested in displaying their innovative activity. In accordance with the recommended methodology for collecting statistical information when classifying products and referring to them as high-tech products, it is necessary to collect a lot of information, which is quite difficult. 2. Methods differentiation of the economic development. The effect of this factor is mainly substantiated by the state of the sectoral structure of economy and the formation of competitive processes on the sectoral market. It is necessary to note that such models take into account competitive processes not the way they are. Instead they are determined indirectly as a result of their development – the rates of profits economic entities get. It happens while in fact the rates of profit for all entities are different, and the market system is far from standard types – perfect competition, monopolistic competition and oligopoly. One of the most wide-spread models is the R. Solow model characterized by the fact that the rate of labor resources’ growth is exogenous, the technological level has a constant index, and the return on two production factors (labor and capital) does not change. A more complex model implies technical progress, too. The next degree of complexity includes models that take into account natural resources. When studying and improving the Solow model, special results were achieved by V.А. Kolemaev [6]. He considered the model in terms of the delay when introducing funds without taking into account the dynamics of the technological progress. Accordingly, he formulated this management task as an accumulation rate in the context of the Solow model. The study of neoclassical models of the economic growth shows that they are not fully adequate taking into account all differentiating factors, competitive processes and differentiating branch structures of the economy. Neoclassical models of the economic growth make it possible to describe specific features of macroeconomic processes, and to consider the economic growth in terms of interchangeable production factors [8]. The Cobb-Douglas model assumes that, at a certain technological level production volumes depend on the amount of capital and labor used. Nevertheless, the technological level is gradually being improved, and therefore temporary factors are introduced into the equation. Based on the changes taking into account the time factor of Jan Tinbergen, the Cobb-Douglas function will be as follows [9]: In order to efficiently assess the impact of innovation activity, it is necessary to make nonlinear multiple regression equations in the form of various multiplicative production functions for statistical estimation of gross domestic product (GDP), gross regional product (GRP), and gross value added (GVA). On the country level, the latter indicator is less than GDP by the difference between food taxes and subsidies. 1. Introduction In general, the growth of economy is associated with the impact of a number of factors: production efficiency, profitability, accumulation rates and investment activity; sectoral and reproduction structure of public products; modification of the absolute size and the relative share of gross domestic product; modification of export and import of raw materials and foreign trade balance. Based on this, in order to reduce the materials specialization of the International Journal of Engineering & Technology 113 r = R / A These variables are related by the following equation: С = g (r) С = g (r) According to the Solow model, saving rates are the main factor characterizing the capital-labor ratio and correspondingly reflecting the level of output. States that have a consistently high saving rate are characterized by a developed level of capital-labor ratio, which, in its turn, provides a more rapid rate of economic growth. The demand in the Solow model is defined as the aggregate of investments (t) and consumption (k) per unit of labor: c = t + k. Thus, the Solow model is the most important starting point for almost any study related to the economic growth. Using this model, it is possible to define the reasons of temporary and permanent (sustainable) economic growth [7]. Consequently, the consumption can be calculated by using the following formula: k = (1 --n) С 2. Methods GVA is presented in statistics as the sum of GRP for all regions, in regions – as the sum of the value added formed by sectors of the economy [12]. Due to the variety of natural and climatic conditions, the agrarian sector of Russian regions is characterized by a wide variety of economic indicators. The GVA indicator allows applying the theory that divides the economic growth into two stages: imitative and innovative. The study of innovations’ spread shows that in Russia there are regions with large agricultural holdings that start innovations, and regions that directly borrow them [14]. Ci = Di ∙Ri γ ∙Ai β ∙Vi γ where Ci is the production volumes, Ci is the production volumes, Ci is the production volumes, Di is the coefficient that shows the development of the scientific and technological progress, Ri γ ∙Ai β ∙Vi γ are the expenditures of labor, capital and natural resources, and The formation of production functions’ models allows making statistical forecasts on the main socio-economic indicators, and ratings of regions by high-tech indicators and the speed of investments use, as well as developing algorithms for the efficient management of accumulation rates (intensity of use). β, γ are the indicators of the elasticity of the production by labor, capital and natural resources. In the context of the above, it is necessary to consider the Solow economic growth model [10]. Thus, for example, Solow introduced an auxiliary variable of labor productivity (C), as well as capital-labor ratio: The Solow model is one of the neoclassical models of the economic growth. Neoclassical models tried to overcome a whole range of possible limitations of Keynesian models and reflect specific characteristics of macroeconomic processes. This model is characterized by the fact that it shows the instability of the dynamic equilibrium of Keynesian models that is the result of the interchangeability of production factors. Instead of the Leontief function, the Cobb-Douglas function where labor and capital are substitutes is used. where [11] Derunova E, Semenov A (2013), Study of the problematic issues of the raw material orientation of the economy: the Dutch disease and its influence on innovative development. World Applied Sciences Journal, 25(9), 1295-1301. nk is the saving rates in the k region, y is the serial number of the economy sectors. In this case the volume of products is calculated as the sum of GVA produced in the regions that pursue conducting imitational or mainly investment policy [12]. [12] Sidorovich AV (2007), Kurs ekonomicheskoy teorii: obshchiye osnovy ekonomicheskoy teorii. Mikroekonomika. Makroekonomika. Osnovy natsional'noy ekonomiki [The course of economic theory: the general foundations of economic theory. Microeconomics. Macroeconomics. Fundamentals of national economy], 3rd edition, Delo i Servis, Moscow. 3. Results and Discussion To obtain an equality formula on the investment inflow and savings under equal market conditions, it is recommended to substitute the consumption formula and the aggregate demand formula. Various models of the economic growth that take into account the fundamentally important factors for the formation of the national economy mainly include such factors as the volumes of capital, physical and intellectual labor, labor productivity, the production return rate, the rate of scientific and technological progress, the population growth rate, demand, production structure, institutional factors, saving rates, etc. t = n ∙ С When adding factors of the innovative development of the production potential to the model, production functions are modified: Almost all models analyze only consequences for the economic development which are the results of changes in various factors. In addition, today there is no single theoretic and practical model that takes into account another important factor of the interregional International Journal of Engineering & Technology 114 C = E (R, A, Q) but also on geographical competitive advantages. References nk ∙( Ry Qy ) αy = (ay + my + qy)( Ry Ay ∙Qy ) ( Ry Qy )αy−1 = ay + my + qy Ay ∙nk Ry αy−1 = Ay αy−1( ay + my + qy Ay ∙nk ) nk ∙( Ry Qy ) αy = (ay + my + qy)( Ry Ay ∙Qy ) ( Ry Qy )αy−1 = ay + my + qy Ay ∙nk Ry αy−1 = Ay αy−1( ay + my + qy Ay ∙nk ) nk ∙( Ry Qy ) αy = (ay + my + qy)( Ry Ay ∙Qy ) ( Ry Qy )αy−1 = ay + my + qy Ay ∙nk Ry αy−1 = Ay αy−1( ay + my + qy Ay ∙nk ) [9] Zhulanov EE (2014), Modelirovaniye razvitiya natsionalnoy sotsialno-ekonomicheskoy sistemy pod vliyaniyem regulirovaniya usloviy vneshneekonomicheskoy deyatelnosti regionalnoy promyshlennosti [Modeling the development of the national socio- economic system as influenced by regulation of the conditions of foreign economic activity of regional industry]. Economic and Humanities, 4(267), 95–104. [10] Zhuravleva GP, Smagina VV (2008), Ekonomicheskaya teoriya i politika rynochnoy sistemy khozyaystvovaniya [Economic theory and policy of the market system of management]. Moscow: Finance and Statistics. where References E is the indicator of labor efficiency that depends on the health, education and qualification of employees, [1] Acemoglu D, Aghion P, Zilibotti F (2006), Distance to frontier, selection, and economic growth. Journal of the European Economic Association 4(1), 37–74. q p y A and Q are the number of conventional units of labor of one employee and the number of employees. The indicators of the capital (r) and labor productivity (c) indexes introduced by Solow are characterized by constant efficiency and are calculated using the following formulas: [2] Dzhumaeva RA, Mukhutdinova TZ (2012), Upravleniye chelovecheskim kapitalom – strategicheskim faktorom razvitiya innovatsionnoy khozyaystvennoy sistemy [Managing human capital as a strategic factor in the development of the innovative economic system]. Bulletin of Kazan Technological University, 2, 84-88. r = R A ∙Q ; c = C A ∙Q y g y [3] Belousov DR (2007), O polozhenii v rossiyskoy ekonomike [On the situation in the Russian economy]. Problems of Forecasting, 3, 21-41. Therefore, [4] Mansurova T (2007), Spetsifika razvitiya syryevogo sektora v rossiyskoy ekonomike [Specificity of the development of the raw materials sector in the Russian economy]. Bulletin of the Kazan Technological University, 5, 189-194. n ∙c = (a + m + q)r [5] Sistema natsionalnykh schetov [System of national accounts], Russian Statistical Yearbook (2017). Moscow, Stat.collection Rosstat, 258-311. where a is the population growth rate, m is the tempo of technical progress, and q is the permanent capital depreciation. [6] Derunova E, Firsova A, Vavilina A et al (2014), The study of the dynamics of innovative development of economy on the endogenous growth through multi-sector extension of the Solow model. Biosciences Biotechnology Research Asia, 11(3), 1581- 1589. In order to more accurately define the economic growth, the Solow model can be improved as follows: ∆r = t −(a + m + q)r [7] Kolemaev VV (2002), Matematicheskaya ekonomika [Mathematical Economics]. Moscow: Unity-Dana. This equation can be transformed taking into account the regional differentiation of economy [11]: [8] Tsaregorodtsev EI (2014), Problemy otsenki innovatsionnoy deyatelnosti regiona [Problems of assessing the innovation activity of the region]. Economics and Management, 2(111), 89-94. 4. Conclusion y [13] Zhulanov EE (2012), Vliyaniye sotsialno-ekonomicheskoy asimmetrii regionov na rezultaty modelirovaniya ekonomicheskogo rosta [Impact of socio-economic asymmetry of regions on the results of economic growth modeling]. Economic Analysis: Theory and Practice, 24(279), 10–20. Thus, GVA produced taking into account the sectoral structure and regional differentiation [13] of economic indicators can be determined by using the following formula: ∑∑Ryk = J j=1 K k=1 ∑∑Qyk( ay + my + qy Ay ∙nk ) 1 αy−1 J j=1 K k=1 where J is the number of industries in the national economy, K is the number of regions in the country. ∑∑Ryk = J j=1 K k=1 ∑∑Qyk( ay + my + qy Ay ∙nk ) 1 αy−1 J j=1 K k=1 where where J is the number of industries in the national economy, K is the number of regions in the country. J is the number of industries in the national economy, K is the number of regions in the country. J is the number of industries in the national economy, K is the number of regions in the country. In the Solow model the dependence of the economy growth rate on the factors of innovative development of the production potential for a country with a developed and regionally differentiated agrarian sector can be fully assessed only taking into account the factor of interregional differences. Thus, the structural reorganization of the economy sectors that will ensure the growth of innovative factors in the production potential development is based on the formation of not only scientific and intellectual, production and technological factors,
https://openalex.org/W4360616242	https://www.mdpi.com/1422-0067/24/7/6038/pdf?version=1679558181	English	null	Butein Inhibits Cell Growth by Blocking the IL-6/IL-6Rα Interaction in Human Ovarian Cancer and by Regulation of the IL-6/STAT3/FoxO3a Pathway	International journal of molecular sciences	2,023	cc-by	12,270	Citation: Park, S.-A.; Seo, Y.J.; Kim, L.K.; Kim, H.J.; Yoon, K.D.; Heo, T.-H. Butein Inhibits Cell Growth by Blocking the IL-6/IL-6Rα Interaction in Human Ovarian Cancer and by Regulation of the IL-6/STAT3/ FoxO3a Pathway. Int. J. Mol. Sci. 2023, 24, 6038. https://doi.org/ 10.3390/ijms24076038 Keywords: ovarian cancer (OC); signaling pathway; interleukin-6 (IL-6); signal transducer and activator of transcription 3 (STAT3); forkhead box O3a (FoxO3a) Butein Inhibits Cell Growth by Blocking the IL-6/IL-6Rα Interaction in Human Ovarian Cancer and by Regulation of the IL-6/STAT3/FoxO3a Pathway Sun-Ae Park 1,† , Young Ju Seo 2,†, Lee Kyung Kim 1 , Hee Jung Kim 1 , Kee Dong Yoon 2,* ung Ju Seo 2,†, Lee Kyung Kim 1 , Hee Jung Kim 1 , Kee Dong Yoon 2,* and Tae-Hwe Heo 1,* 1 Laboratory of Pharmacoimmunology, Integrated Research Institute of Pharmaceutical Sciences and BK21 FOUR Team for Advanced Program for Smart Pharma Leaders, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Bucheon 14662, Republic of Korea 2 College of Pharmacy, The Catholic University of Korea, Bucheon 14662, Republic of Korea * Correspondence: kdyoon@catholic.ac.kr (K.D.Y.); thhur92@catholic.ac.kr (T.-H.H.); Tel.: +82-2-2164-4091 (K.D.Y.); +82-2-2164-4088 (T.-H.H.) † These authors contributed equally to this work. 1 Laboratory of Pharmacoimmunology, Integrated Research Institute of Pharmaceutical Sciences and BK21 FOUR Team for Advanced Program for Smart Pharma Leaders, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Bucheon 14662, Republic of Korea y , J g , , p 2 College of Pharmacy, The Catholic University of Korea, Bucheon 14662, Republic of Korea * Correspondence: kdyoon@catholic.ac.kr (K.D.Y.); thhur92@catholic.ac.kr (T.-H.H.); Tel.: +82-2-2164-4091 (K.D.Y.); +82-2-2164-4088 (T.-H.H.) † These authors contributed equally to this work. Abstract: Butea monosperma (Fabaceae) has been used in traditional Indian medicine to treat a variety of ailments, including abdominal tumors. We aimed to investigate the anti-IL-6 activity of butein in ovarian cancer and elucidate the underlying molecular mechanisms. Butein was isolated and identiﬁed from B. monosperma ﬂowers, and the inhibition of IL-6 signaling was investigated using the HEK-Blue™IL-6 cell line. The surface plasmon resonance assay was used to estimate the binding of butein to IL-6, IL-6Rα, and gp130. After treatment with butein, ovarian cancer cell migration, apoptosis, and tumor growth inhibition were evaluated in vitro and in vivo. Furthermore, we used STAT3 siRNA to identify the mechanistic effects of butein on the IL-6/STAT3/FoxO3a pathway. Butein suppressed downstream signal transduction through higher binding afﬁnity to IL-6. In ovarian cancer, butein inhibited cell proliferation, migration, and invasion, and induced cell cycle arrest and apoptosis. In addition, it decreased the growth of ovarian cancer cells in xenograft tumor models. Butein inhibited STAT3 phosphorylation and induced FoxO3a accumulation in the nucleus by inhibiting IL-6 signaling. The anticancer activity of butein was mediated by blocking the IL-6/IL-6Rα interaction and suppressing IL-6 bioactivity via interfering with the IL-6/STAT3/FoxO3a pathway. International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Academic Editor: Daniel Gabriel Pons Ovarian cancer is the eighth most common cancer in women worldwide and ranks ﬁfth in cancer death among women in the United States. Standard treatment consists of cell reduction surgery and platinum-based chemotherapy. However, it is a malignant cancer with high anticancer drug resistance and recurrence rate, so the 5-year survival rate is only 10–40% [1,2]. Therefore, novel therapeutic agents are required to reduce the chemotherapy resistance and recurrence of ovarian cancer. Received: 27 December 2022 Revised: 17 March 2023 Accepted: 21 March 2023 Published: 23 March 2023 Butea monosperma (Fabaceae) is a medium-sized deciduous tree which grows to about 50 feet and is native to Southeastern Asian nations such as Bangladesh, India, Thailand, and Western Indonesia [3–5]. The ﬂowers of B. monosperma have traditionally been used as an astringent, diuretic, depurative, aphrodisiac, and tonic [6], and a variety of ﬂavonoids (e.g., butein, butin, coreopsin, isobutrin, and monospermoside) have been identiﬁed from this plant. Previous studies have demonstrated that butein exerts anticancer activity and inhibits the proliferation of many human cancers, including colon, breast, hepatocellular, and cervical cancers [7–9]. Butein is involved in cell survival, proliferation, migration, invasion, and angiogenesis, and targets many molecular pathways in various cancers. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 6038. https://doi.org/10.3390/ijms24076038 Int. J. Mol. Sci. 2023, 24, 6038 2 of 18 2 of 18 Butein has also been shown to have immunomodulatory activity by inhibiting the expres- sion of inﬂammatory mediators such as IL-6 and TNF-α in HaCaT cells, a keratinocyte cell line. It most commonly affects the expression of NF-κB and its downstream regula- tors [10,11]. Other important molecular targets include VEGF, STAT3, ERK, JNK, Akt, and p38 [9,12–14]. However, the role of butein in ovarian cancer has not been actively studied; in particular, there have been no studies that clearly explain its molecular mechanism. The fact that it is available as a treatment for ovarian cancer requires better insight into its pharmacological mechanisms. Butein has also been shown to have immunomodulatory activity by inhibiting the expres- sion of inﬂammatory mediators such as IL-6 and TNF-α in HaCaT cells, a keratinocyte cell line. It most commonly affects the expression of NF-κB and its downstream regula- tors [10,11]. Other important molecular targets include VEGF, STAT3, ERK, JNK, Akt, and p38 [9,12–14]. However, the role of butein in ovarian cancer has not been actively studied; in particular, there have been no studies that clearly explain its molecular mechanism. The fact that it is available as a treatment for ovarian cancer requires better insight into its pharmacological mechanisms. Interleukin-6 (IL-6) has been shown to have a direct stimulatory effect on many cancer cells through its action on several cell cycle pathways. IL-6 binds to the non- signaling IL-6 receptor (IL-6R) and then forms a complex with the signaling co-receptor glycoprotein 130 (gp130). IL-6 induced Janus Kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) activation leads to constitutive activation of STAT3, which correlates with enhanced tumor cell growth and chemotherapy resistance [15–17]. This makes the IL-6/IL-6Rα/gp130 signaling pathway an attractive target for therapeutic or preventive intervention. The application of IL-6 blockers as anti-cancer agents has been investigated in many cancer types, but the only currently approved monoclonal antibodies (mAbs) in the United States are tocilizumab (anti-IL-6Rα) and siltuximab (anti-IL-6). These are intended for the treatment of rheumatoid arthritis (RA) and Castleman’s disease, not for use as cancer drugs. Currently, the application of IL-6/IL-6Rα/gp130 blockers as anti-cancer agents has not been extensively studied, much less for ovarian cancer. As disease progression depends on various IL-6-related mechanisms in ovarian cancer, the IL-6 signaling pathway is an ideal target for drug development. It was recently demonstrated that STAT3 regulates the expression of forkhead box class O 3a (FoxO3a) and the cell cycle regulatory proteins p27kip1 and p21waf1 [18,19]. FoxO3a is a transcription factor that mediates several physiological and pathological processes by regulating gene expression in apoptosis, proliferation, cell cycle progression, and DNA damage [20]. Therefore, further studies on the effect of IL-6 blockers in ovarian cancer are needed and elucidating the correlation between IL-6 activated STAT3 phosphorylation and FoxO3a may suggest and exciting new therapeutic directions. g p In this study, we investigated the therapeutic potential of butein isolated from B. monosperma ﬂowers in ovarian cancer. We aimed to elucidate the anti-IL-6 property of butein, which blocks the interaction between IL-6 and IL-6Rα, and to elucidate the underly- ing molecular mechanisms involved in the regulation of the STAT3 pathway. 2. Results 2.1. Identiﬁcation of Compounds 1–14 from Butea monosperma Flowers 2.1. Identiﬁcation of Compounds 1–14 from Butea monosperma Flowers The CH2Cl2- and EtOAc-soluble extract extracts of B. monosperma ﬂowers were sub- jected to multi-step column chromatography to yield 14 known compounds. The chemical structures of these isolates were identiﬁed by spectroscopic evidence to be liquiritigenim- 5′-O-methyl ether (1) [21], (+)-butin (2) [22], (−)-isomonospermoside (3) [23], isocore- opsin (4) [23], liquiritigenin 7-O-beta-D-glucopyranoside (5) [24], (−)-butrin (6) [25], 3′, 4′, 7-trihydroxyﬂavone-7-O-glucoside (7) [26], isoliquilitigenin (8) [27], butein (9) [28], ho- mobutein (10) [29], monospermoside (11) [23], isoliquilitin (12) [28], coreopsin (13) [30], and isobutrin (14) [23] (Figure 1 and Supplementary Information (SI)). Int. J. Mol. Sci. 2023, 24, 6038 3 of 18 Figure 1. Chemical structures of compounds 1–14 isolated from Butea monosperma ﬂowers. Figure 1. Chemical structures of compounds 1–14 isolated from Butea monosperma ﬂowers. Figure 1. Chemical structures of compounds 1–14 isolated from Butea monosperma ﬂowers. Figure 1. Chemical structures of compounds 1–14 isolated from Butea monosperma ﬂowers. 2.2. Characterization of Butein and Anti-IL-6 Activity In Vitro 2.2. Characterization of Butein and Anti-IL-6 Activity In Vitro 2.2. Characterization of Butein and Anti-IL-6 Activity In Vitro 2.2. Characterization of Butein and Anti-IL-6 Activity In Vitro 2.2. Characterization of Butein and Anti IL 6 Activity In Vitro The HEK-Blue™ IL-6 cell line was used to ﬁnd isolates having an inhibitory eﬀect on IL-6 signaling among the 14 isolates from B. monosperma. The cells were treated with in- creasing concentrations of the 14 isolates (0, 3.125, 6.25, 12.5, 25, and 50 µM) in the pres- ence of 10 ng/mL IL-6. After 24 h of reaction, the secreted embryonic alkaline phosphatase (SEAP) signal of HEK-Blue™ IL-6 was inhibited in a concentration-dependent manner by the isolates (Figure S1), with butein (compound 9) achieving the maximum inhibition (Fig- ure 2B). The HEK- Blue™ IL-6 cell bioassay showed that butein blocked IL-6 induced bi- oactivity. We found that butein interfered with the interaction between IL-6 and IL-6Rα and reduced it by 21.7% at 20 µM (Figure S2A). We then performed SPR to conﬁrm if butein could directly bind to IL-6. After each IL-6, IL-6Rα, and gp130 protein was immo- bilized on the CM5 chip, the binding aﬃnity between the immobilized protein and butein was examined. We found that butein had binding aﬃnity to immobilized IL-6 protein (KD = 91.42 µM; Figure 1C). 2.1. Identiﬁcation of Compounds 1–14 from Butea monosperma Flowers In addition, to investigate the association between IL-6Rα and gp130 binding aﬃnity, the RU for each concentration of butein was detected through SPR analysis, and the results were added to Figure S2B. We also compared the binding behav- iors of butein to IL-6Rα with that to gp130 protein. Butein showed binding aﬃnity to im- mobilized IL-6Rα and gp130 proteins (KD = 435.3 µM and KD = 615.7 µM, respectively; Figure S2B). It associates faster with IL-6 than with IL-6Rα and gp130, thus indicating that it has a lower aﬃnity to IL-6Rα and gp130 protein. As a result, butein inhibits downstream i li th h hi h bi di ﬃit t IL 6 The HEK-Blue™IL-6 cell line was used to ﬁnd isolates having an inhibitory effect on IL-6 signaling among the 14 isolates from B. monosperma. The cells were treated with in- creasing concentrations of the 14 isolates (0, 3.125, 6.25, 12.5, 25, and 50 µM) in the presence of 10 ng/mL IL-6. After 24 h of reaction, the secreted embryonic alkaline phosphatase (SEAP) signal of HEK-Blue™IL-6 was inhibited in a concentration-dependent manner by the isolates (Figure S1), with butein (compound 9) achieving the maximum inhibition (Figure 2B). The HEK- Blue™IL-6 cell bioassay showed that butein blocked IL-6 induced bioactivity. We found that butein interfered with the interaction between IL-6 and IL-6Rα and reduced it by 21.7% at 20 µM (Figure S2A). We then performed SPR to conﬁrm if butein could directly bind to IL-6. After each IL-6, IL-6Rα, and gp130 protein was immobi- lized on the CM5 chip, the binding afﬁnity between the immobilized protein and butein was examined. We found that butein had binding afﬁnity to immobilized IL-6 protein (KD = 91.42 µM; Figure 1C). In addition, to investigate the association between IL-6Rα and gp130 binding afﬁnity, the RU for each concentration of butein was detected through SPR analysis, and the results were added to Figure S2B. We also compared the binding behaviors of butein to IL-6Rα with that to gp130 protein. Butein showed binding afﬁnity to im-mobilized IL-6Rα and gp130 proteins (KD = 435.3 µM and KD = 615.7 µM, respectively; Figure S2B). It associates faster with IL-6 than with IL-6Rα and gp130, thus indicating that it has a lower afﬁnity to IL-6Rα and gp130 protein. As a result, butein inhibits downstream signaling through higher binding afﬁnity to IL-6. signaling through higher binding aﬃnity to IL 6. 2.3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian Cancer Cells Characterization of butein and its anti-IL-6 activity in vitro. (A) Chemical structure of E l I ll d h f b f h h Figure 2. Characterization of butein and its anti-IL-6 activity in vitro. (A) Chemical structure of igure 2. Characterization of butein and its anti-IL-6 activity in vitro. (A) Chemical structure of utein. (B) HEK-Blue™ IL-6 cells were treated with various concentration of butein for 24 h. The ctivation of IL-6 was measured by a SEAP activity assay after the treatment of HEK-Blue™ IL-6 ells with the diﬀerent indicated concentrations of butein for 1 h in the presence or absence of IL-6 or 24 h (* p < 0.05, ** p < 0.01). (C) For SPR analysis, IL-6 was immobilized on a CM5 sensor chip, nd butein was injected into the ﬂow cells. T-200 BIAevaluation software (v3.0) was used to subtract he references and determine the steady-state KD. IL-6, interleukin 6. Figure 2. Characterization of butein and its anti-IL-6 activity in vitro. (A) Chemical structure of butein. (B) HEK-Blue™IL-6 cells were treated with various concentration of butein for 24 h. The activation of IL-6 was measured by a SEAP activity assay after the treatment of HEK-Blue™IL-6 cells with the different indicated concentrations of butein for 1 h in the presence or absence of IL-6 for 24 h (* p < 0.05, ** p < 0.01). (C) For SPR analysis, IL-6 was immobilized on a CM5 sensor chip, and butein was injected into the ﬂow cells. T-200 BIAevaluation software (v3.0) was used to subtract the references and determine the steady-state KD. IL-6, interleukin 6. igure 2. Characterization of butein and its anti-IL-6 activity in vitro. (A) Chemical structure of utein. (B) HEK-Blue™ IL-6 cells were treated with various concentration of butein for 24 h. The ctivation of IL-6 was measured by a SEAP activity assay after the treatment of HEK-Blue™ IL-6 ells with the diﬀerent indicated concentrations of butein for 1 h in the presence or absence of IL-6 or 24 h (* p < 0.05, ** p < 0.01). (C) For SPR analysis, IL-6 was immobilized on a CM5 sensor chip, nd butein was injected into the ﬂow cells. T-200 BIAevaluation software (v3.0) was used to subtract he references and determine the steady-state KD. IL-6, interleukin 6. Figure 2. Characterization of butein and its anti-IL-6 activity in vitro. (A) Chemical structure of butein. signaling through higher binding aﬃnity to IL 6. 2.3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian Cancer Cells (B) HEK-Blue™IL-6 cells were treated with various concentration of butein for 24 h. The activation of IL-6 was measured by a SEAP activity assay after the treatment of HEK-Blue™IL-6 cells with the different indicated concentrations of butein for 1 h in the presence or absence of IL-6 for 24 h (* p < 0.05, ** p < 0.01). (C) For SPR analysis, IL-6 was immobilized on a CM5 sensor chip, and butein was injected into the ﬂow cells. T-200 BIAevaluation software (v3.0) was used to subtract the references and determine the steady-state KD. IL-6, interleukin 6. signaling through higher binding aﬃnity to IL 6. 2.3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian Cancer Cells sig a i g t oug ig e bi di g a ity to I 6 2.3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian Cancer Cells To investigate the effects of butein on ovarian cancer in vitro, A2780 and SKOV3 cell lines were used. Cell viability of A2780 and SKOV3 cells was inhibited with IC50 values of 64.7 ± 6.27 µM and 175.3 ± 61.95 µM, respectively, upon butein treatment (Figure 3A). Similarly, their clonogenicity was signiﬁcantly inhibited in the butein-treated cells compared with the control siltuximab (anti-IL-6)-treated cells (Figure 3B). The wound healing assay indicated that butein reduced cell migration at 24 h and 48 h in a concentration-dependent manner compared with the control, 0.1% DMSO-, and siltuximab-treated group (Figure 3C). Cell inﬁltration was similarly affected as observed in the Matrigel cell invasion assay (Figure 3D). Int. J. Mol. Sci. 2023, 24, 6038 4 of 18 Figure 2. Characterization of butein and its anti-IL-6 activity in vitro. (A) Chemical structure of butein. (B) HEK-Blue™ IL-6 cells were treated with various concentration of butein for 24 h. The activation of IL-6 was measured by a SEAP activity assay after the treatment of HEK-Blue™ IL-6 cells with the diﬀerent indicated concentrations of butein for 1 h in the presence or absence of IL-6 for 24 h (* p < 0.05, ** p < 0.01). (C) For SPR analysis, IL-6 was immobilized on a CM5 sensor chip, and butein was injected into the ﬂow cells. T-200 BIAevaluation software (v3.0) was used to subtract the references and determine the steady-state KD. IL-6, interleukin 6. Figure 2. Characterization of butein and its anti-IL-6 activity in vitro. (A) Chemical structure of butein. (B) HEK-Blue™IL-6 cells were treated with various concentration of butein for 24 h. The activation of IL-6 was measured by a SEAP activity assay after the treatment of HEK-Blue™IL-6 cells with the different indicated concentrations of butein for 1 h in the presence or absence of IL-6 for 24 h (* p < 0.05, ** p < 0.01). (C) For SPR analysis, IL-6 was immobilized on a CM5 sensor chip, and butein was injected into the ﬂow cells. T-200 BIAevaluation software (v3.0) was used to subtract the references and determine the steady-state KD. IL-6, interleukin 6. Characterization of butein and its anti-IL-6 activity 2. Characterization of butein and its anti-IL-6 activi gure 2. 3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian T i i h ﬀ f b i i i i A27 2.4. Butein Induced Ovarian Cancer Cell Cycle Arrest and Cell Apoptosis (B) Clonogen assay revealed that butein dramatically reduced the colony growth of ovarian cancer cells (* p < 0.0 Figure 3. Butein inhibited cell viability, proliferation, migration, and invasion of ovarian (A) A2780 and SKOV3 cells were treated with butein at the indicated concentrations in t 48 h and processed for MTT assay to analyze cell viability (* p < 0.05, p < 0.01). (B) assay revealed that butein dramatically reduced the colony growth of ovarian cancer cell p < 0.01, *** p < 0.001). (C) A2780 and SKOV3 cells were treated with butein and allowed to the scratched area for 24 h and 48 h. Yellow color lines indicate a gap in the scratche percentage of migrating area in the wound-healing assay quantiﬁed in A2780 and SKO shown (* p < 0.05, p < 0.01, and * p < 0.001). (D) Matrigel invasion assay was used to the invasion of A2780 and SKOV3 cells after 48 h of butein treatment. Bars indicate mean deviation of three independent experiments performed in triplicate ( p < 0.01 and * p Figure 3. Butein inhibited cell viability, proliferation, migration, and invasion of ovarian cancer (A) A2780 and SKOV3 cells were treated with butein at the indicated concentrations in triplica 48 h and processed for MTT assay to analyze cell viability (* p < 0.05, ** p < 0.01). (B) Clono assay revealed that butein dramatically reduced the colony growth of ovarian cancer cells (* p < Figure 3. Butein inhibited cell viability, proliferation, migration, and invasion of ovari (A) A2780 and SKOV3 cells were treated with butein at the indicated concentrations 48 h and processed for MTT assay to analyze cell viability (* p < 0.05, p < 0.01). ( assay revealed that butein dramatically reduced the colony growth of ovarian cancer p < 0.01, *** p < 0.001). (C) A2780 and SKOV3 cells were treated with butein and allo to the scratched area for 24 h and 48 h. Yellow color lines indicate a gap in the scrat percentage of migrating area in the wound-healing assay quantiﬁed in A2780 and shown (* p < 0.05, p < 0.01, and * p < 0.001). (D) Matrigel invasion assay was use the invasion of A2780 and SKOV3 cells after 48 h of butein treatment. Bars indicate me Figure 3. 3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian T i i h ﬀ f b i i i i A27 2.4. Butein Induced Ovarian Cancer Cell Cycle Arrest and Cell Apoptosis To investigate the eﬀects of butein on ovarian cancer in vitro, A2780 and SKOV3 cell nes were used. Cell viability of A2780 and SKOV3 cells was inhibited with IC50 values f 64.7 ± 6.27 µM and 175.3 ± 61.95 µM, respectively, upon butein treatment (Figure 3A). imilarly, their clonogenicity was signiﬁcantly inhibited in the butein-treated cells com- ared with the control siltuximab (anti-IL-6)-treated cells (Figure 3B). The wound healing ssay indicated that butein reduced cell migration at 24 h and 48 h in a concentration- ependent manner compared with the control, 0.1% DMSO-, and siltuximab-treated roup (Figure 3C). Cell inﬁltration was similarly aﬀected as observed in the Matrigel cell nvasion assay (Figure 3D). Cell cycle progression was monitored using ﬂow cytometry. Exposure to butein resulted in an increase in G1-phase cells along with a decrease in S-phase cells. The effect was observed with 25 µM butein treatment, leading to 55.9% of A2780 cells in G1-phase vs. 45.3% under control conditions. Similarly, 65.8% of SKOV3 cells upon butein treatment and 58.3% under control conditions were in G1-phase (Figure 4A). Butein was also found to signiﬁcantly increase total apoptosis in both A2780 and SKOV3 cell lines in a dose- dependent manner (p < 0.005) (Figure 4B). Consequently, our results demonstrated that treatment with butein inhibits the growth of ovarian cancer cells by inducing cell cycle arrest and apoptosis. In accordance with the above data, Western blot analysis indicated that the expression levels of the cell cycle proteins CDK4, CDK6, and Cyclin D1 were reduced, while that of p27kip1 was enhanced, in the butein-treated A2780 and SKOV3 cell lines in a dose- dependent manner (Figure 4C). Meanwhile, the pro-apoptosis protein Bax was upregulated while Bcl-2 and Mcl-1 were downregulated in the butein-treated A2780 and SKOV3 cell lines in a dose-dependent manner (Figure 4D). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S3. These data suggest that butein regulates cell fate by modulating the expression of cell cycle and apoptosis proteins. Int. J. Mol. Sci. 2023, 24, 6038 Int. J. Mol. Sci. 2023, 24, x 5 of 18 Figure 3. Butein inhibited cell viability, proliferation, migration, and invasion of ovarian cancer cell (A) A2780 and SKOV3 cells were treated with butein at the indicated concentrations in triplicate fo 48 h and processed for MTT assay to analyze cell viability (* p < 0.05, ** p < 0.01). 3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian T i i h ﬀ f b i i i i A27 2.4. Butein Induced Ovarian Cancer Cell Cycle Arrest and Cell Apoptosis Butein inhibited cell viability, proliferation, migration, and invasion of ovarian cancer cells. (A) A2780 and SKOV3 cells were treated with butein at the indicated concentrations in triplicate for 48 h and processed for MTT assay to analyze cell viability (* p < 0.05, ** p < 0.01). (B) Clonogenic assay revealed that butein dramatically reduced the colony growth of ovarian cancer cells (* p < 0.05, Figure 3. Butein inhibited cell viability, proliferation, migration, and invasion of ovarian cancer cells. (A) A2780 and SKOV3 cells were treated with butein at the indicated concentrations in triplicate for 48 h and processed for MTT assay to analyze cell viability (* p < 0.05, ** p < 0.01). (B) Clonogenic assay revealed that butein dramatically reduced the colony growth of ovarian cancer cells (* p < 0.05, p < 0.01, * p < 0.001). (C) A2780 and SKOV3 cells were treated with butein and allowed to migrate to the scratched area for 24 h and 48 h. Yellow color lines indicate a gap in the scratched area. The percentage of migrating area in the wound-healing assay quantiﬁed in A2780 and SKOV3 cells is shown (* p < 0.05, p < 0.01, and * p < 0.001). (D) Matrigel invasion assay was used to determine the invasion of A2780 and SKOV3 cells after 48 h of butein treatment. Bars indicate mean ± standard deviation of three independent experiments performed in triplicate ( p < 0.01 and * p < 0.001). Int. J. Mol. Sci. 2023, 24, 6038 Int. J. 6 of 18 7 of 2 Figure 4. Butein induced growth arrest and apoptosis of ovarian cancer cells. (A) Butein cycle arrest in ovarian cancer cells, which were stained by PI, and cell cycle distribution was a by ﬂow cytometry. (B) A2780 and SKOV3 cells were treated with various concentrations of b triplicate for 48 h to detect cell apoptosis using the FITC Annexin V apoptosis Kit ( p < 0 * p < 0.001). (C) Butein decreased the expression of Cyclin D1, CDK 4, CDK 6, and increa expression of p27kip1 in A2780 and SKOV3 cells. (D) Butein decreased the expression of Bcl- and increased the expression of Bax in A2780 and SKOV3 cells. Figure 4. Butein induced growth arrest and apoptosis of ovarian cancer cells. 3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian T i i h ﬀ f b i i i i A27 2.4. Butein Induced Ovarian Cancer Cell Cycle Arrest and Cell Apoptosis (A) Butein induced cycle arrest in ovarian cancer cells, which were stained by PI, and cell cycle distribution was analyzed by ﬂow cytometry. (B) A2780 and SKOV3 cells were treated with various concentrations of butein in triplicate for 48 h to detect cell apoptosis using the FITC Annexin V apoptosis Kit ( p < 0.01 and * p < 0.001). (C) Butein decreased the expression of Cyclin D1, CDK 4, CDK 6, and increased the expression of p27kip1 in A2780 and SKOV3 cells. (D) Butein decreased the expression of Bcl-2, Mcl-1, and increased the expression of Bax in A2780 and SKOV3 cells. Int. J. Mol. Sci. 2023, 24, 6038 7 of 18 d in on of 7 of 18 d i on of 2.5. Butein Inhibited STAT3 Phosphorylation and Induced Intranuclear Accumulation of FoxO3a through Inhibition of IL-6 Signaling .5. Butein Inhibited STAT3 Phosphorylation and Induced Intranuclear Accumulation of FoxO3a hrough Inhibition of IL-6 Signaling 2.5. Butein Inhibited STAT3 Phosphorylation and Induced Intranuclear Accumulation of FoxO3a through Inhibition of IL-6 Signaling 5. Butein Inhibited STAT3 Phosphorylation and Induced Intranuclear Accumulation of FoxO3a hrough Inhibition of IL-6 Signaling To further elucidate the effect of IL-6 inhibition by butein, the expression of the downstream gene, STAT3, was analyzed by Western blotting of proteins from the A2780 and SKOV3 cell lines after treatment with varying concentrations of butein. No signiﬁcant change was observed in the total amount of STAT3. However, the phosphorylation of STAT3 was found to be inhibited in a concentration-dependent manner in the butein-treated cells relative to the IL-6 treated cells (Figure 5A). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S4. Western blot analysis of the nuclear and cytoplasmic protein extracts revealed that butein also induced FoxO3a accumulation in the nucleus and decreased cytoplasmic FoxO3a. Consistent with this, expression of the proliferation-related gene p27kip1 was also found to be enhanced in the nucleus (Figure 5B). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S5. These results indicate that butein induces STAT3 inactivation through the inhibition of IL-6 signaling in ovarian cancer and increases FoxO3a and p27kip1 levels in the nucleus following STAT3 inactivation. 3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian T i i h ﬀ f b i i i i A27 2.4. Butein Induced Ovarian Cancer Cell Cycle Arrest and Cell Apoptosis To further elucidate the eﬀect of IL-6 inhibition by butein, the expression of the ownstream gene, STAT3, was analyzed by Western blotting of proteins from the A2780 nd SKOV3 cell lines after treatment with varying concentrations of butein. No signiﬁcant hange was observed in the total amount of STAT3. However, the phosphorylation of TAT3 was found to be inhibited in a concentration-dependent manner in the butein- eated cells relative to the IL-6 treated cells (Figure 5A). Whole membrane and protein xpression levels detected by Western blotting are shown in Figure S4. Western blot anal- sis of the nuclear and cytoplasmic protein extracts revealed that butein also induced oxO3a accumulation in the nucleus and decreased cytoplasmic FoxO3a. Consistent with his, expression of the proliferation-related gene p27kip1 was also found to be enhanced in he nucleus (Figure 5B). Whole membrane and protein expression levels detected by West- rn blotting are shown in Figure S5. These results indicate that butein induces STAT3 in- ctivation through the inhibition of IL-6 signaling in ovarian cancer and increases FoxO3a nd p27kip1 levels in the nucleus following STAT3 inactivation. Figure 5. Butein inhibited STAT3 phosphorylation and induced intranuclear accumulation of FoxO3 through inhibition of IL-6 signaling. (A) The phosphorylation levels of STAT3 were decreased in the butein-treated group. (B) The upregulated levels of FoxO3a and p27kip1 in the nucleus and the Figure 5. Butein inhibited STAT3 phosphorylation and induced intranuclear accumulation of FoxO3 through inhibition of IL-6 signaling. (A) The phosphorylation levels of STAT3 were decreased in the butein-treated group. (B) The upregulated levels of FoxO3a and p27kip1 in the nucleus and the downregulated levels of p27kip1 in the cytoplasm were determined by Western blotting of A2780 and SKOV3 cell extracts. β-actin served as the loading control. gure 5. Butein inhibited STAT3 phosphorylation and induced intranuclear accumulation of FoxO3 rough inhibition of IL-6 signaling. (A) The phosphorylation levels of STAT3 were decreased in the utein-treated group. (B) The upregulated levels of FoxO3a and p27kip1 in the nucleus and the Figure 5. Butein inhibited STAT3 phosphorylation and induced intranuclear accumulation of FoxO3 through inhibition of IL-6 signaling. (A) The phosphorylation levels of STAT3 were decreased in the butein-treated group. (B) The upregulated levels of FoxO3a and p27kip1 in the nucleus and the downregulated levels of p27kip1 in the cytoplasm were determined by Western blotting of A2780 and SKOV3 cell extracts. β-actin served as the loading control. Int. J. 2.6. Butein Increased Protein Expression of FoxO3a and p27kip1 through Inactivation of STAT3 2.6. Butein Increased Protein Expression of FoxO3a and p27kip1 through Inactivation of STAT p f p g f To elucidate the mechanism of butein’s effect on FoxO3a and p27kip1, we used siRNA- mediated knock-down of STAT3. Based on the Western blot results, siSTAT3-5—which had the highest knock-down efﬁciency among the siSTAT3 primers—was selected (Figure 6A). When the ovarian cancer cell line was treated with siSTAT3, the cell growth inhibition was similar to that in the butein-treated group. The inhibitory effect was enhanced when butein and siSTAT3 were administered together (Figure 6B). Treatment of butein and siSTAT3 collectively in ovarian cancer cell lines inhibited colony formation, while no signiﬁcant difference was found between groups treated separately with siSTAT3 and butein (Figure 6C). These data demonstrate that butein exerts antiproliferative effects on ovarian cancer cell lines through STAT3. Additionally, FoxO3a and p27kip1 protein levels were found to be upregulated upon STAT3 knock-down, as in the butein-treated group (Figure 6D). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S6. These data show that butein treatment not only mimics the downstream effects of STAT3 suppression but also enhances them, suggesting that butein affects FoxO3a and p27kip1 through STAT3 inactivation. 3. Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian T i i h ﬀ f b i i i i A27 2.4. Butein Induced Ovarian Cancer Cell Cycle Arrest and Cell Apoptosis Mol. Sci. 2023, 24, 6038 8 of 18 2.6. Butein Increased Protein Expression of FoxO3a and p27kip1 through Inactivation of STAT3 2.7. Butein Exerts an Antitumor Effect In Vivo on Ovarian Cancer Cells To test the effect of butein on tumor growth inhibition in vivo, we generated xenograft mice using the A2780 cell line. The butein-treated group showed a signiﬁcant inhibi- tion of tumor growth compared with the vehicle as well as the siltuximab-treated group (Figure 7A). The tumor mass also showed a similar trend (Figure 7B,C). There was no dif- ference in the total mouse weight during the experimental period (Figure 7D). To evaluate the production of IL-6, IL-1β, and TNF-α in serum in mouse blood, the total amount of IL-6, IL-1β, and TNF-α was normalized to the total amount of vehicle (Figure 7E). In the vehicle group, tumor cells were closely arranged into complete and atypical structures. In contrast, the group treated with a high concentration of butein displayed a greater degree of tumor cell death, characterized by an incomplete cell membrane, a pyknotic nucleus, and condensed cytoplasm. Supporting the in vitro experimental results, we identiﬁed similar changes in protein expression upon butein treatment in vivo (Figure 7G,H). Butein- mediated inhibition of STAT3 phosphorylation as well as increased nuclear FoxO3a were observed in mouse tumor tissues. Whole membrane and protein expression levels detected by Western blotting are shown in Figure S7. These data suggested that butein treatment inhibited tumor growth in ovarian cancer cells by increasing the nuclear accumulation of FoxO3a through the inhibition of STAT3 phosphorylation in vivo. Int. J. Mol. Sci. 2023, 24, 6038 Int. J. Mol. S 9 of 18 of 20 038 Figure 6. Butein increased protein expression of FoxO3a and p27kip1 through inactivati (A) Western blot showing STAT3 protein expression following siSTAT3 treatment. β-actin a loading control. (B) Cell proliferation upon butein and siSTAT3 treatment was detected CCK-8 assay ( p < 0.01 and * p < 0.001; NS, not signiﬁcant). (C) Cell growth following siSTAT3 treatment was detected by colony formation assay (* p < 0.05, p < 0.01, and NS, not signiﬁcant). Data represent the mean ± SD of three replicates. (D) Expression lev FoxO3a, and p27kip1 proteins in ovarian cancer cells with the combination of butein a treatment. β-actin was used as a loading control. Figure 6. Butein increased protein expression of FoxO3a and p27kip1 through inactivation of STAT3. (A) Western blot showing STAT3 protein expression following siSTAT3 treatment. β-actin was used as a loading control. 2.7. Butein Exerts an Antitumor Effect In Vivo on Ovarian Cancer Cells (B) Cell proliferation upon butein and siSTAT3 treatment was detected through the CCK-8 assay ( p < 0.01 and *** p < 0.001; NS, not signiﬁcant). (C) Cell growth following butein and siSTAT3 treatment was detected by colony formation assay (* p < 0.05, p < 0.01, and * p < 0.001; NS, not signiﬁcant). Data represent the mean ± SD of three replicates. (D) Expression levels of STAT3, FoxO3a, and p27kip1 proteins in ovarian cancer cells with the combination of butein and siSTAT3 treatment. β-actin was used as a loading control. 10 of 18 Int. J. Mol. Sci. 2023, 24, 6038 Int. J. Mol. Sci. 2023, 24, x FO 10 of 18 Figure 7. Butein inhibited A2780 and tumor growth in vivo. A2780 cells (1 × 107) were injected sub- cutaneously into BALB/c nude mice with an equal volume of PBS. When tumors reached a volume of 150 mm3, siltuximab (10 mg/kg) or butein (2 or 4 mg/kg) in a vehicle of 0.05% CMC and 5% DMSO was administered by intraperitoneal injection ﬁve times a week. (A) Tumor volumes were calculated from caliper measurements (* p < 0.05, *** p < 0.001). (B) The tumor masses were excised for compar- ison between the groups. (C) After 21 days of treatment, all mice were sacriﬁced, and the total mass Figure 7. Butein inhibited A2780 and tumor growth in vivo. A2780 cells (1 × 107) w subcutaneously into BALB/c nude mice with an equal volume of PBS. When tumo volume of 150 mm3, siltuximab (10 mg/kg) or butein (2 or 4 mg/kg) in a vehicle of 0.0 5% DMSO was administered by intraperitoneal injection ﬁve times a week. (A) Tumor v calculated from caliper measurements (* p < 0.05, * p < 0.001). (B) The tumor masses for comparison between the groups. (C) After 21 days of treatment, all mice were sacri total mass of each tumor was determined at autopsy (n = 5 mice per treatment group * p < 0.001). (D) The mouse body weight measured on the days indicated. (E) To asse and TNF-α production in serum in mouse blood, the total amount of IL-6, IL-1β, and normalized to the vehicle (* p < 0.05, ** p < 0.01). (F) H&E staining results show the antica butein on ovarian cancer (G) The phosphorylation levels of STAT3 were determined u Figure 7. 2.7. Butein Exerts an Antitumor Effect In Vivo on Ovarian Cancer Cells Butein inhibited A2780 and tumor growth in vivo. A2780 cells (1 × 107) were injected sub- cutaneously into BALB/c nude mice with an equal volume of PBS. When tumors reached a volume of 150 mm3, siltuximab (10 mg/kg) or butein (2 or 4 mg/kg) in a vehicle of 0.05% CMC and 5% DMSO was administered by intraperitoneal injection ﬁve times a week. (A) Tumor volumes were calculated from caliper measurements (* p < 0.05, *** p < 0.001). (B) The tumor masses were excised for compar- ison between the groups. (C) After 21 days of treatment, all mice were sacriﬁced, and the total mass Figure 7. Butein inhibited A2780 and tumor growth in vivo. A2780 cells (1 × 107) were injected subcutaneously into BALB/c nude mice with an equal volume of PBS. When tumors reached a volume of 150 mm3, siltuximab (10 mg/kg) or butein (2 or 4 mg/kg) in a vehicle of 0.05% CMC and 5% DMSO was administered by intraperitoneal injection ﬁve times a week. (A) Tumor volumes were calculated from caliper measurements (* p < 0.05, * p < 0.001). (B) The tumor masses were excised for comparison between the groups. (C) After 21 days of treatment, all mice were sacriﬁced, and the total mass of each tumor was determined at autopsy (n = 5 mice per treatment group) ( p < 0.01, *** p < 0.001). (D) The mouse body weight measured on the days indicated. (E) To assess IL-6, IL-1β, and TNF-α production in serum in mouse blood, the total amount of IL-6, IL-1β, and TNF-α were normalized to the vehicle (* p < 0.05, ** p < 0.01). (F) H&E staining results show the anticancer effect of butein on ovarian cancer. (G) The phosphorylation levels of STAT3 were determined using Western blotting of the harvested tumor tissue. β-actin served as a loading control. (H) The upregulated levels of FoxO3a and p27kip1 were determined using Western blotting of nuclear extracts of the harvested tumor tissue. β-actin and Lamin B1 served as loading controls. Figure 7. Butein inhibited A2780 and tumor growth in vivo. A2780 cells (1 × 107) were injected sub- cutaneously into BALB/c nude mice with an equal volume of PBS. 3. Discussion Ovarian cancer is the most lethal gynecological malignancy, and inﬂammation has been shown to play a large role in ovarian cancer growth. IL-6 is a cytokine that acts on chronic inﬂammation as a major tumor-promoting inﬂammatory mediator. IL-6 has been shown to activate signaling pathways leading to tumor proliferation, the most studied of which are the JAK and STAT3 pathways. Many drugs were found to inhibit IL-6 signaling, including siltuximab and sirukumab [31], although, none of them currently show promising outcomes in ovarian cancer treatment. Therefore, we aimed to discover new small molecule inhibitors of IL-6 signaling and to elucidate their mechanisms of action. g g The 14 compounds isolated and identiﬁed from Butea monosperma ﬂowers are in accordance with previous studies [21–30]. Of these, butein shows anti-inﬂammatory activity and has been shown to be a potential therapeutic agent for the treatment of chronic inﬂammatory diseases and cancers [12]. Recent evidence suggests that it inhibits the activities of anti-inﬂammatory cytokines such as IL-6, IL-1β, and TNF-α [13]. However, whether it directly inhibits anti-inﬂammatory cytokines has not been studied. Using the SPR assay, we conﬁrmed that butein binds to IL-6, IL-6Rα, and gp130 through intermolecular interactions. We further validated that the binding force between butein and IL-6 was higher, and that butein inhibited IL-6 downstream signaling using the HEK-Blue™IL-6 cell line. Ours is the ﬁrst study to report that butein binds to IL-6 and inhibits its downstream signaling. Furthermore, our data indicating the low expression of these cytokines in the mouse blood serum following butein treatment supports these previous ﬁndings. g pp p g Our results showed that survival, migration, and invasion of ovarian cancer cells were inhibited by butein, and that this occurred in a concentration-dependent manner. Furthermore, treatment with increasing concentrations of butein resulted in increased cell cycle arrest as well as increased apoptosis. Further investigation revealed that butein affects the expression levels of cell cycle proteins as well as apoptosis proteins. Along similar lines, previous studies have shown that butein inhibits the activation of various oncogenes through many signaling mechanisms [12,32]. A recent report revealed that butein can exert a chemosensitizing effect through the miR-186-5p-TWIST1 axis, suggesting that butein exerts its chemosensitizing effect, at least in part, through microRNA modulation [33]. exerts its chemosensitizing effect, at least in part, through microRNA modulation [33]. 2.7. Butein Exerts an Antitumor Effect In Vivo on Ovarian Cancer Cells When tumors reached a volume of 150 mm3, siltuximab (10 mg/kg) or butein (2 or 4 mg/kg) in a vehicle of 0.05% CMC and 5% DMSO was administered by intraperitoneal injection ﬁve times a week. (A) Tumor volumes were calculated from caliper measurements (* p < 0.05, *** p < 0.001). (B) The tumor masses were excised for compar- ison between the groups. (C) After 21 days of treatment, all mice were sacriﬁced, and the total mass Figure 7. Butein inhibited A2780 and tumor growth in vivo. A2780 cells (1 × 107) were injected subcutaneously into BALB/c nude mice with an equal volume of PBS. When tumors reached a volume of 150 mm3, siltuximab (10 mg/kg) or butein (2 or 4 mg/kg) in a vehicle of 0.05% CMC and 5% DMSO was administered by intraperitoneal injection ﬁve times a week. (A) Tumor volumes were calculated from caliper measurements (* p < 0.05, * p < 0.001). (B) The tumor masses were excised for comparison between the groups. (C) After 21 days of treatment, all mice were sacriﬁced, and the total mass of each tumor was determined at autopsy (n = 5 mice per treatment group) ( p < 0.01, *** p < 0.001). (D) The mouse body weight measured on the days indicated. (E) To assess IL-6, IL-1β, and TNF-α production in serum in mouse blood, the total amount of IL-6, IL-1β, and TNF-α were normalized to the vehicle (* p < 0.05, ** p < 0.01). (F) H&E staining results show the anticancer effect of butein on ovarian cancer. (G) The phosphorylation levels of STAT3 were determined using Western blotting of the harvested tumor tissue. β-actin served as a loading control. (H) The upregulated levels of FoxO3a and p27kip1 were determined using Western blotting of nuclear extracts of the harvested tumor tissue. β-actin and Lamin B1 served as loading controls. Int. J. Mol. Sci. 2023, 24, 6038 11 of 18 11 of 18 3. Discussion Butein is known to exhibit anticancer effects by the inhibition of STAT3, Akt, and PI3K signaling in other cancers as well [9,13,34]. Our in vitro as well as in vivo data suggests that the same molecular pathways are affected by butein via similar mechanisms in ovarian cancer. Additionally, it has been reported that, following IL-6 signaling, phosphorylated STAT3 regulates the nuclear translocation of FoxO3a, leading to increased expression of p27kip1 in T cells [19], similar to the mechanisms we found in ovarian cancer cells. Furthermore, recent evidence suggests that butein leads to increased p27kip1 expression by promoting not only the nuclear localization of FoxO3a, but also by enhancing its binding to the p27kip1 promoter, resulting in cell cycle arrest and thereby inhibiting cell proliferation [18,35]. We found that butein increased the expression of p27kip1 by regulating the mechanism of STAT3 and FoxO3a in ovarian cancer. According to a recent study, FoxO3a is an important regulator during the development of drug resistance and may show great potential as a novel biomarker for prognostic evaluation and therapeutic targets in cancer patients [36–38]. Additionally, FoxO3a exhibits great therapeutic potential due to its essential role in cancer progression, particularly in drug resistance. In summary, butein from Butea monosperma ﬂower isolates show excellent therapeutic potential in ovarian cancer cells. It has shown anti-IL-6 activity, which induces inactivation of STAT3 through IL-6 binding and nuclear accumulation of FoxO3a in ovarian cancer cells. This suggests that butein, along with current front-line chemotherapy drugs, may represent a promising approach towards ovarian cancer treatment; however, this requires further research. Int. J. Mol. Sci. 2023, 24, 6038 12 of 18 12 of 18 4.1. General Experimental Procedures Preparative HPLC was performed on a Gilson HPLC system (Middleton, WI, USA) which consisted of a binary pump, a liquid handler, a UV/Vis detector and a Luna C18(2) column (21.2 × 250 mm I.D., 5 µm, phenomenex, Torrance, CA, USA). Silica gel 60 (40–63 µm, Merck, Germany) and ZEOprep 90 C18 (40–63 µm, Zeochme, Uetikon, Switzerland) were used for column chromatography. The semi-preparative scale high-performance countercurrent chromatography (HPCCC) instrument used was a Spectrum instrument (Dynamic Extractions, Berkshire, UK). The HPCCC system was composed of an IOTA 300 s pump (ECOM, Prague, Czech Republic), a Sapphire 600 UV–VIS detector (ECOM, Prague, Czech Republic) and a Foxy R2 fraction collector (Teledyne Isco, Lincoln, NE, USA). LC–MS data were obtained using an Agilent 6530 Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA). One-dimensional (1D) and two-dimensional (2D) NMR data were recorded on an Avance 500 spectrometer (Bruker, Karlsruhe, Germany). Organic solvents were purchased from Dae-Jung Chemical Co., Ltd. (Seoul, Republic of Korea). 4.2. Plant Material Flowers of B. monosperma were collected from Popa Mountain National Park in March 2014, and the specimen of B. monosperma was identiﬁed by Khin Myo Htwe (Popa Mountain National Park). A voucher specimen (#PopaButea_M 032013) of this plant was deposited at the Herbarium of the College of Pharmacy, the Catholic University of Korea. 4.3. Extraction and Isolation 2023, 24, 6038 13 of 18 13 of 18 4.4. IL-6 Inhibitory Bioassay with HEK-Blue™IL-6 Cells 4.4. IL-6 Inhibitory Bioassay with HEK-Blue™IL-6 Cells HEK-Blue™IL-6 cells were obtained from InvivoGen (San Diego, CA, USA). In a 96-well plate, 20 µL butein at various concentrations (0, 3.125, 6.25, 12.5, 25, and 50 µM) were added to the cell suspension (50,000 cells/200 µL). Following this, the recombinant human IL-6 (BioLegend, San Diego, CA, USA) was administered at 10 ng/mL. After 24 h, 20 µL of the IL-6-treated cell suspension was added to 180 µL of QUANTI-Blue™Solution (JnvivoGen, San Diego, CA, USA) in a new 96-well plate. After 2 h of incubation at 37 ◦C, the spectrophotometric absorbance between 620 and 655 nm was recorded. HEK-Blue™IL-6 cells were obtained from InvivoGen (San Diego, CA, USA). In a 96-well plate, 20 µL butein at various concentrations (0, 3.125, 6.25, 12.5, 25, and 50 µM) were added to the cell suspension (50,000 cells/200 µL). Following this, the recombinant human IL-6 (BioLegend, San Diego, CA, USA) was administered at 10 ng/mL. After 24 h, 20 µL of the IL-6-treated cell suspension was added to 180 µL of QUANTI-Blue™Solution (JnvivoGen, San Diego, CA, USA) in a new 96-well plate. After 2 h of incubation at 37 ◦C, the spectrophotometric absorbance between 620 and 655 nm was recorded. 4.7. Clonogenic Formation Assay A2780 and SKOV3 cell lines were seeded at approximately 5000 cells/well in 24-well plates. Butein was added to the cell medium at various concentrations (5, 10, and 25 µM) together with control siltuximab (CTNO328; EUSA Pharma, Inc., Hemel Hempstead, UK) for 12 h. After 2 weeks, the colonies formed were ﬁxed with ﬁxer buffer, stained with crystal violet, and imaged with a ChemiDoc imaging system (Bio-Rad, Herles, CA, USA). 4.6. Analysis of Cell Viability by the MTT Assay A2780 and SKOV3 cell lines were purchased from the European Collection of Cell Cultures (ECACC, London, UK) and Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) respectively. Each cell line was seeded in a 96-well plate at an appropriate cell number (3000 cells/100 µL in each well). After 24 h, butein was added to the plates at each of the following concentrations, 0, 3.125, 6.25, 12.5, 25, and 50 µM, in triplicate in the presence of serum medium. After 48 h, 100 µL of N, N-dimethylformamide (Sigma-Aldrich, St. Louis, MO, USA) solubilizing solution was added to each well and incubated for 4 h. The solution was then aspirated, and 100 µL of DMSO was added for cell lysis. The spectrophotometric absorbance was measured at 540 nm. Half of the maximum inhibitory concentration (IC50) was determined using GraphPad Prism software (version 7.0). 4.5. Surface Plasmon Resonance Assay The surface plasmon resonance (SPR) assay was measured using the BIAcore T200 model (GE Healthcare, Chicago, IL, USA) with HBS-EP buffer (1×HBS-EP, GE Healthcare, Chicago, IL, USA) containing 5% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). The pH scouting for IL-6 (PeproTech, Rocky Hill, NJ, USA), IL-6Rα (PeproTech, Rocky Hill, NJ, USA), and gp130 (Sino Biological, Beijing, China, or ANRT, Daejeon, Korea) immobilization was performed in 10 mM acetate buffer at pH 4.5. IL-6, IL-6Rα, and gp130 were immobilized on a CM5 chip at 750, 1200, and 2000 response units (RU). Butein was injected into the IL-6, IL-6Rα and gp130-immobilized ﬂow cell at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µM with a 25 µL/min ﬂow rate for 150 s and allowed to dissociate for 300 s. Steady-state KD was determined via the T-200 BIAevaluation software (GE Healthcare, Chicago, IL, USA). 4.3. Extraction and Isolation The dried ﬂowers of B. monosperma (157 g) were ground into a ﬁne powder and ex- tracted with methanol (2 L× 90 min × three times) to give a methanol-soluble extract (80.4 g). The methanol-soluble extract was suspended in H2O (1 L) and partitioned with organic solvents to produce CH2Cl2- (4.2 g), EtOAc- (4.3 g), and n-BuOH- (49.6 g) soluble extracts The CH2Cl2-soluble extract was subjected to silica gel medium-pressure column chromatography (MPLC) using a CH2Cl2–MeOH mixture (40:1 →0:100 (v/v)) to give 16 sub-fractions (BMD1-BMD16). BMD8 (114.8 mg) was puriﬁed by RP-HPLC using a MeCN–H2O mixture (45:55 (v/v)) to yield 1 (21.8 mg). BMD10 (142.2 mg) was puriﬁed by RP-HPLC using 48:52 (v/v) of a MeCN–H2O mixture to produce 8 (35.2 mg). BMD13 (270.1 mg) was subjected to RP-MPLC using a gradient elution of a MeCN–H2O mixture (20:80 (v/v) →90:10 (v/v)) to yield 9 (124.6 mg). BMD14 (424 mg) was subjected to RP– MPLC with a gradient elution of a MeCN–H2O mixture (10:90 (v/v) →70:30 (v/v)) to yield 2 (145.6 mg). BMD6 (593.3 mg) was resolved into seven sub-fractions (BMD6-1 to BMD6-7) using RP-MPLC (MeCN–H2O, 10:90 (v/v) →90:10 (v/v)). BMD6-4 (20.4 mg) was puriﬁed by RP-HPLC using a MeCN–H2O mixture (48:52 (v/v)) to produce 10 (7.3 mg). The EtOAc- soluble extract (4.27g) was subjected to silica gel-MPLC with a mixture of CH2Cl2–MeOH (5:1 v/v), resulting in four sub-fractions (BME1-BME4). BME3 was separated by RP-MPLC using a gradient elution of a MeCN–H2O mixture (15:85 (v/v) →50:50 (v/v)) to obtain eight sub-fractions (BME3-1 to BME3-8). BME3-4 (39.3 mg) was puriﬁed by RP-HPLC (MeCN–H2O, 25:75 (v/v)) to yield 3 (19.7 mg). BME3-5 (106 mg) was subjected to RP-MPLC with 20:80 (v/v) of a MeCN–H2O mixture, resulting in three sub-fractions (BME3-5-1 to BME3-5-3). Compounds 11 (15.6 mg) and 12 (3 mg) were puriﬁed from BME3-5-3 using RP- HPLC with 35:65 (v/v) of a MeCN–H2O mixture. Compounds 7 (8.9 mg), 4 (16.6 mg) and 5 (0.5 mg) were separated by RP-HPLC using isocratic elution with a MeCN–H2O mix- ture (23:77 (v/v)). BME4 (32.8 mg) was resolved by RP-HPLC using a MeCN–H2O mix- ture to yield 13 (11.6 mg). The n-BuOH-soluble extract (200 mg) was subjected to high- performance column chromatography using a biphasic solvent system (EtOACn–BuOH– H2O, 5:5:10 (v/v/v); rotation speed—1500 rpm; ﬂow rate—3 mL/min; RP mode) to yield 6 (20.3 mg) and 14 (18.4 mg). Int. J. Mol. Sci. 4.8. Wound-Healing Assay A2780 and SKOV3 cells were cultured to 90% conﬂuence in 6-well plates. After changing to a serum-free medium, the cell monolayer was scraped to artiﬁcially form homogeneous wound with uniform thickness. The drug treatment for each well was carried out in the following manner: siltuximab at 50 µM; butein at 10, 25, and 50 µM. The cells were imaged at 0, 24, and 48 h. Int. J. Mol. Sci. 2023, 24, 6038 14 of 18 14 of 18 4.10. Flow Cytometric Analysis of Cell Cycle and Apoptosis A2780 and SKOV3 cell lines were treated with control (siltuximab 10 µM) and butein (5, 10, and 25 µM). After 6 h, the cells were ﬁxed with 70% ethanol for cell cycle analysis and stained with propidium iodide (PI). Cell death was analyzed using the FITC Annexin V Apoptosis Detection kit with 7-AAD (Cat.no. 640922, BioLegend, San Diego, CA, USA). Cells were sorted using a FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA) and cell cycle and apoptosis were analyzed using BD FACS Diva software version 6.2. Analysis (BD Biosciences, Franklin Lakes, NJ, USA) was performed in triplicate. 4.11. Small Interfering RNA (siRNA) Transfection The siRNA siNC was used as a negative control, and siSTAT3 was used as a target siRNA. The siRNAs used were produced by Genolution (Genolution Pharmaceutical lnc., Seoul, Korea) and were transfected according to the manufacturer’s instructions. Target sequences for siRNAs are listed in Table S1. 4.9. Matrigel Invasion Assay Matrigel invasion assays were performed using a BioCoat Matrigel Invasion Chamber (BD Bioscience, Bedford, MA, USA). A2780 and SKOV3 cells (50,000 cells) were placed inside the chamber in serum-free medium containing 500 µL of drugs, and 700 µL of medium containing serum were placed outside the chamber. After incubation for 48 h, the inﬁltrating cells were stained using the Differential Quick Stain Kit (Cat.no. 26096-50, Electron Microscopy Sciences, Hatﬁeld, PA, USA). Cells were imaged with a Slide Scanner (Aperio CS2, Leica Microsystems, Wetzlar, Germany) and the number of invading cells was quantiﬁed using Image J. 4.15. Enzyme-Linked Immunosorbent Assay (ELISA) An amount of 0.5 µg/mL rhIL-6 protein (Cat.no.570806, BioLegend) was coated onto 96-well ELISA plates at 4 ◦C overnight, and then the plates were washed with PBST (1 × PBS containing 0.05% Tween-20), followed by the addition of PBSA (1 × PBS con- taining 1% bovine serum albumin (BSA)) (Bovogen, East Keilor, Australia) for 1 h at room temperature. After blocking, the FDA-approved compounds (20 uM) (Cat.no. L-1300, Sell- eckchem, Houston, TX, USA) were incubated for 2 h at room temperature. After washing three times with PBST, 0.2 µg/mL recombinant IL-6Ra protein (Cat.no.ILR-H5259, Acro biosystems, Newark, DE, USA) was added to the plates, which were incubated for 1 h. After washing three times with PBST, HRP-conjugated goat anti-human IgG Ab (Cat.no. 10-035-098, Jackson Immno, Hatﬁeld, West Grove, PA, USA) was added to the plates and incubated for 1 h. After washing three times with PBST, TMB solution (Cat.no.11684302001, Roche, Mannheim, Germany) was added to the plates and incubated for 15 min. Then, stop solution (2N HCl) was added to the plates. The absorbance was measured at 450 nm using the Epoch microplate spectrophotometer (BioTek instruments, Winooski, VT, USA). Concentrations of IL-6, Interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) in mouse blood serum were measured using an ELISA (mouse IL-6, IL-1β, and TNF-α uncoated ELISA Kit, Invitrogen, MA, USA) kit according to the manufacturer’s protocol. All ELISA analyses were performed in triplicate. 4.12. Western Blotting After each drug treatment of the cells, the cells were dissolved in 100 µL of radioim- munoprecipitation assay (RIPA) buffer (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA) and centrifuged to separate the supernatant containing protein. Isolated protein concen- trations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA). Aliquots of proteins quantiﬁed at 30 ng were boiled at 100 ◦C in 5× sample buffer, separated on a 12% SDS-polyacrylamide gel and electrophoresed onto polyvinylidene diﬂuoride (PDVF) membranes (Millipore, Billerica, MA, USA). The PDVF membranes were blocked for 1 h at 25 ◦C with 5% BSA in 1× tris buffered saline containing 0.05% Tween20 (TBST) (Sigma-Aldrich, St. Louis, MO, USA). The membrane was incu- bated in the primary antibody at 4 ◦C overnight. PDVF membranes were washed with 1× TBST and incubated with AfﬁniPure goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) for 1 h at room temperature. After washing again with TBST buffer, enhanced signals were detected using the SuperSignal™West Femto Maximum Sensitivity Substrate kit (cat.no. 34094, Thermo Scientiﬁc, Rockford, IL, USA). The blot was imaged using the ChemiDoc imaging system (Bio-Rad, Herles, CA, USA), and protein expression was quantiﬁed with the ImageJ software (v1.8.0). p p q g The following primary antibodies were used: anti-β-actin (cat. No. 8457S; dilution 1:1000), anti-Bcl-2 (cat. no. 3498S; 1:500), anti-Bax (cat. no. 2772S; 1:1000), anti-Mcl-1 (cat. no. 4572S; 1:1000), anti-Cyclin D1 (cat. no. 2978T; 1:1000), anti-CDK4 (cat. no. 12790T; 1:1000), anti-CDK6 (cat. no. 3136T; 1:1000), anti-p27kipl (cat. no. 3686T; 1:1000), anti-phospho-STAT3 (Tyr705) (cat. no. 9131S; 1:500), anti-STAT3 (cat.no. 12640S; 1:500), anti-FoxO3a (cat. no. 2497S; 1:1000), and Lamin B1 (cat.no. 12586S, 1:1000) (all from Cell Signaling Technologies, Danvers, MA, USA). Int. J. Mol. Sci. 2023, 24, 6038 15 of 18 15 of 18 4.14. Hematoxylin and Eosin (H&E) Staining in Mouse Tumor Tissues After the experiment was completed, mice were sacriﬁced and tumor tissues were collected. Collected tumor tissues were ﬁxed in 4% paraformaldehyde for 24 h. Fixed tissues were washed in 1 × PBS and embedded in parafﬁn. The parafﬁn block was sectioned at a thickness of 2 µm and sections placed on a slide glass. After hematoxylin and eosin staining, the sections were dehydrated, deparafﬁnized with mineral oil, and a cover slip was added. 4.13. Mouse Xenografts 4.13. Mouse Xenografts Adult BALB/c nude mice aged 5 weeks (n = 5, body weight 17−19 g) were purchased from Orient Bio (Seongnam, Republic of Korea) and reared in aseptic conditions with 55 ± 10% humidity and 25 ± 2 ◦C temperature under a 12 h/12 h light-dark cycle (Catholic University protocol). All animal experimental work was carried out in compliance with the legal obligations and federal guidelines and legal obligations for the care and maintenance of laboratory animals. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Catholic University of Korea (ap- proval number: CUK-IACUC-2019-026-01). A2780 cell suspension (1 × 107 cells/200 µL in 1 × PBS) was injected subcutaneously into the dorsal scapula area of each mouse. The tumor developed 14 days after implantation to a size of approximately 150 mm3. The mice were then divided randomly into vehicle control (5% DMSO), siltuximab (10 mg/kg), and butein (2 or 4 mg/kg) groups. All drugs were dissolved in 0.05% carboxymethylcellulose sodium salt (CMC) (Sigma-Aldrich, St. Louis, MO, USA) and injected intraperitoneally ﬁve times a week for three weeks. The tumor size was measured using calipers once every 2 days. The tumor volume was calculated using a simplified equation (length × width2 × 0.5). Each tumor was harvested 22 days post-treatment. 4.16. Data Analysis All data, except that from the cell cycle analysis, were evaluated using GraphPad Prism software (version 7.00; GraphPad Software, Inc., San Diego, CA, USA). Results are presented as mean ± standard deviation (SD). Following two-way ANOVA, Bonferroni’s post hoc tests were performed with GraphPad Prism 7.0. Int. J. Mol. Sci. 2023, 24, 6038 16 of 18 5. Conclusions Based on our ﬁndings, we can conclude that butein from Butea monosperma ﬂower isolates inhibits ovarian cancer growth by binding to IL-6 and suppressing its activity, which results in inactivation of STAT3 and nuclear accumulation of FoxO3a and p27kip1, thereby limiting tumor growth. Our work highlights butein as a promising therapeutic agent for ovarian cancer treatment. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24076038/s1. Author Contributions: Conceptualization, T.-H.H. and K.D.Y.; methodology, L.K.K. and H.J.K.; software, S.-A.P. and Y.J.S.; validation, S.-A.P. and L.K.K.; investigation, S.-A.P.; data curation, S.-A.P., Y.J.S., H.J.K., T.-H.H. and K.D.Y.; writing—original draft preparation, S.-A.P. and Y.J.S.; writing—review and editing, S.-A.P. and H.J.K.; visualization, S.-A.P., Y.J.S., T.-H.H. and K.D.Y.; supervision, T.-H.H. and K.D.Y.; project administration, T.-H.H. and K.D.Y.; funding acquisition, T.-H.H. and K.D.Y. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Basic Sciences Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Tech- nology (grant nos. NRF-2022R1A2C2009911, 2018R1A6A1A03025108, 2021R1A2C2009782, and 2021R1A6A3A1303840811). This work was supported by the Catholic University of Korea Research Fund, 2022. Funding: This work was supported by the Basic Sciences Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Tech- nology (grant nos. NRF-2022R1A2C2009911, 2018R1A6A1A03025108, 2021R1A2C2009782, and 2021R1A6A3A1303840811). This work was supported by the Catholic University of Korea Research Fund, 2022. Institutional Review Board Statement: This study was approved by the Institutional animal care and use committee (IACUC) of the Catholic University of Korea (approval number: CUK-IACUC-2019-026-01). Institutional Review Board Statement: This study was approved by the Institutional animal care and use committee (IACUC) of the Catholic University of Korea (approval number: CUK-IACUC-2019-026-01). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest for all the authors. Abbreviations IL-6: Interleukin-6; IL-6R: Interleukin-6 receptor; HPLC: high-performance liquid chromatogra- phy; HPLCC: high-performance countercurrent chromatography; SPR: surface plasmon resonance; RIPA: radioimmunoprecipitation assay; STAT3: signal transducer and activator of transcription 3; FoxO3a: Forkhead box O3a; NRF: National Research Foundation of Korea. 9. Bai, X.; Ma, Y.; Zhang, G. Butein suppresses cervical cancer growth through the PI3K/AKT/mTOR pathway. Oncol. Rep. 2015, 33, 3085–3092. [CrossRef] 10. Seo, W.Y.; Youn, G.S.; Choi, S.Y.; Park, J. Butein, a tetrahydroxychalcone, suppresses pro-inﬂammatory responses in HaCaT keratinocytes. BMB Rep. 2015, 48, 495. [CrossRef] m, S.I.; Kim, H.-S.; Kim, S.J.; Song, Y.S. Tumor evolution and chemoresistance in ovarian cancer. NPJ Precis CrossRef] [PubMed] 2. James, N.E.; Woodman, M.; Ribeiro, J.R. Prognostic immunologic signatures in epithelial ovarian cancer. Oncogene 2022, 41, 1389–1396. [CrossRef] [PubMed] 2. James, N.E.; Woodman, M.; Ribeiro, J.R. Prognostic immunologic signatures in epithelial ovarian cancer. Oncogene 2022, 41, 1389–1396. [CrossRef] [PubMed] 3. Sutariya, B.K.; Saraf, M.N. A comprehensive review on pharmacological proﬁle of Butea monosperma (Lam.) Taub. J. Appl. Pharm. Sci. 2015, 5, 159–166. [CrossRef] 4 S i R K S Si h i A K A idi b i i l f B i Fi i 77 86 90 [C R f] 3. Sutariya, B.K.; Saraf, M.N. A comprehensive review on pharmacological proﬁle of Butea monosperma (Lam.) Taub. J. Appl. Pharm. Sci. 2015, 5, 159–166. [CrossRef] S S S h d b l f i i [C f] 4. Somani, R.; Kasture, S.; Singhai, A.K. Antidiabetic potential of Butea monosperma in rats. Fitoterapia 2006, 77, 86–90. [CrossRef] 5. Gupta, A.; Singh, S.; Gaur, K.; Singh, A.; Kumar, L. A review on pharmacognostic study of Butea monosperma. Int. J. Res. Ayurveda Pharm. 2017, 8, 196–199. [CrossRef] 4. Somani, R.; Kasture, S.; Singhai, A.K. Antidiabetic potential of Butea monosperma in rats. Fitoterapia 200 5. Gupta, A.; Singh, S.; Gaur, K.; Singh, A.; Kumar, L. A review on pharmacognostic study of Butea monosp Pharm. 2017, 8, 196–199. [CrossRef] 6. Chokchaisiri, R.; Suaisom, C.; Sriphota, S.; Chindaduang, A.; Chuprajob, T.; Suksamrarn, A. Bioactive ﬂavonoids of the ﬂowers of Butea monosperma. Chem. Pharm. Bull. 2009, 57, 428–432. [CrossRef] 6. Chokchaisiri, R.; Suaisom, C.; Sriphota, S.; Chindaduang, A.; Chuprajob, T.; Suksamrarn, A. Bioactive ﬂavonoids of the ﬂowers of Butea monosperma. Chem. Pharm. Bull. 2009, 57, 428–432. [CrossRef] p 7. Yang, P.Y.; Hu, D.N.; Kao, Y.H.; Lin, I.; Liu, F.S. Butein induces apoptotic cell death of human cervical cancer cells. Oncol. Lett. 2018, 16, 6615–6623. [CrossRef] 8. Yang, P.-Y.; Hu, D.-N.; Lin, I.-C.; Liu, F.-S. Butein shows cytotoxic effects and induces apoptosis in human ovarian cancer cells. Am. J. Chin. Med. 2015, 43, 769–782. [CrossRef] 9. Bai, X.; Ma, Y.; Zhang, G. Butein suppresses cervical cancer growth through the PI3K/AKT/mTOR pathway. Oncol. Rep. 2015, 33, 3085–3092. [CrossRef] , , [ ] 10. Seo, W.Y.; Youn, G.S.; Choi, S.Y.; Park, J. Butein, a tetrahydroxychalcone, suppresses pro-inﬂammatory responses in HaCaT keratinocytes. BMB Rep. 2015, 48, 495. [CrossRef] Int. J. Mol. Sci. 2023, 24, 6038 17 of 18 17 of 18 11. m, S.I.; Kim, H.-S.; Kim, S.J.; Song, Y.S. Tumor evolution and chemoresistance in ovarian cancer. NPJ Precis CrossRef] [PubMed] Cioce, M.; Sacconi, A.; Pass, H.I.; Canino, C.; Strano, S.; Blandino, G.; Fazio, V.M. Insights into intra-tumoral heterogeneity: Transcriptional proﬁling of chemoresistant MPM cell subpopulations reveals involvement of NFkB and DNA repair pathways and contributes a prognostic signature. Int. J. Mol. Sci. 2021, 22, 12071. [CrossRef] [PubMed] p g g J 12. Padmavathi, G.; Roy, N.K.; Bordoloi, D.; Arfuso, F.; Mishra, S.; Sethi, G.; Bishayee, A.; Kunnumakka disease: A comprehensive review. Phytomedicine 2017, 25, 118–127. [CrossRef] [PubMed] p y hoe, J.W.; Lee, B.J.; Kang, M.H.; Joo, M.K.; Kim, J.H.; Yeon, J.E.; Park, J.-J.; Kim, J.S.; Bak, Y.-T. Butein ef 13. Lee, S.D.; Choe, J.W.; Lee, B.J.; Kang, M.H.; Joo, M.K.; Kim, J.H.; Yeon, J.E.; Park, J.-J.; Kim, J.S.; Bak, Y.-T. Butein effects in colitis and interleukin-6/signal transducer and activator of transcription 3 expression. World J. Gastroenterol. WJG 2015, 21, 465. [CrossRef] [PubMed] 14. Ansari, M.Y.; Ahmad, N.; Haqqi, T.M. Butein activates autophagy through AMPK/TSC2/ULK1/mTOR pathway to inhibit IL-6 expression in IL-1β stimulated human chondrocytes. Cell. Physiol. Biochem. 2018, 49, 932–946. [CrossRef] [PubMed] 15. Taher, M.Y.; Davies, D.M.; Maher, J. The role of the interleukin (IL)-6/IL-6 receptor axis in ca 2018, 46, 1449–1462. [CrossRef] s, D.M.; Maher, J. The role of the interleukin (IL)-6/IL-6 receptor axis in cancer. Biochem. Soc. Trans [CrossRef] 16. Johnson, D.E.; O’Keefe, R.A.; Grandis, J.R. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat. Rev. Clin. Oncol. 2018, 15, 234–248. [CrossRef] 17. Browning, L.; Patel, M.; Bring Horvath, E.; Tawara, K.; Jorcyk, C. IL-6 and ovarian cancer: Inﬂammatory cytokines in promotion of metastasis. Cancer Manag. Res. 2018, 10, 6685–6693. [CrossRef] 18. Tang, Y.-L.; Huang, L.-B.; Lin, W.-H.; Wang, L.-N.; Tian, Y.; Shi, D.; Wang, J.; Qin, G.; Li, A.; Liang, Y.-N. Butein inhibits cell proliferation and induces cell cycle arrest in acute lymphoblastic leukemia via FOXO3a/p27kip1 pathway. Oncotarget 2016, 7, 18651. [CrossRef] 19. Oh, H.-M.; Yu, C.-R.; Golestaneh, N.; Amadi-Obi, A.; Lee, Y.S.; Eseonu, A.; Mahdi, R.M.; Egwuagu, C.E. STAT3 protein promotes T-cell survival and inhibits interleukin-2 production through up-regulation of Class O Forkhead transcription factors. J. Biol. Chem. 2011, 286, 30888–30897. [CrossRef] [ ] 20. Liu, Y.; Ao, X.; Ding, W.; Ponnusamy, M.; Wu, W.; Hao, X.; Yu, W.; Wang, Y.; Li, P.; Wang, J. Critical role of FOXO3a in carcinogenesis. Mol. Cancer 2018, 17, 104. [CrossRef] g 21. Cao, J.; Yang, J.-N.; Zhou, X.-Q.; Zhang, Y.-Y.; Zhu, X.-Y.; Yue, R.-M.; Hui, Y.; Chen, W.-H. m, S.I.; Kim, H.-S.; Kim, S.J.; Song, Y.S. Tumor evolution and chemoresistance in ovarian cancer. NPJ Precis CrossRef] [PubMed] Chemical Constituents of Psychotria hainanensis. Chem. Nat. Compd. 2020, 56, 533–534. [CrossRef] p 22. Junior, G.M.V.; de M. Sousa, C.M.; Cavalheiro, A.J.; Lago, J.H.G.; Chaves, M.H. Phenolic derivatives from fruits of Dipteryx lacunifera Ducke and evaluation of their antiradical activities. Helv. Chim. Acta 2008, 91, 2159–2167. [CrossRef] 23. Than, N.N.; Yangon, M.; Fiebig, H.-H.; Laatsch, H. Investigation of Bioactive Constituents and the Antitumour principle from Butea monosperma. Head Neck 2016, 4, 21. 24. Jin, Q.; Lee, C.; Lee, J.-W.; Lee, I.-S.; Lee, M.-K.; Jeon, W.-K.; Hwang, B.-Y. Chemical constituents from the fruits of Prunus mume. Nat. Prod. Sci. 2012, 18, 200–203. 25. Jassbi, A.R.; Singh, P.; Krishna, V.; Gupta, P.K.; Tahara, S. Antioxidant study and assignments of NMR spectral data for 3′, 4′, 7-trihydroxyﬂavanone 3′, 7-di-O-β-D-glucopyranoside (butrin) and its hydrolyzed product. Chem. Nat. Compd. 2004, 40, 250–253. [CrossRef] 26. Do Nascimento, A.M.; de Oliveira, D.C. 5-deoxyﬂavone glycoside from Calea uniﬂora L. (Asteraceae). Biochem. Syst. Ecol. 2004, 32, 1079–1081. [CrossRef] 27. Ryu, Y.B.; Kim, J.H.; Park, S.-J.; Chang, J.S.; Rho, M.-C.; Bae, K.-H.; Park, K.H.; Lee, W.S. Inhibition of n polyphenol compounds isolated from the roots of Glycyrrhiza uralensis. Bioorganic Med. Chem. Lett. 201 y g y y polyphenol compounds isolated from the roots of Glycyrrhiza uralensis. Bioorganic Med. Chem. Lett. 2010, 20, 971–974. [CrossRef] 28. Prachayasittikul, S.; Buraparuangsang, P.; Worachartcheewan, A.; Isarankura-Na-Ayudhya, C.; Ruchirawat, S.; Prachayasit- tikul, V. Antimicrobial and antioxidative activities of bioactive constituents from Hydnophytum formicarum Jack. Molecules 2008, 13, 904–921. [CrossRef] 28. Prachayasittikul, S.; Buraparuangsang, P.; Worachartcheewan, A.; Isarankura-Na-Ayudhya, C.; Ruchirawat, S.; Prachayasit- tikul, V. Antimicrobial and antioxidative activities of bioactive constituents from Hydnophytum formicarum Jack. Molecules 2008, 13, 904–921. [CrossRef] 29. Roh, K.; Lee, J.-h.; Kang, H.; Park, K.W.; Song, Y.; Lee, S.; Ku, J.-M. Synthesis and evaluation of butein de in vivo inﬂammatory response suppression in lymphedema. Eur. J. Med. Chem. 2020, 197, 112280. [Cro Lee, J.-h.; Kang, H.; Park, K.W.; Song, Y.; Lee, S.; Ku, J.-M. Synthesis and evaluation of butein derivatives for i ﬂammatory response suppression in lymphedema. Eur. J. Med. Chem. 2020, 197, 112280. [CrossRef] 30. Lima, T.C.; Souza, R.J.; Santos, A.D.; Moraes, M.H.; Biondo, N.E.; Barison, A.; Steindel, M.; Biavatti, M.W. Evaluation of leishmanicidal and trypanocidal activities of phenolic compounds from Calea uniﬂora Less. Nat. Prod. Res. 2016, 30, 551–557. [CrossRef] 31. Heo, T.-H.; Wahler, J.; Suh, N. Potential therapeutic implications of IL-6/IL-6R/gp130-targeting agents in breast cancer. Oncotarget 2016, 7, 15460. 36. Wang, J.; Sun, T.; Meng, Z.; Wang, L.; Li, M.; Chen, J.; Qin, T.; Yu, J.; Zhang, M.; Bie, Z. XPO1 inhibition synergizes with PARP1 inhibition in small cell lung cancer by targeting nuclear transport of FOXO3a. Cancer Lett. 2021, 503, 197–212. [CrossRef] g y g g p 37. Hui, R.C.; Francis, R.E.; Guest, S.K.; Costa, J.R.; Gomes, A.R.; Myatt, S.S.; Brosens, J.J.; Lam, E.W. Doxorubicin activates FOXO3a to induce the expression of multidrug resistance gene ABCB1 (MDR1) in K562 leukemic cells. Mol. Cancer Ther. 2008, 7, 670–678. [CrossRef] m, S.I.; Kim, H.-S.; Kim, S.J.; Song, Y.S. Tumor evolution and chemoresistance in ovarian cancer. NPJ Precis CrossRef] [PubMed] [CrossRef] [PubMed] 31. Heo, T.-H.; Wahler, J.; Suh, N. Potential therapeutic implications of IL-6/IL-6R/gp130-targeting agents in breast cancer. Oncotarget 2016, 7, 15460. [CrossRef] [PubMed] 32 Padmavathi G ; Rathnakaram S R ; Monisha J ; Bordoloi D ; Roy N K ; Kunnumakkara A B Potential of butein a tetrahydroxy- hnakaram, S.R.; Monisha, J.; Bordoloi, D.; Roy, N.K.; Kunnumakkara, A.B. Potential of butein, a tetrahydroxy te cancer. Phytomedicine 2015, 22, 1163–1171. [CrossRef] [PubMed] 32. Padmavathi, G.; Rathnakaram, S.R.; Monisha, J.; Bordoloi, D.; Roy, N.K.; Kunnumakkara, A.B. Potential chalcone to obliterate cancer. Phytomedicine 2015, 22, 1163–1171. [CrossRef] [PubMed] 33. Cioce, M.; Rutigliano, D.; Puglielli, A.; Fazio, V.M. Butein-instigated miR-186-5p-dependent modulation of TWIST1 affects resistance to cisplatin and bioenergetics of Malignant Pleural Mesothelioma cells. Cancer Drug Resist. 2022, 5, 814–828. [CrossRef] [PubMed] 34. Choi, H.S.; Kim, M.K.; Choi, Y.K.; Shin, Y.C.; Cho, S.-G.; Ko, S.-G. Rhus verniciﬂua Stokes (RVS) and butein induce apoptosis of paclitaxel-resistant SKOV-3/PAX ovarian cancer cells through inhibition of AKT phosphorylation. BMC Complement. Altern. Med. 2016, 16, 122. [CrossRef] 35. Zhang, L.; Yang, X.; Li, X.; Li, C.; Zhao, L.; Zhou, Y.; Hou, H. Butein sensitizes HeLa cells to cisplatin through the AKT and ERK/p38 MAPK pathways by targeting FoxO3a. Int. J. Mol. Med. 2015, 36, 957–966. [CrossRef] Int. J. Mol. Sci. 2023, 24, 6038 18 of 18 36. Wang, J.; Sun, T.; Meng, Z.; Wang, L.; Li, M.; Chen, J.; Qin, T.; Yu, J.; Zhang, M.; Bie, Z. XPO1 inhibition synergizes with PARP1 inhibition in small cell lung cancer by targeting nuclear transport of FOXO3a. Cancer Lett. 2021, 503, 197–212. [CrossRef] g y g g p 37. Hui, R.C.; Francis, R.E.; Guest, S.K.; Costa, J.R.; Gomes, A.R.; Myatt, S.S.; Brosens, J.J.; Lam, E.W. Doxorubicin activates FOXO3a to induce the expression of multidrug resistance gene ABCB1 (MDR1) in K562 leukemic cells. Mol. Cancer Ther. 2008, 7, 670–678. [CrossRef] [ ] 38. Chang, E.M.; Lim, E.; Yoon, S.; Jeong, K.; Bae, S.; Lee, D.R.; Yoon, T.K.; Choi, Y.; Lee, W.S. Cisplatin induces overactivation of the dormant primordial follicle through PTEN/AKT/FOXO3a pathway which leads to loss of ovarian reserve in mice. PLoS ONE 2015, 10, e0144245. [CrossRef] 38. Chang, E.M.; Lim, E.; Yoon, S.; Jeong, K.; Bae, S.; Lee, D.R.; Yoon, T.K.; Choi, Y.; Lee, W.S. Cisplatin induces overactivation of the dormant primordial follicle through PTEN/AKT/FOXO3a pathway which leads to loss of ovarian reserve in mice. PLoS ONE 2015, 10, e0144245. m, S.I.; Kim, H.-S.; Kim, S.J.; Song, Y.S. Tumor evolution and chemoresistance in ovarian cancer. NPJ Precis CrossRef] [PubMed] [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4254880235	https://www.qeios.com/read/SQCOMA/pdf	English	null	Detection Limit	Definitions	2,020	cc-by	53	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Detection Limit National Cancer Institute Qeios ID: SQCOMA · https://doi.org/10.32388/SQCOMA Source National Cancer Institute. Detection Limit. NCI Thesaurus. Code C105701. The smallest concentration of an analyte that can be determined with a stated precision or confidence. Qeios ID: SQCOMA · https://doi.org/10.32388/SQCOMA 1/1
https://openalex.org/W4387451961	https://www.biorxiv.org/content/biorxiv/early/2023/10/09/2023.10.06.561254.full.pdf	English	null	NEDD4-binding protein 1 suppresses HBV replication by degrading pgRNA	bioRxiv (Cold Spring Harbor Laboratory)	2,023	cc-by	17,853	. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 1 1 Title 2 Full title: NEDD4-binding protein 1 suppresses HBV replication by degrading pgRNA 3 Short title: Effect of NEDD4-binding protein 1 on the replication of HBV 4 5 Authors 6 Nobuhiro Kobayashi1,2, Saori Suzuki2,3, Yuki Sakamoto2, Rigel Suzuki2,3, Tomoya Saito1,2, Takuma 7 Izumi4, Kisho Noda5, Daisuke Okuzaki6, Yumi Kanegae7, Sanae Hayashi8, Yasuhito Tanaka8, 8 Yoshiharu Matsuura9,10, Osamu Takeuchi11, Tomokazu Tamura2,3, Akinobu Taketomi1, Takasuke 9 Fukuhara2,3,10,12 10 11 Affiliations 12 1　 Department of Gastroenterological Surgery 1, Graduate School of Medicine, Hokkaido University, 13 Hokkaido, Japan 14 2 　 Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido 15 University, Hokkaido, Japan 16 3　Institute for Vaccine Research and Development: HU-IVReD, Hokkaido University, Hokkaido, 17 Japan 18 4　Department of Surgery, Matsuyama Red Cross Hospital, Ehime, Japan 19 5　Tokyo Bay Urayasu Ichikawa Medical Center, Chiba, Japan 1 Title 18 4　Department of Surgery, Matsuyama Red Cross Hospital, Ehime, Japan . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 2 20 6 　Genome Information Research Center, Research Institute for Microbial Diseases, Osaka 21 University, Osaka, Japan 22 7　Core Research Facilities, Research Center for Medical Sciences, The Jikei University School of 23 Medicine, Tokyo, Japan 24 8　Department of Gastroenterology and Hepatology, Faculty of Life Sciences, Kumamoto University, 25 Kumamoto, Japan 26 9　Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 27 Osaka, Japan 28 10 　Laboratory of Virus Control, Center for Infectious Disease Education and Research, Osaka 29 University, Osaka, Japan 30 11　Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, 31 Japan 32 12　AMED-CREST, Japan Agency for Medical Research and Development (AMED), Tokyo, Japan 33 34 Abstract 35 Chronic infection with hepatitis B virus (HBV) places patients at increased risk for liver cirrhosis 36 and hepatocellular carcinoma. Although nucleos(t)ide analogs are mainly used for the treatment of 37 HBV, they require long-term administration and may lead to the emergence of drug resistant mutants. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 3 38 Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV- 39 suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We 40 screened 132 RBPs using an expression plasmid library by measuring HBV relaxed circular DNA 41 (rcDNA) levels in hepatocellular carcinoma. After we identified one RBP which suppressed rcDNA, 42 the domain-deficient mutants were generated to determine the region contributing to the anti-HBV 43 effect. Also, we measured pregenomic RNA (pgRNA) and covalently closed circular DNA (cccDNA) 44 level in the RBP transfected cells and confirmed the degradation of pgRNA by Northern blotting. Our 45 screen identified NEDD4-binding protein 1 (N4BP1) having an anti-HBV effect. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 1 Title In hepatocellular 46 carcinoma cell lines transfected or infected with HBV, overexpression of N4BP1 decreased rcDNA 47 levels while knockdown or knockout of N4BP1 expression rescued rcDNA levels. We found that both 48 the KH-like and RNase domains of N4BP1 were required for the protein’s anti-HBV effect. N4BP1 49 suppressed HBV replication by promoting pgRNA, PreS1 and PreS2/S degradation. In summary, we 50 found that N4BP1 is a newly identified host factor able to counteract HBV production by reducing 51 HBV RNA levels. 52 53 Author summary 54 There is still a large number of HBV-infected people in the world today because of no curative 55 treatment for HBV infection. In this study, we focused on and screened RNA-binding proteins to . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 4 56 identify new host factors which inhibit HBV replication. As a result, we found that NEDD4-binding 57 protein 1 (N4BP1) expression suppresses rcDNA production by promoting the degradation of 58 pregenomic RNA, 2.4kb and 2.1kb HBV RNA. Furthermore, KH-like domain or RNase domain of 59 N4BP1 were involved in this anti-HBV effect. In addition, the N4BP1 levels were lower in HCC 60 resection samples of exacerbated patients, suggesting that individual N4BP1 levels might be related 61 to HCC progression. This novel factor can potentially become a key to new HBV treatments. 62 63 Introduction 64 Hepatitis B virus (HBV) infection causes either acute or chronic hepatitis, with approximately 65 296 million people worldwide suffering from chronic hepatitis B and an estimated 1.5 million new 66 cases arising each year. Chronically infected individuals are at increased risk for liver disease, and 67 annually ~820,000 die from cirrhosis and hepatocellular carcinoma (HCC) caused by HBV 68 infection[1]. Although there is a prophylactic vaccine for HBV, there are still many new cases of HBV 69 infection due to the large number of chronic HBV infected patients still in Africa, Asia, and Eastern 70 Europe and the low vaccination rates in these highly endemic areas[2]. 71 HBV is an enveloped DNA virus with a small, partially double-stranded genome of 3.2 kbp. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 1 Title 72 Entry into hepatocytes is mediated by binding to the bile acid transporter sodium-taurocholate 56 identify new host factors which inhibit HBV replication. As a result, we found that NEDD4-binding 57 protein 1 (N4BP1) expression suppresses rcDNA production by promoting the degradation of 58 pregenomic RNA, 2.4kb and 2.1kb HBV RNA. Furthermore, KH-like domain or RNase domain of 59 N4BP1 were involved in this anti-HBV effect. In addition, the N4BP1 levels were lower in HCC 60 resection samples of exacerbated patients, suggesting that individual N4BP1 levels might be related 61 to HCC progression. This novel factor can potentially become a key to new HBV treatments. 63 Introduction 64 Hepatitis B virus (HBV) infection causes either acute or chronic hepatitis, with approximately 65 296 million people worldwide suffering from chronic hepatitis B and an estimated 1.5 million new 66 cases arising each year. Chronically infected individuals are at increased risk for liver disease, and 67 annually ~820,000 die from cirrhosis and hepatocellular carcinoma (HCC) caused by HBV 68 infection[1]. Although there is a prophylactic vaccine for HBV, there are still many new cases of HBV 69 infection due to the large number of chronic HBV infected patients still in Africa, Asia, and Eastern 70 Europe and the low vaccination rates in these highly endemic areas[2]. 71 HBV is an enveloped DNA virus with a small, partially double-stranded genome of 3.2 kbp. 72 Entry into hepatocytes is mediated by binding to the bile acid transporter sodium-taurocholate 73 cotransporting polypeptide (NTCP). After being released into the nucleus, the HBV genome is 71 HBV is an enveloped DNA virus with a small, partially double-stranded genome of 3.2 kbp. 72 Entry into hepatocytes is mediated by binding to the bile acid transporter sodium-taurocholate 73 cotransporting polypeptide (NTCP). After being released into the nucleus, the HBV genome is . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 5 77 strand DNA first and then the positive strand is synthesized to generate rcDNA. 78 Interferon (IFN) and nucleos(t)ide analogs (NAs) are currently used as treatments for HBV 79 infection, and both have shown efficacy in controlling viremia[7, 8]. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 1 Title IFNs are involved in the 80 inhibition of HBV replication, transcription, and other important processes by inducing the expression 81 of various IFN-stimulated genes (ISGs) in host cells[9]. However, IFN treatment can cause 82 considerable side effects, including liver damage, and is thus unsuitable for patients with severe liver 83 dysfunction[10]. 84 In contrast, NAs impair further HBV intrahepatic spreading by blocking production of new 85 virions. But the HBV antigens HBs or HBe are not eliminated with treatment as NAs inhibit the reverse 86 transcription step that occurs after these antigens have already been translated. Furthermore, NAs do 87 not directly target cccDNA, allowing the virus to persist in hepatocytes[11, 12]. Consequently, to 88 prevent relapse due to reactivation of HBV after discontinuing NAs, long-term treatment is 89 recommended[13]. 90 In efforts to overcome the limitations of these current therapeutic options, research has looked to 91 identify host factors with an antiviral role. Understanding the mechanism of action of such factors . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 6 92 could aid in the development of new antiviral treatments. Several host factors are known to inhibit 93 HBV replication, including RNA-binding proteins (RBPs). For example, the CCCH zinc finger 94 antiviral protein ZAP (ZC3HAV1) binds to HBV RNA with its four zinc finger motifs at the N- 95 terminus, promoting pgRNA degradation[14]. Similarly, MCPIP1 (ZC3H12A, regnase-1) has also 96 been reported to inhibit HBV replication by binding to HBV pgRNA through its zinc finger motif, 97 directly degrading pgRNA via its RNase activity[15]. Furthermore, PUF60 is a host factor that not 98 only positively regulates HBV 3.5 kb precore plus pregenomic RNA expression by binding to the 99 HBV enhancer region II with the transcription factor TCF7L2 but can also promote degradation of the 100 HBV 3.5 kb RNA by binding via its N-terminal RNA recognition motif[16]. Despite these descriptions, 101 HBV produces high levels of HBV RNAs in infected primary human hepatocytes (PHHs) and in vivo, 102 so the antiviral effects of these host factors appears limited. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 1 Title 103 To determine if other RBPs may have an antiviral role, we performed a comprehensive screen 104 using an expression library of RBPs. We identified NEDD4-binding protein 1 (N4BP1) as a host factor 105 with anti-HBV activity. We subsequently elucidated the mechanism of how N4BP1 suppresses HBV 106 replication by promoting pgRNA degradation. 107 108 Results 109 Screen for RNA-binding proteins that inhibit HBV propagation 92 could aid in the development of new antiviral treatments. Several host factors are known to inhibit 93 HBV replication, including RNA-binding proteins (RBPs). For example, the CCCH zinc finger 94 antiviral protein ZAP (ZC3HAV1) binds to HBV RNA with its four zinc finger motifs at the N- 95 terminus, promoting pgRNA degradation[14]. Similarly, MCPIP1 (ZC3H12A, regnase-1) has also 96 been reported to inhibit HBV replication by binding to HBV pgRNA through its zinc finger motif, 97 directly degrading pgRNA via its RNase activity[15]. Furthermore, PUF60 is a host factor that not 98 only positively regulates HBV 3.5 kb precore plus pregenomic RNA expression by binding to the 99 HBV enhancer region II with the transcription factor TCF7L2 but can also promote degradation of the 100 HBV 3.5 kb RNA by binding via its N-terminal RNA recognition motif[16]. Despite these descriptions, 101 HBV produces high levels of HBV RNAs in infected primary human hepatocytes (PHHs) and in vivo, 102 so the antiviral effects of these host factors appears limited. 103 To determine if other RBPs may have an antiviral role, we performed a comprehensive screen . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 7 110 To identify novel RBPs involved in HBV propagation, we screened 132 RBPs using an 111 expression plasmid library. Huh7 cells were co-transfected with an RBP expression plasmid and a 1.3- 112 fold-overlength HBV genome (genotype C). At 3 days post-transfection (dpt), cells were collected to 113 measure intracellular rcDNA levels. As shown in Fig 1A, 23 out of 132 RBPs suppressed rcDNA 114 levels by at least 50% compared to the control. To identify novel RBPs involved in HBV propagation, we screened 132 RBPs using an 110 pression plasmid library. 1 Title Huh7 cells were co-transfected with an RBP expression plasmid and a 1.3- 114 levels by at least 50% compared to the control. 115 As a secondary screen, we then examined the effect of these 23 RBPs on HBV entry and 116 replication. HepG2-hNTCP C4 cells expressing each of the 23 RBPs were infected with HBV 117 (genotype D), and intracellular rcDNA measured by quantitative PCR (qPCR) at 14 days post- 118 infection (dpi). Seven of the 23 RBPs (N4BP1, TIA-1, ZC3H12B, PJA1, HNRNPC, ZCCHC10, and 119 KHNYN) reduced rcDNA levels in comparison with the control (Fig 1B). These results were 120 consistent with previous reports that found TIA-1 and PJA1 inhibit expression of HBs antigen and 121 HBV transcription and replication, respectively[17, 18]. 122 Of the five remaining factors, we found by western blot that three (N4BP1, HNRNPC, and 123 ZCCHC10) are endogenously expressed in Huh7 and HepG2-hNTCP C4 cells (Fig 1C). To test 121 HBV transcription and replication, respectively[17, 18]. 122 Of the five remaining factors, we found by western blot that three (N4BP1, HNRNPC, and 123 ZCCHC10) are endogenously expressed in Huh7 and HepG2-hNTCP C4 cells (Fig 1C). To test 124 whether these proteins affect HBV replication, we knocked down each one and assessed rcDNA levels 125 following transfection with the 1.3-fold-overlength genome of HBV genotype C. The antiviral drug 126 entecavir, a nucleoside analog, was used as a positive control. Knockdown of each factor was 127 confirmed by western blotting (WB), but unlike knockdown of N4BP1, knockdown of HNRNPC or . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 8 128 ZCCHC10 did not consistently increase the levels of rcDNA depending on the siRNA used (Fig 2A- 129 2C). Thus, N4BP1 became the focus of our study. 130 131 N4BP1 is a suppressive host factor of HBV replication 132 Next, to determine whether N4BP1 also had an antiviral effect against other HBV genotypes, we 133 co-transfected Huh7 cells with a plasmid expressing N4BP1 and a plasmid containing 1.3-fold- 134 overlength genomes of HBV genotypes A, B, and C. At 3 dpt, rcDNA levels were decreased in cells 135 transfected with each genotype (Fig 3A). 1 Title 136 To further elucidate the role of N4BP1 in HBV replication, we constructed Huh7 N4BP1 137 knockout (KO) cells using CRISPR/Cas9. We generated two clones of N4BP1 KO Huh7 cells (Fig 138 3B) and once more assessed whether intracellular rcDNA levels changed at 3 dpt with a plasmid 139 containing the 1.3-fold-overlength genome of HBV genotype C. In both KO lines, rcDNA levels 140 increased approximately 3-fold compared to control cells (Fig 3B). After rescuing N4BP1 expression 141 in our KO line, rcDNA levels became significantly decreased again (Fig 3C, 3D), suggesting that 142 N4BP1 is a suppressive host factor of HBV replication. In addition, we performed a transcriptomic 143 analysis to determine whether host factors are involved in the anti-HBV activity of N4BP1. As 144 visualized by heat maps, host factors differentially expressed between N4BP1-overexpressing PHHs 145 and control PHHs (S1 Fig A). We found 25 genes that were upregulated by 2-fold or more in N4BP1- . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 131 N4BP1 is a suppressive host factor of HBV replication 136 To further elucidate the role of N4BP1 in HBV replication, we constructed Huh7 N4BP1 137 knockout (KO) cells using CRISPR/Cas9. We generated two clones of N4BP1 KO Huh7 cells (Fig 138 3B) and once more assessed whether intracellular rcDNA levels changed at 3 dpt with a plasmid 139 containing the 1.3-fold-overlength genome of HBV genotype C. In both KO lines, rcDNA levels 140 increased approximately 3-fold compared to control cells (Fig 3B). After rescuing N4BP1 expression 141 in our KO line, rcDNA levels became significantly decreased again (Fig 3C, 3D), suggesting that 142 N4BP1 is a suppressive host factor of HBV replication. In addition, we performed a transcriptomic 143 analysis to determine whether host factors are involved in the anti-HBV activity of N4BP1. As 144 visualized by heat maps, host factors differentially expressed between N4BP1-overexpressing PHHs 145 and control PHHs (S1 Fig A). We found 25 genes that were upregulated by 2-fold or more in N4BP1- . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 9 146 vs control PHHs and 17 genes that were downregulated by 0.5-fold or more (S1 Fig B, S1 Table). 147 None of these factors were related to immune pathways nor had been previously reported to be 148 involved in HBV replication. 149 150 N4BP1 expression is not induced by HBV 151 We then evaluated whether N4BP1 expression level was increased by HBV infection. A plasmid 152 containing the 1.3-fold-overlength genome of HBV genotype C was transfected into Huh7 cells, and 153 N4BP1 expression assessed by WB and qPCR at 3 dpt. Transfection of the HBV genome did not 154 significantly affect N4BP1 expression at either the transcriptional or protein level (Fig 4A, 4B). 155 N4BP1 expression also did not significantly change in HepG2-hNTCP C4 cells after infection with 156 HBV (Fig 4A, 4C). 131 N4BP1 is a suppressive host factor of HBV replication Finally, we measured N4BP1 expression in HepAD38.7 cells that stably secrete 157 HBV particles unless cultured in the presence of tetracycline. In this system, removal of tetracycline 158 and induction of HBV particle production also did not result in any change in N4BP1 expression (Fig 159 4A, 4D). Together, these results indicate that N4BP1 expression is not affected by HBV infection. 160 161 The KH-like and RNase domains of N4BP1 are critical for the anti-HBV effect of N4BP1 162 As shown in Fig 5A, N4BP1 is composed of 896 amino acids and contains a KH-like domain at . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint The KH-like and RNase domains of N4BP1 are critical for the anti-HBV effect of N4BP1 162 As shown in Fig 5A, N4BP1 is composed of 896 amino acids and contains a KH-like domain at 163 its N terminus side and an RNase domain at its C terminus[19]. To examine which N4BP1 domains . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 10 164 are needed to suppress HBV replication, we constructed deletion mutants lacking either the KH-like 165 (ΔKH; deletion of amino acids 59-143) or the RNase domain (ΔRNase; deletion of amino acids 617- 166 769). 167 The plasmid encoding wild-type or mutant N4BP1 was respectively co-transfected into naïve 168 Huh7 cells with the plasmid containing the 1.3-fold-overlength genome of HBV genotype C and the 169 rcDNA levels measured. The expression of the deletion mutants was confirmed by WB using an HA- 170 tagged antibody (Fig 5B, upper). The rcDNA level in N4BP1 overexpressing cells was significantly 171 reduced, while the rcDNA levels in the ΔKH- or ΔRNase-expressing cells were comparable to those 172 in the control cells (Fig 5B, lower). 173 Residue D623 in the RNase domain of N4BP1 (Fig 5A) has been shown to be important for the 174 protein’s RNase activity[20]. A D623N substitution causes loss of RNase activity without changing 175 the structure of N4BP1. Co-transfecting the Huh7 cells with a plasmid expressing this N4BP1 D623 176 mutant and the HBV overlength genome also did not lead to a decrease in rcDNA levels (Fig 5C). 177 These results suggest that both the KH-like and RNase domains are critical for the anti-HBV effect of 178 N4BP1. 179 180 N4BP1 promotes pgRNA degradation 181 Having shown the effect of N4BP1 on rcDNA, we then sought to determine more specifically . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 11 182 which step of HBV replication N4BP1 suppresses. To that end, we assessed the impact of N4BP1 183 overexpression on cccDNA and pgRNA levels. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 217 significantly inducing N4BP1 expression in PHHs. The KH-like and RNase domains of N4BP1 are critical for the anti-HBV effect of N4BP1 HepG2-hNTCP C4 cells were infected with HBV and 184 cells were harvested at 14 dpi. While cccDNA levels were not affected by overexpression of N4BP1, 185 pgRNA and rcDNA levels were both significantly reduced (Fig 6A-6C). 186 For further verification, we performed the same experiments using PHHs. Adenovirus vectors 187 were used to overexpress N4BP1 in PHHs (Fig 6D), and the cells were then infected with HBV 188 genotype C. As shown in Fig 6E-6G, even in PHHs, cccDNA levels were comparable to control cells, 189 but pgRNA and rcDNA levels were both significantly decreased. Collectively, these results indicate 190 that N4BP1 augmented pgRNA degradation. 191 To elucidate the mechanism of pgRNA suppression by N4BP1, we turned to HepAD38.7 cells. 192 First, the cells were cultured for 2 days in media without tetracycline to induce HBV replication. Then 193 N4BP1 or the domain deletion mutant plasmids were transfected, and tetracycline added at 2 dpt to 194 stop viral replication. Cells were collected starting 2 days after adding tetracycline to measure the 195 pgRNA levels (Fig 6H). As shown in Fig 6I, the expression of wild-type N4BP1 augmented the rate 196 of pgRNA degradation. In contrast, both domain deficient mutants resulted in pgRNA degradation at 197 a rate similar to the control, suggesting that N4BP1 affects the degradation rate of pgRNA. Next, we 198 examined whether N4BP1 expression also suppresses HBV RNAs other than pgRNA. We co- 199 transfected Huh7 cells with a plasmid expressing N4BP1 and a plasmid containing 1.3-fold-overlength 191 To elucidate the mechanism of pgRNA suppression by N4BP1, we turned to HepAD38.7 cells. 192 First, the cells were cultured for 2 days in media without tetracycline to induce HBV replication. Then 193 N4BP1 or the domain deletion mutant plasmids were transfected, and tetracycline added at 2 dpt to 194 stop viral replication. Cells were collected starting 2 days after adding tetracycline to measure the 195 pgRNA levels (Fig 6H). As shown in Fig 6I, the expression of wild-type N4BP1 augmented the rate 196 of pgRNA degradation. In contrast, both domain deficient mutants resulted in pgRNA degradation at 197 a rate similar to the control, suggesting that N4BP1 affects the degradation rate of pgRNA. Next, we 198 examined whether N4BP1 expression also suppresses HBV RNAs other than pgRNA. We co- 199 transfected Huh7 cells with a plasmid expressing N4BP1 and a plasmid containing 1.3-fold-overlength . The KH-like and RNase domains of N4BP1 are critical for the anti-HBV effect of N4BP1 CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 12 200 genomes of HBV genotype C. At 3 dpt, cells were collected and HBV RNA expression was measured 201 by Northern blotting. Results showed that control Huh7 cells showed intense expression of 3.5kb, 202 2.4kb, and 2.1kb HBV RNA, whereas N4BP1 overexpression Huh7 cells showed weak expression of 203 all these RNAs (Fig 6J). 204 205 N4BP1 expression is induced by IFN-α/λ in PHHs 206 It has been reported that N4BP1 expression is increased 3- to 5-fold with IFN-α treatment in 207 Jurkat cells and THP-1 cells[20]. To see if this was the case in PHHs and hepatoma cell lines, we 208 treated Huh7, HepG2-hNTCP C4, and PHHs with 1000 U/ml of IFN-α and 100 ng/ml of IFN-λ, which 209 are known to also inhibit HBV replication. Cells were collected at 24 and 48 hours post-drug treatment 210 and N4BP1 was examined by qPCR and WB (Fig 7A). The ISG15 was included as a positive control. 211 As shown in Fig 7B and 7C, levels of N4BP1 mRNA as measured by qPCR were slightly increased 212 in both Huh7 and HepG2-hNTCP C4 cells by IFN-α or IFN-λ. WB also showed that IFN-α or IFN-λ 213 treatments had no effect on N4BP1 protein levels in Huh7 cells and HepG2-hNTCP C4 cells. In 214 contrast, IFN-α and IFN-λ treatment in PHHs resulted in an approximately 2-fold increase in N4BP1 215 mRNA expression and protein level was also increased. Collectively, these results indicate that IFN- 216 α and IFN-λ treatment slightly induce N4BP1 expression in Huh7 and HepG2-hNTCP C4 cells while 217 significantly inducing N4BP1 expression in PHHs. 201 by Northern blotting. Results showed that control Huh7 cells showed intense expression of 3.5kb, 202 2.4kb, and 2.1kb HBV RNA, whereas N4BP1 overexpression Huh7 cells showed weak expression of 203 all these RNAs (Fig 6J). 205 N4BP1 expression is induced by IFN-α/λ in PHHs 206 It has been reported that N4BP1 expression is increased 3- to 5-fold with IFN-α treatment in 207 Jurkat cells and THP-1 cells[20]. To see if this was the case in PHHs and hepatoma cell lines, we 208 treated Huh7, HepG2-hNTCP C4, and PHHs with 1000 U/ml of IFN-α and 100 ng/ml of IFN-λ, which 209 are known to also inhibit HBV replication. Cells were collected at 24 and 48 hours post-drug treatment 210 and N4BP1 was examined by qPCR and WB (Fig 7A). The ISG15 was included as a positive control. 211 As shown in Fig 7B and 7C, levels of N4BP1 mRNA as measured by qPCR were slightly increased 212 in both Huh7 and HepG2-hNTCP C4 cells by IFN-α or IFN-λ. WB also showed that IFN-α or IFN-λ 213 treatments had no effect on N4BP1 protein levels in Huh7 cells and HepG2-hNTCP C4 cells. In 214 contrast, IFN-α and IFN-λ treatment in PHHs resulted in an approximately 2-fold increase in N4BP1 215 mRNA expression and protein level was also increased. Collectively, these results indicate that IFN- 216 α and IFN-λ treatment slightly induce N4BP1 expression in Huh7 and HepG2-hNTCP C4 cells while 217 significantly inducing N4BP1 expression in PHHs. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 13 218 Since N4BP1 expression was induced by IFN treatment in the primary cells and not the hepatoma 219 cell lines, we thought that N4BP1 expression may differ depending on the immune status of the human 220 liver. Therefore, we assessed ISG15 and N4BP1 mRNA expression in HBV-HCC resection human 221 liver tissue samples. ISG15 (Fig 8A) and N4BP1 (Fig 8B) mRNA expression in the HBV DNA 222 negative group tended to be higher than in the HBV DNA positive group. 223 224 Discussion 225 NAs are generally well tolerated, very effective in inhibiting HBV replication, and are currently 226 the most commonly used treatment for chronic hepatitis B. However, since NAs inhibit reverse 227 transcription, HBV cccDNA remains in the nucleus of hepatocytes so the patient is never completely 228 cured. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 205 N4BP1 expression is induced by IFN-α/λ in PHHs On the other hand, the RNase domain of N4BP1 is also involved in regulating 241 genome stability and repressing the expression of target genes through mRNA strand breaks[26]. Thus, 242 it is possible that N4BP1 binds to HBV RNA through its KH-like domain and degrades HBV RNA by 243 the RNase activity of its RNase domain. In fact, in this study, N4BP1 suppressed HBV replication at 244 the step of pgRNA production in the HBV life cycle (Fig 6B, 6F, 6I). We also found that N4BP1 245 suppresses not only the 3.5 kb RNA of HBV, but also the 2.4 kb and 2.1 kb RNAs by NB (Fig 6J), 246 which means N4BP1 suppresses pgRNA, PreS1 and PreS2/S. 247 N4BP1 is known to be an inhibitor of innate immunity and inflammation-mediated cytokine 248 production. NF-κB is an important transcription factor for the elicitation of cytokine responses, and 249 its activation is necessary for many immune responses. N4BP1 interacts with the NF-κB signal 250 essential modulator (NEMO, also known as IκB kinase γ) to suppress Toll-like receptor (TLR)– 251 dependent NF-κB activation [27]. Furthermore, even in vivo, it has been reported that N4BP1-/- mice 252 develop mild inflammation and have exacerbated TLR-dependent inflammatory responses[28]. 253 Recently, N4BP1 was found as a new hotspot gene for HBV integration[29]. Although the significance 237 We found that both the KH-like domain and the RNase domain of N4BP1 were essential for the 238 protein’s anti-HBV effect (Fig 5B). It has been reported that the KH-like domain binds to RNA or 239 single-strand DNA and is involved in splicing, transcriptional regulation, and translational 240 regulation[19, 24, 25]. On the other hand, the RNase domain of N4BP1 is also involved in regulating 241 genome stability and repressing the expression of target genes through mRNA strand breaks[26]. Thus, 242 it is possible that N4BP1 binds to HBV RNA through its KH-like domain and degrades HBV RNA by 243 the RNase activity of its RNase domain. In fact, in this study, N4BP1 suppressed HBV replication at 244 the step of pgRNA production in the HBV life cycle (Fig 6B, 6F, 6I). We also found that N4BP1 245 suppresses not only the 3.5 kb RNA of HBV, but also the 2.4 kb and 2.1 kb RNAs by NB (Fig 6J), 246 which means N4BP1 suppresses pgRNA, PreS1 and PreS2/S. 246 which means N4BP1 suppresses pgRNA, PreS1 and PreS2/S. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 205 N4BP1 expression is induced by IFN-α/λ in PHHs Indeed, patients who discontinue NA treatment can experience virologic relapse and thus must 229 continue NA therapy for extended periods. Long-term treatment with NA is associated with the 230 development of antiviral drug resistance; for example, mutations in the HBV polymerase gene, which 231 is a target of NAs, have been reported[21-23]. Resistance to NA therapy can lead to hepatitis relapse 232 and even death. Thus, we sought to identify host factors involved in HBV proliferation that would be 233 distinct from the viral sites targeted by NAs. In a library of 132 RBPs containing proteins with known 234 RNA-binding domains such as RNA recognition motif domains or zinc finger domains, we found that 235 N4BP1 inhibited HBV replication, was negatively correlated with HBV rcDNA levels in hepatoma 218 223 224 Discussion 225 NAs are generally well tolerated, very effective in inhibiting HBV replication, and are currently 226 the most commonly used treatment for chronic hepatitis B. However, since NAs inhibit reverse 227 transcription, HBV cccDNA remains in the nucleus of hepatocytes so the patient is never completely 228 cured. Indeed, patients who discontinue NA treatment can experience virologic relapse and thus must 229 continue NA therapy for extended periods. Long-term treatment with NA is associated with the 230 development of antiviral drug resistance; for example, mutations in the HBV polymerase gene, which 231 is a target of NAs, have been reported[21-23]. Resistance to NA therapy can lead to hepatitis relapse 232 and even death. Thus, we sought to identify host factors involved in HBV proliferation that would be 233 distinct from the viral sites targeted by NAs. In a library of 132 RBPs containing proteins with known 234 RNA-binding domains such as RNA recognition motif domains or zinc finger domains, we found that 235 N4BP1 inhibited HBV replication, was negatively correlated with HBV rcDNA levels in hepatoma 14 cell lines and suppressed HBV replication by promoting pgRNA degradation. 236 cell lines and suppressed HBV replication by promoting pgRNA degradation. 237 We found that both the KH-like domain and the RNase domain of N4BP1 were essential for the 238 protein’s anti-HBV effect (Fig 5B). It has been reported that the KH-like domain binds to RNA or 239 single-strand DNA and is involved in splicing, transcriptional regulation, and translational 240 regulation[19, 24, 25]. 205 N4BP1 expression is induced by IFN-α/λ in PHHs 247 N4BP1 is known to be an inhibitor of innate immunity and inflammation-mediated cytokine 248 production. NF-κB is an important transcription factor for the elicitation of cytokine responses, and 249 its activation is necessary for many immune responses. N4BP1 interacts with the NF-κB signal 250 essential modulator (NEMO, also known as IκB kinase γ) to suppress Toll-like receptor (TLR)– 251 dependent NF-κB activation [27]. Furthermore, even in vivo, it has been reported that N4BP1-/- mice 252 develop mild inflammation and have exacerbated TLR-dependent inflammatory responses[28]. 253 Recently, N4BP1 was found as a new hotspot gene for HBV integration[29]. Although the significance . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 15 254 of HBV integration to N4BP1 remains unknown, it is interesting that the virus integration occurred to 255 N4BP1 which has anti-HBV activity. Regarding virus replication, N4BP1 is known to directly 256 recognize and degrade retroviruses such as HIV-1[20] and to be involved in replication of the Porcine 257 Reproductive and Respiratory Syndrome Virus[30]. In both cases, the RNase activity of N4BP1 was 258 found to inhibit viral replication by directly degrading viral RNA, consistent with our data. In addition, 259 the RNase domain of N4BP1 has a similar structure to that of MCPIP1[31], which has been reported 260 to recognize and degrade HBV pgRNA directly[15]. However, MCPIP1 is a cytoplasmic RNase, 261 whereas N4BP1 is mainly localized on the nucleus. Furthermore, MCPIP1 has not been reported to 262 suppress 2.4 kb and 2.1 kb HBV RNAs, whereas we have demonstrated that N4BP1 suppresses these 263 RNAs. These results suggest that MCPIP1 and N4BP1 may regulate HBV degrading mRNAs through 264 different processes. The mechanism behind our finding that the KH-like domain of N4BP1 was also 265 important for the anti-HBV effect of this protein is presumably due to its binding to HBV RNA but 266 further work is needed to elucidate the mechanism more precisely. 267 Although N4BP1 expression has been reported to be induced by type I IFN[20], stimulation of 268 N4BP1 expression by IFN-α (type I IFN) and IFN-λ (type III IFN) treatment varied between the PHHs 269 and hepatoma cell lines we tested. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 205 N4BP1 expression is induced by IFN-α/λ in PHHs CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 17 17 290 JCRB0403 and JCRB9068). HepG2-hNTCP C4 cells and HepAD38.7 cells were kindly provided by 291 Takaji Wakita (NIID, Tokyo, Japan). PHHs were obtained from Phoenix Bio (Hiroshima, Japan). 292 Huh7 cells and HEK293T cells were maintained in DMEM (Nacalai Tesque, Kyoto, Japan) 293 supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin (Nacalai Tesque) and 10% fetal 294 bovine serum (FBS, Sigma, MO, USA). HepG2-hNTCP C4 cells were maintained in the 295 aforementioned media containing 400 μg/ml G418 (Nacalai Tesque). HepAD38.7 cells were 296 maintained in DMEM/Ham’s F-12 media (Nacalai Tesque) supplemented with 10% FBS, 100 U/ml 297 of penicillin, 100 μg/ml of streptomycin, 400 μg/ml of G418, 5 μg/ml of insulin (Nacalai Tesque), and 298 400 ng/ml of tetracycline (Nacalai Tesque). PHHs were maintained in PHH-specific media provided 299 by the manufacture (Phoenix Bio). 300 301 Generation of RBP overexpressing cells 302 RBP-overexpressing Huh7 cells were generated by transfection of RBP expression plasmids 303 using TransIT LT-1 transfection reagent (Mirus, WI, USA). Briefly, Huh7 cells were seeded in 24- 304 well plates (Greiner, Bad Haller, Austria) 1 day prior to the transfection. The following day, 1 μl of 305 TransIT LT-1 transfection reagent, 0.3 μg of RBP expression plasmid, and 0.1 μg of a plasmid 306 containing a 1.3-fold-overlength genome of HBV (genotype C) were mixed in 50 μl of Opti-MEM 307 (Thermo Fisher Scientific, MA, USA). After 15 minutes incubation at room temperature, the mixture 290 JCRB0403 and JCRB9068). HepG2-hNTCP C4 cells and HepAD38.7 cells were kindly provided by 291 Takaji Wakita (NIID, Tokyo, Japan). PHHs were obtained from Phoenix Bio (Hiroshima, Japan). 292 Huh7 cells and HEK293T cells were maintained in DMEM (Nacalai Tesque, Kyoto, Japan) 293 supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin (Nacalai Tesque) and 10% fetal 294 bovine serum (FBS, Sigma, MO, USA). HepG2-hNTCP C4 cells were maintained in the 295 aforementioned media containing 400 μg/ml G418 (Nacalai Tesque). . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 205 N4BP1 expression is induced by IFN-α/λ in PHHs This could be due to the disrupted IFN signaling reported in the cell 267 Although N4BP1 expression has been reported to be induced by type I IFN[20], stimulation of 268 N4BP1 expression by IFN-α (type I IFN) and IFN-λ (type III IFN) treatment varied between the PHHs 269 and hepatoma cell lines we tested. This could be due to the disrupted IFN signaling reported in the cell 270 lines Huh7 and HepG2 that result in them being less sensitive to IFN[32, 33]. Also, ISG15 is an 271 inducible factor by IFN-α and IFN-λ[34, 35]. In characterizing N4BP1 expression, we found that . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 16 272 N4BP1 expression was not induced by HBV infection or transfection in hepatoma cell lines (Fig 4A- 273 4D). Furthermore, in clinical liver tissue samples, N4BP1 expression was significantly lower in HBV- 274 DNA positive vs HBV-DNA negative samples (Fig 8B). These results suggest that N4BP1 expression 275 may differ according to each individual and that the inherent expression level of N4BP1 is related to 276 HBV DNA negative conversion. Otherwise, it is thought that factors upstream of N4BP1 may cause 277 differences in N4BP1 expression and affect HBV DNA negative conversion. Regardless, a greater of 278 samples needs to be examined in order to further understand the relationship between the expression 279 of N4BP1 and HBV. 280 In conclusion, to investigate different treatments against HBV infection from currently approved 281 HBV therapeutics, we focused on RBPs. We found that N4BP1 is a RBP with novel anti-HBV activity, 282 inhibiting HBV replication via degradation of HBV pgRNA. Although the detailed molecular 283 mechanism behind the anti-HBV effect of N4BP1 remains to be uncovered, understanding the 284 association between N4BP1 and HBV could contribute to the development of new HBV treatments. 285 286 Materials and methods 287 Cell culture 288 All cells were grown at 37℃ in a 5% (v/v) CO2 environment. Huh7 cells and HEK293T cells 289 were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB, Osaka, Japan, . 205 N4BP1 expression is induced by IFN-α/λ in PHHs HepAD38.7 cells were 296 maintained in DMEM/Ham’s F-12 media (Nacalai Tesque) supplemented with 10% FBS, 100 U/ml 297 of penicillin, 100 μg/ml of streptomycin, 400 μg/ml of G418, 5 μg/ml of insulin (Nacalai Tesque), and 298 400 ng/ml of tetracycline (Nacalai Tesque). PHHs were maintained in PHH-specific media provided 299 by the manufacture (Phoenix Bio). 300 301 Generation of RBP overexpressing cells 302 RBP-overexpressing Huh7 cells were generated by transfection of RBP expression plasmids 303 using TransIT LT-1 transfection reagent (Mirus, WI, USA). Briefly, Huh7 cells were seeded in 24- 304 well plates (Greiner, Bad Haller, Austria) 1 day prior to the transfection. The following day, 1 μl of 305 TransIT LT-1 transfection reagent, 0.3 μg of RBP expression plasmid, and 0.1 μg of a plasmid 306 containing a 1.3-fold-overlength genome of HBV (genotype C) were mixed in 50 μl of Opti-MEM 307 (Thermo Fisher Scientific, MA, USA). After 15 minutes incubation at room temperature, the mixture . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 18 308 was added into the supernatant of the cultured Huh7 cells. Entecavir (Fujifilm, Tokyo, Japan) was 309 added at 5 μM the day after transfection as a positive control. 309 added at 5 μM the day after transfection as a positive control. 310 RBP-overexpressing HepG2-hNTCP C4 cells were made by lentiviral transduction. Lentiviruses 311 were made by transfecting HIV gag/pol, VSV-G, REV, and pcs-II EF RBP expression plasmids into 312 HEK293T cells using TransIT LT-1 transfection reagent. At 2 dpt, the supernatant of HEK293T was 313 collected and spun at 4,200 rpm for 2 minutes at room temperature and the supernatant added to 314 HepG2-hNTCP C4 cells. Entecavir or heparin was used as a positive control. Entecavir was added at 315 5 μM the day after infection. Heparin (Mochida Pharmaceutical Co., Tokyo, Japan) was added at 5 316 U/ml 30 min before infection. 317 To express N4BP1 in PHHs, we used an adenovirus vector (AdV). . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 325 (MOI) of 5 two days before HBV infection. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells 327 Generation of RBP knockdown cells 328 RBP knockdown cell lines were generated using Silencer Select siRNA (Thermo Fisher 329 Scientific) and Lipofectamine RNAiMAX (Thermo Fisher Scientific) following the manufacturer’s 330 protocol. Briefly, 1.5 μl of Lipofectamine RNAiMAX and 5 pg of siRNA were mixed in 50 μl of Opti- 331 MEM. After incubating for 5 minutes at room temperature, the mixture was gently added to the cell 332 supernatant. The following siRNA reagents were used in this study: control siRNA (Silencer Select 333 Negative Control No.1 siRNA, 4390843), ZCCHC10 siRNA#1 (s29507), ZCCHC10 siRNA#2 334 (s29508), ZCCHC10 siRNA#3 (s29509), HNRNPC siRNA#1 (s6719), HNRNPC siRNA#2 (s6720), 335 HNRNPC siRNA#3 (s6721), N4BP1 siRNA#1 (s18638), N4BP1 siRNA#2 (s18639). 336 337 Generation of N4BP1 knockout cells 338 N4BP1 KO Huh7 cells were generated by CRISPR/Cas9 using pX330-U6-Chimeric_BB-CBh- 339 hSpCas9 (pX330, Addgene, MA, USA) and HR110PA-1 (System Biosciences, CA, USA). The 340 pX330-puro vector was constructed by integrating the puromycin resistance gene sequence from the 341 HR110PA-1 vector into the pX330 vector. The pX330-puro vector was digested using BbsI (Biolabs, 342 MA, USA) and the target sequence was subcloned. The plasmid was transfected into Huh7 cells using 343 TransIT LT-1 transfection reagent and after selection with puromycin dihydrochloride (Invivogen, CA, 328 RBP knockdown cell lines were generated using Silencer Select siRNA (Thermo Fisher 329 Scientific) and Lipofectamine RNAiMAX (Thermo Fisher Scientific) following the manufacturer’s 330 protocol. Briefly, 1.5 μl of Lipofectamine RNAiMAX and 5 pg of siRNA were mixed in 50 μl of Opti- 331 MEM. After incubating for 5 minutes at room temperature, the mixture was gently added to the cell 332 supernatant. The following siRNA reagents were used in this study: control siRNA (Silencer Select 333 Negative Control No.1 siRNA, 4390843), ZCCHC10 siRNA#1 (s29507), ZCCHC10 siRNA#2 334 (s29508), ZCCHC10 siRNA#3 (s29509), HNRNPC siRNA#1 (s6719), HNRNPC siRNA#2 (s6720), 335 HNRNPC siRNA#3 (s6721), N4BP1 siRNA#1 (s18638), N4BP1 siRNA#2 (s18639). 337 Generation of N4BP1 knockout cells 338 N4BP1 KO Huh7 cells were generated by CRISPR/Cas9 using pX330-U6-Chimeric_BB-CBh- 339 hSpCas9 (pX330, Addgene, MA, USA) and HR110PA-1 (System Biosciences, CA, USA). The 340 pX330-puro vector was constructed by integrating the puromycin resistance gene sequence from the 341 HR110PA-1 vector into the pX330 vector. The pX330-puro vector was digested using BbsI (Biolabs, 342 MA, USA) and the target sequence was subcloned. 205 N4BP1 expression is induced by IFN-α/λ in PHHs The N4BP1expressing 318 adenovirus vector (N4BP1 AdV) was constructed using the cosmid cassette pAxEFwit2, which has a 319 cloning site SwaI flanked by the EF1α promoter and rabbit β-globin polyA signal in the E1-deleted 320 region of the adenovirus genome. The N4BP1 cDNA was prepared from the pCS2-EF-N4BP1-HA 321 plasmid and inserted into the SwaI cloning site. The AdV was prepared as previously described[36, 322 37]. The AdV was titrated using the methods described by Pei et al[38]. Briefly, the copy numbers of 323 viral genome successfully transduced into target cells were measured by qPCR. For generating 324 N4BP1-overexpressing PHHs, cells were infected with N4BP1 AdV at a multiplicity of infection 325 (MOI) of 5 two days before HBV infection. 317 To express N4BP1 in PHHs, we used an adenovirus vector (AdV). The N4BP1expressing 318 adenovirus vector (N4BP1 AdV) was constructed using the cosmid cassette pAxEFwit2, which has a 319 cloning site SwaI flanked by the EF1α promoter and rabbit β-globin polyA signal in the E1-deleted 320 region of the adenovirus genome. The N4BP1 cDNA was prepared from the pCS2-EF-N4BP1-HA 321 plasmid and inserted into the SwaI cloning site. The AdV was prepared as previously described[36, 322 37]. The AdV was titrated using the methods described by Pei et al[38]. Briefly, the copy numbers of 323 viral genome successfully transduced into target cells were measured by qPCR. For generating 324 N4BP1-overexpressing PHHs, cells were infected with N4BP1 AdV at a multiplicity of infection 325 (MOI) f 5 d b f HBV i f i 19 326 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells The plasmid was transfected into Huh7 cells using 343 TransIT LT-1 transfection reagent and after selection with puromycin dihydrochloride (Invivogen, CA, . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 20 344 USA), a single colony was isolated using cloning cylinders (Merck, Darmstadt, Germany) and 345 propagated. KO of the target gene was confirmed by WB and DNA sequencing. 346 347 Plasmid Constructions 348 We used the following plasmids containing 1.3-fold-overlength genomes of different HBV 349 genotypes: pUC19-Ae_us (genotype A, accession no. AB246337), pGEM-Bj-JPN56 (genotype B, 350 accession no. AB246342), and pUC19-C_JPNAT (genotype C, accession no. AB246345). 351 Each RBP expression plasmid had a C-terminus HA tag and was inserted between the XhoI and 352 XbaI sites of the pcs-II EF vector. The KH-like domain deletion mutant (ΔKH), RNase domain 353 deletion mutant (ΔRNase), and D623N mutant were generated from the N4BP1 expression plasmid. 354 ΔKH plasmid was generated by deleting amino acids 59-143 from N4BP1; ΔRNase was generated by 355 deleting amino acids 617-769 N4BP1; and D623N was generated by changing the 623rd amino acid 356 from asparagine acid to asparagine. To insert these mutated N4BP1 genes into the pcs-II EF vector 357 between the XhoI and XbaI sites, we used the following primer sets and In-Fusion Snap Assembly 358 Master Mix (Takara Bio, Shiga, Japan) per the manufacturer’s directions. Each N4BP1 mutant plasmid 359 had a C-terminus HA tag. 360 ΔKH: 5’- CGCTACCGGTCTCGAGATGGCGGCCCGGGCGGTGCT -3’ (forward) 361 5’- GGGTAGGTTCTCTTTCGCCCCGCAGAGCTGCAGCC -3’ (reverse) , and 344 USA), a single colony was isolated using cloning cylinders (Merck, Darmstadt, Germany) and 344 USA), a single colony was isolated using cloning cylinders (Merck, Darmstadt, Germany) and 360 ΔKH: 5’- CGCTACCGGTCTCGAGATGGCGGCCCGGGCGGTGCT -3’ (forward) 21 21 362 5’- CAGCTCTGCGGGGCGAAAGAGAACCTACCCAGTAG -3’ (forward) 363 5’- GGTACATGGTCTCGAGATCCAACACCATGGCAGAAA -3’ (reverse) 364 ΔRNase: 5’- CGCTACCGGTCTCGAGATGGCGGCCCGGGCGGTGCT -3’ (forward) 365 5’- TTCCTTCTGAAGAAAATCCGTTCTCCCTGGTTCAT -3’ (reverse) , and 366 5’- CCAGGGAGAACGGATTTTCTTCAGAAGGAAGTCTG -3’ (forward) 367 5’- GGGTAGGTTCTCTTTCGCCCCGCAGAGCTGCAGCC -3’ (reverse) 368 D623N: 5’- CGCTACCGGTCTCGAGATGGCGGCCCGGGCGGTGCT -3’ (forward) 369 5’- AACATTGCTCCCATTTATAACAATGTGTTTCAAAT -3’ (reverse) , and 370 5’- AATGGGAGCAATGTTGCAATTACCCATGGTCTGAA -3’ (forward) 371 5’- GGTACATGGTCTCGAGATCCAACACCATGGCAGAAA -3’ (reverse) 372 373 Viruses 374 The production of HBV particles (genotype D) was adapted from a previous report[39]. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells Briefly, 375 HepAD38.7 cells were cultured under tetracycline (400 ng/mL) for maintenance. To produce HBV 376 particles, tetracycline was removed and the supernatant collected after 6 and 9 days. The collected 377 supernatant was mixed with 30% polyethylene glycol (PEG) 8000 (Sigma) and incubated overnight 378 at 4℃. Then it was spun at 3,000 rpm for 20 minutes at 4℃ for concentration. Before HBV infection, 379 HepG2-hNTCP C4 cells were seeded in collagen type I-coated 24-well plates (Iwaki, Tokyo, Japan) . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 22 380 and transduced with lentivirus to express RBP (see “Generation of RBP-overexpressing cells” above). 381 At 2 days post-transduction, the cells were infected with 5,000 genome equivalents (GEq)/cell of HBV 382 in DMEM containing 3% DMSO (Nacalai Tesque) and 4% PEG 8000, and the media was changed 383 every 2-3 days until 14 dpi. 384 HBV genotype C for PHHs infection was purchased from Phoenix Bio. The infection into PHHs 385 was performed at 5 Geq/cell in PHH-specific media containing 40% PEG 8000 following the 386 manufacturer’s protocol. The culture media was replaced every 4 days. Cells were harvested at 14 dpi 387 for WB and reverse transcription quantitative PCR (RT-qPCR). 388 389 Western blotting 390 Cells were lysed with 2 × Laemmli sample buffer (Bio Rad Laboratories, CA, USA) containing 391 10% 2-mercaptoethanol (Sigma) and boiled for 10 minutes at 100℃. Then lysates were loaded on a 392 10-20% gradient SDS-PAGE gel (Atto, Tokyo, Japan) and transferred onto a polyvinylidene fluoride 393 transfer membrane (Millipore, MA, USA). Membranes were immersed in Block Ace (Kac, Kyoto, 394 Japan) containing 1% bovine serum albumin (Sigma) and incubated for 1 hour at room temperature. 395 The blocking solution was then decanted, and replaced with Can Get Signal Solution 1 (Toyobo, Osaka, 396 Japan) containing the appropriate antibodies described below and incubated for 1 hour. The 397 membranes were washed with 1 × PBS (Fujifilm) containing 0.1% polyoxyethylene (20) sorbitan 389 Western blotting 390 Cells were lysed with 2 × Laemmli sample buffer (Bio Rad Laboratories, CA, USA) containing 391 10% 2-mercaptoethanol (Sigma) and boiled for 10 minutes at 100℃. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells Then lysates were loaded on a 392 10-20% gradient SDS-PAGE gel (Atto, Tokyo, Japan) and transferred onto a polyvinylidene fluoride 393 transfer membrane (Millipore, MA, USA). Membranes were immersed in Block Ace (Kac, Kyoto, 394 Japan) containing 1% bovine serum albumin (Sigma) and incubated for 1 hour at room temperature. 395 The blocking solution was then decanted, and replaced with Can Get Signal Solution 1 (Toyobo, Osaka, 396 Japan) containing the appropriate antibodies described below and incubated for 1 hour. The 397 membranes were washed with 1 × PBS (Fujifilm) containing 0.1% polyoxyethylene (20) sorbitan . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 23 398 monolaurate (Fujifilm) and incubated with Can Get Signal Solution 2 (Toyobo) containing horseradish 399 peroxidase-conjugated antibody against rabbit or mouse immunoglobulins for 1 hour. After addition 400 of Amersham ECL Prime Western Blotting Detection Reagents (Cytiva, Tokyo, Japan), the 401 membranes were subsequently imaged via LuminoGraph I (Atto). 401 membranes were subsequently imaged via LuminoGraph I (Atto). 402 The following antibodies were used in this study: anti-N4BP1 (Bethyl Laboratories, TX, USA), 403 anti-ZC3H12B (Gene Tex, CA, USA), anti-HNRNPC (abcam, Cambridgeshire, UK), anti-ZCCHC10 404 (Sigma), anti-KHNYN (Fujifilm), anti-HA tag (Biolegend, CA, USA), anti-rabbit IgG (H+L) HRP 405 (Jackson Immuno Research Laboratories, MD, USA), anti-mouse IgG (H+L) HRP (Thermo Fisher 406 Scientific), and anti-GAPDH (Fujifilm). The antibodies used in this study are listed in S2 Table. 407 408 Purification of intracellular HBV rcDNA 409 To extract intracellular HBV DNA, cells were washed twice with PBS, and cells were lysed in 410 lysis buffer (100 mM Tris-HCl [pH 8.0], 0.2% NP-40) for 15 minutes at 4℃. After spinning at 13,000 411 rpm for 1 minute at room temperature, the supernatant was collected and incubated with 1 M MgCl2, 412 0.2 mg/ml of DNase I (Sigma), and 10 mg/ml of RNase A (Nacalai Tesque) for at least 3 hours at 413 37℃. After adding 500 mM EDTA and 5 M NaCl, the lysates were digested with 20 mg/ml of 414 proteinase K (Kanto Chemical, Tokyo, Japan) and 10% SDS for 5 hours at 55℃. 327 Generation of RBP knockdown cells The extracted HBV 415 DNA was purified using phenol-chloroform-isoamyl alcohol (Sigma), then precipitated with 402 The following antibodies were used in this study: anti-N4BP1 (Bethyl Laboratories, TX, USA), 403 anti-ZC3H12B (Gene Tex, CA, USA), anti-HNRNPC (abcam, Cambridgeshire, UK), anti-ZCCHC10 404 (Sigma), anti-KHNYN (Fujifilm), anti-HA tag (Biolegend, CA, USA), anti-rabbit IgG (H+L) HRP 405 (Jackson Immuno Research Laboratories, MD, USA), anti-mouse IgG (H+L) HRP (Thermo Fisher 406 Scientific), and anti-GAPDH (Fujifilm). The antibodies used in this study are listed in S2 Table. 409 To extract intracellular HBV DNA, cells were washed twice with PBS, and cells were lysed in 410 lysis buffer (100 mM Tris-HCl [pH 8.0], 0.2% NP-40) for 15 minutes at 4℃. After spinning at 13,000 411 rpm for 1 minute at room temperature, the supernatant was collected and incubated with 1 M MgCl2, 412 0.2 mg/ml of DNase I (Sigma), and 10 mg/ml of RNase A (Nacalai Tesque) for at least 3 hours at 413 37℃. After adding 500 mM EDTA and 5 M NaCl, the lysates were digested with 20 mg/ml of 414 proteinase K (Kanto Chemical, Tokyo, Japan) and 10% SDS for 5 hours at 55℃. The extracted HBV 415 DNA was purified using phenol-chloroform-isoamyl alcohol (Sigma), then precipitated with . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 24 416 isopropanol, 0.5 mg/ml of glycogen, and 3M sodium acetate. After washing with 70% ethanol, the 417 purified HBV DNA was resolved in 50 μl pure water. HBV rcDNA was detected by qPCR using 418 Power SYBR Green PCR Master Mix (Applied Biosystems, CA, USA). As a standard for rcDNA, 419 pUC19-C_JPNAT was used. qPCR procedures were following the manufacturer's protocol. Briefly, 420 the samples were incubated at 50℃ for 2 minutes and at 95℃ for 10 minutes to initial denaturation. 421 Then, the samples were incubated at 95℃ for 15 seconds and at 60℃ for 1 minute for 40 cycles to 422 polymerase reaction. The following primer pair was used in this study. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells 423 rcDNA: 5’- GGAGGGATACATAGAGGTTCCTTGA -3’ (forward) 424 5’- GTTGCCCGTTTGTCCTCTAATTC -3’ (reverse) 425 426 Quantification of intracellular HBV cccDNA level 427 To extract intracellular HBV cccDNA, the cells were lysed by lysis buffer (10mM Tris-HCl [pH 428 7.4], 10mM EDTA, 0.5% SDS) and 5M NaCl was added. After incubation overnight at 4℃, the 429 samples were spun at 15,000 rpm for 30 minutes at 4℃ and the supernatant was collected to isolate 430 protein-free DNA. The DNA was purified a total of three times by phenol (Nacalai Tesque), phenol- 431 chloroform-isoamyl, and chloroform (Fujifilm). After precipitation with isopropanol and ethanol, 432 Plasmid-Safe ATP-Dependent DNase (Lucigen, WI, USA) was added to the purified DNA to remove 433 double-stranded and circular DNA. The Purified DNA was extracted in 30 μl of 10mM Tris-Cl (pH . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 25 434 8.5) using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan). HBV cccDNA 435 was measured by qPCR using TaqMan Fast Advanced Master Mix (Applied Biosystems, CA, USA). 436 As a standard for cccDNA, pUC19-C_JPNAT was used. qPCR procedures were done per the 437 manufacturer’s protocol. In brief, the samples were incubated at 50℃ for 2 minutes and at 95℃ for 10 438 minutes to initiate denaturation. Then, the samples were incubated at 95℃ for 15 seconds and at 60℃ 439 for 1 minute for 50 cycles for the polymerase reaction. The following primer pair and probe were used 440 in this study. 441 cccDNA: 5’- CGTCTGTGCCTTCTCATCTGC -3’ (forward) 442 5’- GCACAGCTTGGAGGCTTGAA -3’ (reverse) 443 5’- CTGTAGGCATAAATTGGT -3’ (Probe) 444 445 Measurement of intracellular HBV pgRNA 446 Total RNA including pgRNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, 447 Germany) following the manufacturer’s protocol. The extracted RNA was resolved in 50 μl pure water 448 and measured by RT-qPCR using Power SYBR Green RNA-to-Ct 1-step Kit (Applied Biosystems). 449 The pgRNA expression level was normalized against the expression level of GAPDH as a 450 housekeeping gene and calculated by the ΔΔCT method. RT-qPCR procedures were done following 451 manufacturer’s protocol. 327 Generation of RBP knockdown cells First, the samples were incubated at 50℃ for 2 minutes and at 95℃ for 10 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 26 452 minutes to intiate denaturation. Then, the samples were incubated at 95℃ for 15 seconds and at 60℃ 453 for 1 minute for 40 cycles for the polymerase reaction. Samples were then incubated at 95℃ for 15 454 seconds, 60℃ for 1 minute, and 95℃ for 15 seconds for the melting curve measurement. The following 455 primer pairs were used in this study. 456 pgRNA: 5’- TCCCTCGCCTCGCAGACG -3’ (forward) 457 5’- GTTTCCCACCTTATGAGTC -3’ (reverse) 458 GAPDH: 5’- AGAAGGCTGGGGCTCATTTG -3’ (forward) 459 5’- AGGGGCCATCCACAGTCTTC -3’ (reverse) 460 461 pgRNA degradation assay 462 HepAD38.7 cells were seeded in collagen type I-coated 12-well plates (Iwaki) and cultured 463 overnight in media containing tetracycline hydrochloride. After washing twice with PBS, cells were 464 cultured without tetracycline for 48 hours. Then, the cells were transfected with N4BP1 wild-type 465 expression plasmid, ΔKH expression plasmid, ΔRNase expression plasmid, or control vector. These 466 plasmids were transfected as follows: 100 μl of Opti-MEM, 6 μl of TransIT LT-1, and 3μg of 467 expression plasmid. After transfection of plasmids, cells were cultured for 48 hours and changed to a 468 tetracycline-containing media in order to stop HBV production. Cells were collected at 2, 3, and 4 469 days after changing to tetracycline-containing media. Total RNA, including pgRNA, was extracted as 27 70 described above for RT-qPCR. 471 472 Drug treatment assay 473 Huh7 cells, HepG2-hNTCP C4 cells, and PHHs were stimulated with 1000 U/ml of IFN-α (PBL 474 Assay Science, NJ, USA) or 100 ng/ml of IFN-λ (Pepro Tech, NJ, USA). At 24 and 48 hours post- 475 drug stimulation, total RNA in cells was extracted for qPCR using the RNeasy Mini Kit, and cDNA 476 was synthesized using SuperScript IV VILO Master Mix (Invitrogen). For WB, the cells were lysed 477 with 2 × Laemmli sample buffer containing 10% 2-mercaptoethanol and processed as described above. 478 qPCR was performed using Power SYBR Green PCR Master Mix. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells The mRNA expression levels of 479 N4BP1 and ISG15 were normalized against the expression level of GAPDH as a housekeeping gene 480 and calculated by the ΔΔCT method. The following primer pairs were used in this study. 481 N4BP1: 5’- CCCGATGATCCTCTGGGAAG -3’ (forward) 482 5’- TTTGGCAGGGCACTGAGTAG -3’ (reverse) 483 ISG15: 5’- ACTCATCTTTGCCAGTACAGGAG -3’ (forward) 484 5’- CAGCATCTTCACCGTCAGGTC -3’ (reverse) 485 GAPDH: see above 486 487 Analysis of N4BP1 expression in clinical liver samples . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 28 28 488 To assess the N4BP1 expression of HBV-HCC resection samples, we confirmed that all 489 specimens were HBs-Ag (+), HBe-Ag (-), and HBe-Ab (+) as documented in the patient medical 490 records. The samples were divided into an HBV DNA positive group (n=9) and an HBV DNA negative 491 group (n=8). In all cases, we used tissue from non-cancerous areas. RNA was extracted from the 492 resected tissues using the RNeasy Mini Kit and N4BP1 expression was measured by qPCR as 493 described above. This study was approved by the Institutional Review Board of the Graduate School 494 of Medicine, Hokkaido University (No. 14-015, 022-0195). All patients participated in this study 495 provided written informed consent for the use of their samples and clinical data and they were 496 informed of the opportunity to opt out. 497 498 Northern blotting 499 Huh7 cells were co-transfected with a plasmid containing the 1.3-fold-overlength genome of 500 HBV genotype C and N4BP1 using TransIT LT-1 transfection reagent, and cells were collected at 3 501 dpt. Total RNA was extracted using Isogen (Nippon Gene, Tokyo, Japan) and subjected to 502 electrophoresis in 1% agarose-formaldehyde gel and morpholinepropanesulfonic acid buffer (Nacalai 503 Tesque). Ribosomal RNA was visualized by ethidium bromide staining, and electrophoresed RNA 504 was transferred onto a positively charged nylon membrane (Roche, Basel-Stadt, Switzerland). An 505 HBV RNA probe and GAPDH RNA probe were linearized and synthesized by digoxigenin RNA 488 To assess the N4BP1 expression of HBV-HCC resection samples, we confirmed that all 489 specimens were HBs-Ag (+), HBe-Ag (-), and HBe-Ab (+) as documented in the patient medical 490 records. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells The samples were divided into an HBV DNA positive group (n=9) and an HBV DNA negative 491 group (n=8). In all cases, we used tissue from non-cancerous areas. RNA was extracted from the 492 resected tissues using the RNeasy Mini Kit and N4BP1 expression was measured by qPCR as 493 described above. This study was approved by the Institutional Review Board of the Graduate School 494 of Medicine, Hokkaido University (No. 14-015, 022-0195). All patients participated in this study 495 provided written informed consent for the use of their samples and clinical data and they were 496 informed of the opportunity to opt out. 488 498 Northern blotting 499 Huh7 cells were co-transfected with a plasmid containing the 1.3-fold-overlength genome of 500 HBV genotype C and N4BP1 using TransIT LT-1 transfection reagent, and cells were collected at 3 501 dpt. Total RNA was extracted using Isogen (Nippon Gene, Tokyo, Japan) and subjected to 502 electrophoresis in 1% agarose-formaldehyde gel and morpholinepropanesulfonic acid buffer (Nacalai 503 Tesque). Ribosomal RNA was visualized by ethidium bromide staining, and electrophoresed RNA 504 was transferred onto a positively charged nylon membrane (Roche, Basel-Stadt, Switzerland). An 505 HBV RNA probe and GAPDH RNA probe were linearized and synthesized by digoxigenin RNA . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 29 506 labeling kit (Roche). Hybridization and detection were performed using the DIG Northern Starter kit 507 (Roche) following the manufacturer's protocol. The signals were detected by LuminoGraph I (Atto). 508 509 RNA sequencing 510 PHHs were analyzed for RNA sequencing. N4BP1-overexpressing PHHs were infected with HBV 511 genotype C (5 Geq/cell) as described above. At 3 dpi, total RNA in PHHs was extracted using RNeasy 512 Mini Kit following the manufacturer's protocol. RNA sequencing was performed by Osaka University 513 (Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, 514 Osaka, Japan). We ran 2 well (N4BP1-overexpressing) vs 2 well (control) samples. Whole 515 transcriptome sequencing was applied to RNA samples using the Illumina NovaSeq 6000 platform in 516 a 101 bp single-end mode. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells Sequenced reads were mapped to the human reference genome sequence 517 (hg19) using TopHat version 2.2.1 in combination with Bowtie 2 version 2.2.3 and SAMtools version 518 1.0. The number of fragments per kilobase of exon per million mapped fragments was calculated using 519 Cufflinks version 2.2.1. The obtained data were analyzed by iDEP.951[40]. The cutoff value of gene 520 expression was set at fold change >2 or < 0.5 and FDR < 0.05 compared to control cells. To review 521 GEO data, access https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE225322. The accession 522 No is GSE225322 523 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 30 524 Statistical analysis 525 Data are averages of three or more independent experiments. All data in figures are presented as 526 the mean ± standard deviation (SD). Evaluation of data was performed by Mann-Whitney U test. P- 527 value of < 0.05 was considered statistically significant. Statistical analysis was performed using JMP 528 Pro version 16. 529 530 Acknowledgements 531 We thank Haruko Kubo, Mizuho Tetsuka, and Saya Shimamura for their secretarial work. We 532 are grateful to Takaji Wakita (NIID) for providing HepG2-hNTCP C4 cells and HepAD38.7 cells. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells 533 534 Authors contributions 535 Conceptualization: Yoshiharu Matsuura, Osamu Takeuchi, Akinobu Taketomi, Takasuke Fukuhara 536 Data curation: Nobuhiro Kobayashi, Saori Suzuki, Rigel Suzuki, Daisuke Okuzaki, Tomokazu 537 Tamura, Takasuke Fukuhara 538 Formal analysis: Nobuhiro Kobayashi, Saori Suzuki, Rigel Suzuki, Daisuke Okuzaki, Tomokazu 539 Tamura, Takasuke Fukuhara 540 Funding acquisition: Takasuke Fukuhara 541 Investigation: Nobuhiro Kobayashi, Saori Suzuki, Yuki Sakamoto, Takuma Izumi, Kisho Noda, 541 Investigation: Nobuhiro Kobayashi, Saori Suzuki, Yuki Sakamoto, Takuma Izumi, Kisho Noda, 31 542 Daisuke Okuzaki, Yumi Kanegae 543 Methodology: Nobuhiro Kobayashi, Saori Suzuki, Rigel Suzuki, Sanae Hayashi, Yasuhito Tanaka, 544 Tomokazu Tamura, Akinobu Taketomi, Takasuke Fukuhara 545 Project administration: Akinobu Taketomi, Takasuke Fukuhara 546 Resources: Daisuke Okuzaki, Yumi Kanegae, Yoshiharu Matsuura, Osamu Takeuchi, Akinobu 547 Taketomi, Takasuke Fukuhara 548 Software: Daisuke Okuzaki 549 Supervision: Akinobu Taketomi, Takasuke Fukuhara 550 Validation: Nobuhiro Kobayashi, Saori Suzuki, Yuki Sakamoto, Tomoya Saito, Takuma Izumi, 551 Kisho Noda 552 Visualization: Nobuhiro Kobayashi, Saori Suzuki, Takasuke Fukuhara 553 Writing - original draft: Nobuhiro Kobayashi, Saori Suzuki, Daisuke Okuzaki, Yumi Kanegae, 554 Takasuke Fukuhara 555 Writing - review & editing: Nobuhiro Kobayashi, Saori Suzuki, Yuki Sakamoto, Rigel Suzuki, 556 Tomoya Saito, Takuma Izumi, Kisho Noda, Daisuke Okuzaki, Yumi Kanegae, Sanae Hayashi, 557 Yasuhito Tanaka, Yoshiharu Matsuura, Osamu Takeuchi, Tomokazu Tamura, Akinobu Taketomi, 558 Takasuke Fukuhara 559 All authors have approved the submitted version of the manuscript and agreed to be accountable 559 All authors have approved the submitted version of the manuscript and agreed to be accountable . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 32 560 for any part of the work. 561 562 References 563 1. World Health Organisation. Hepatitis B Fact Sheet 2021 [updated 24 June 202213 Feb 564 2023]. Available from: https://www.who.int/en/news-room/fact-sheets/detail/hepatitis-b. 565 2. World Health Organisation. Immunization Coverage 2021 [updated 14 July 202213 Feb 566 2023]. Available from: https://www.who.int/news-room/fact-sheets/detail/immunization-coverage 567 3. Koniger C, Wingert I, Marsmann M, Rosler C, Beck J, Nassal M. Involvement of the host 568 DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir 569 of hepatitis B viruses. Proc Natl Acad Sci U S A. 2014;111(40):E4244-53. Epub 2014/09/10. doi: 570 10.1073/pnas.1409986111. PubMed PMID: 25201958; PubMed Central PMCID: PMCPMC4209993. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells 571 4. Qi Y, Gao Z, Xu G, Peng B, Liu C, Yan H, et al. DNA Polymerase kappa Is a Key Cellular 572 Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus. PLoS Pathog. 573 2016;12(10):e1005893. Epub 2016/10/27. doi: 10.1371/journal.ppat.1005893. PubMed PMID: 574 27783675; PubMed Central PMCID: PMCPMC5081172. 575 5. Sheraz M, Cheng J, Tang L, Chang J, Guo JT. Cellular DNA Topoisomerases Are Required 576 for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA. J Virol. 2019;93(11). Epub 577 2019/03/15. doi: 10.1128/jvi.02230-18. PubMed PMID: 30867306; PubMed Central PMCID: 575 5. Sheraz M, Cheng J, Tang L, Chang J, Guo JT. Cellular DNA Topoisomerases Are Required 576 for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA. J Virol. 2019;93(11). Epub 577 2019/03/15. doi: 10.1128/jvi.02230-18. PubMed PMID: 30867306; PubMed Central PMCID: . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 33 578 PMCPMC6532102. 578 PMCPMC6532102. 579 6. Kitamura K, Que L, Shimadu M, Koura M, Ishihara Y, Wakae K, et al. Flap endonuclease 580 1 is involved in cccDNA formation in the hepatitis B virus. PLoS Pathog. 2018;14(6):e1007124. Epub 581 2018/06/22. doi: 10.1371/journal.ppat.1007124. PubMed PMID: 29928064; PubMed Central PMCID: 582 PMCPMC6013022. 583 7. Rijckborst V, Janssen HL. The Role of Interferon in Hepatitis B Therapy. Curr Hepat Rep. 584 2010;9(4):231-8. Epub 2010/10/16. doi: 10.1007/s11901-010-0055-1. PubMed PMID: 20949114; 585 PubMed Central PMCID: PMCPMC2945466. 586 8. Doong SL, Tsai CH, Schinazi RF, Liotta DC, Cheng YC. Inhibition of the replication of 587 hepatitis B virus in vitro by 2',3'-dideoxy-3'-thiacytidine and related analogues. Proc Natl Acad Sci U 588 S A. 1991;88(19):8495-9. Epub 1991/10/01. doi: 10.1073/pnas.88.19.8495. PubMed PMID: 1656445; 589 PubMed Central PMCID: PMCPMC52535. 590 9. Li Q, Sun B, Zhuo Y, Jiang Z, Li R, Lin C, et al. Interferon and interferon-stimulated genes 591 in HBV treatment. Front Immunol. 2022;13:1034968. Epub 2022/12/20. doi: 592 10.3389/fimmu.2022.1034968. PubMed PMID: 36531993; PubMed Central PMCID: 593 PMCPMC9751411. 594 10. Tang LSY, Covert E, Wilson E, Kottilil S. Chronic Hepatitis B Infection: A Review. JAMA. 595 2018;319(17):1802-13. Epub 2018/05/02. doi: 10.1001/jama.2018.3795. PubMed PMID: 29715359. 594 10. Tang LSY, Covert E, Wilson E, Kottilil S. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells Chronic Hepatitis B Infection: A Review. JAMA. 595 2018;319(17):1802-13. Epub 2018/05/02. doi: 10.1001/jama.2018.3795. PubMed PMID: 29715359. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 34 596 11. Dienstag JL, Perrillo RP, Schiff ER, Bartholomew M, Vicary C, Rubin M. A preliminary 597 trial of lamivudine for chronic hepatitis B infection. N Engl J Med. 1995;333(25):1657-61. Epub 598 1995/12/21. doi: 10.1056/nejm199512213332501. PubMed PMID: 7477217. 599 12. Nowak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas HC, McDade H. Viral dynamics 600 in hepatitis B virus infection. Proc Natl Acad Sci U S A. 1996;93(9):4398-402. Epub 1996/04/30. doi: 601 10.1073/pnas.93.9.4398. PubMed PMID: 8633078; PubMed Central PMCID: PMCPMC39549. 602 13. Suzuki F, Akuta N, Suzuki Y, Sezaki H, Arase Y, Hosaka T, et al. Clinical and virological 603 features of non-breakthrough and severe exacerbation due to lamivudine-resistant hepatitis B virus 604 mutants. J Med Virol. 2006;78(3):341-52. Epub 2006/01/19. doi: 10.1002/jmv.20546. PubMed PMID: 605 16419116. 606 14. Mao R, Nie H, Cai D, Zhang J, Liu H, Yan R, et al. Inhibition of hepatitis B virus replication 607 by the host zinc finger antiviral protein. PLoS Pathog. 2013;9(7):e1003494. Epub 2013/07/16. doi: 608 10.1371/journal.ppat.1003494. PubMed PMID: 23853601; PubMed Central PMCID: 609 PMCPMC3708887. 610 15. Li M, Yang J, Zhao Y, Song Y, Yin S, Guo J, et al. MCPIP1 inhibits Hepatitis B virus 611 replication by destabilizing viral RNA and negatively regulates the virus-induced innate inflammatory 612 responses. Antiviral Res. 2020;174:104705. Epub 2020/01/12. doi: 10.1016/j.antiviral.2020.104705. 613 PubMed PMID: 31926181. 596 11. Dienstag JL, Perrillo RP, Schiff ER, Bartholomew M, Vicary C, Rubin M. A preliminary 597 trial of lamivudine for chronic hepatitis B infection. N Engl J Med. 1995;333(25):1657-61. Epub 598 1995/12/21. doi: 10.1056/nejm199512213332501. PubMed PMID: 7477217. 610 15. Li M, Yang J, Zhao Y, Song Y, Yin S, Guo J, et al. MCPIP1 inhibits Hepatitis B virus 611 replication by destabilizing viral RNA and negatively regulates the virus-induced innate inflammatory 612 responses. Antiviral Res. 2020;174:104705. Epub 2020/01/12. doi: 10.1016/j.antiviral.2020.104705. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 35 614 16. Sun S, Nakashima K, Ito M, Li Y, Chida T, Takahashi H, et al. Involvement of PUF60 in 615 Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression. 616 Sci Rep. 2017;7(1):12874. Epub 2017/10/11. doi: 10.1038/s41598-017-12497-y. PubMed PMID: 617 28993636; PubMed Central PMCID: PMCPMC5634508. 618 17. Tang H, Huang Y, Chen J, Yu C, Huang AL. Cellular protein TIA-1 regulates the expression 619 of HBV surface antigen by binding the HBV posttranscriptional regulatory element. Intervirology. 620 2008;51(3):203-9. Epub 2008/08/30. doi: 10.1159/000151632. PubMed PMID: 18753794. 621 18. Xu W, Ma C, Zhang Q, Zhao R, Hu D, Zhang X, et al. PJA1 Coordinates with the SMC5/6 622 Complex To Restrict DNA Viruses and Episomal Genes in an Interferon-Independent Manner. J Virol. 623 2018;92(22). Epub 2018/09/07. doi: 10.1128/jvi.00825-18. PubMed PMID: 30185588; PubMed 624 Central PMCID: PMCPMC6206484. 625 19. Cai J, Yang L, Wang B, Huang Y, Tang J, Lu Y, et al. Identification of a novel N4BP1-like 626 gene from grass carp (Ctenopharyngodon idella) in response to GCRV infection. Fish Shellfish 627 Immunol. 2014;36(1):223-8. Epub 2013/11/14. doi: 10.1016/j.fsi.2013.11.003. PubMed PMID: 628 24220004. 629 20. Yamasoba D, Sato K, Ichinose T, Imamura T, Koepke L, Joas S, et al. N4BP1 restricts HIV- 630 1 and its inactivation by MALT1 promotes viral reactivation. Nat Microbiol. 2019;4(9):1532-44. Epub 631 2019/05/28. doi: 10.1038/s41564-019-0460-3. PubMed PMID: 31133753. 614 16. Sun S, Nakashima K, Ito M, Li Y, Chida T, Takahashi H, et al. Involvement of PUF60 in 615 Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression. 616 Sci Rep. 2017;7(1):12874. Epub 2017/10/11. doi: 10.1038/s41598-017-12497-y. PubMed PMID: 617 28993636; PubMed Central PMCID: PMCPMC5634508. 993636; PubMed Central PMCID: PMCPMC5634508. 620 2008;51(3):203-9. Epub 2008/08/30. doi: 10.1159/000151632. PubMed PMID: 18753794. 621 18. Xu W, Ma C, Zhang Q, Zhao R, Hu D, Zhang X, et al. PJA1 Coordinates with the SMC5/6 622 Complex To Restrict DNA Viruses and Episomal Genes in an Interferon-Independent Manner. J Virol. 623 2018;92(22). Epub 2018/09/07. doi: 10.1128/jvi.00825-18. PubMed PMID: 30185588; PubMed . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 36 632 21. Honkoop P, Niesters HG, de Man RA, Osterhaus AD, Schalm SW. Lamivudine resistance 633 in immunocompetent chronic hepatitis B. Incidence and patterns. J Hepatol. 1997;26(6):1393-5. Epub 634 1997/06/01. doi: 10.1016/s0168-8278(97)80476-x. PubMed PMID: 9210628. 635 22. Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DL. Mutation in HBV 636 RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology. 637 1996;24(3):714-7. Epub 1996/09/01. doi: 10.1002/hep.510240340. PubMed PMID: 8781348. 638 23. Bartholomew MM, Jansen RW, Jeffers LJ, Reddy KR, Johnson LC, Bunzendahl H, et al. 639 Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver 640 transplantation. Lancet. 1997;349(9044):20-2. Epub 1997/01/04. doi: 10.1016/S0140-6736(96)02266- 641 0. PubMed PMID: 8988118. 642 24. Valverde R, Edwards L, Regan L. Structure and function of KH domains. FEBS J. 643 2008;275(11):2712-26. Epub 2008/04/22. doi: 10.1111/j.1742-4658.2008.06411.x. PubMed PMID: 644 18422648. 645 25. Hollingworth D, Candel AM, Nicastro G, Martin SR, Briata P, Gherzi R, et al. KH domains 646 with impaired nucleic acid binding as a tool for functional analysis. Nucleic Acids Res. 647 2012;40(14):6873-86. Epub 2012/05/02. doi: 10.1093/nar/gks368. PubMed PMID: 22547390; 648 PubMed Central PMCID: PMCPMC3413153. 649 26. Anantharaman V, Aravind L. The NYN domains: novel predicted RNAses with a PIN 634 1997/06/01. doi: 10.1016/s0168-8278(97)80476-x. PubMed PMID: 9210628. 645 25. Hollingworth D, Candel AM, Nicastro G, Martin SR, Briata P, Gherzi R, et al. KH domains 646 with impaired nucleic acid binding as a tool for functional analysis. Nucleic Acids Res. 647 2012;40(14):6873-86. Epub 2012/05/02. doi: 10.1093/nar/gks368. PubMed PMID: 22547390; 648 PubMed Central PMCID: PMCPMC3413153. 649 26. Anantharaman V, Aravind L. The NYN domains: novel predicted RNAses with a PIN 649 26. Anantharaman V, Aravind L. The NYN domains: novel predicted RNAses with a PIN 649 26. Anantharaman V, Aravind L. The NYN domains: novel predicted RNAses with a PIN . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 37 650 domain-like fold. RNA Biol. 2006;3(1):18-27. Epub 2006/11/23. doi: 10.4161/rna.3.1.2548. PubMed 651 PMID: 17114934. 652 27. Shi H, Sun L, Wang Y, Liu A, Zhan X, Li X, et al. N4BP1 negatively regulates NF-kappaB 653 by binding and inhibiting NEMO oligomerization. Nat Commun. 327 Generation of RBP knockdown cells 2021;12(1):1379. Epub 2021/03/04. 654 doi: 10.1038/s41467-021-21711-5. PubMed PMID: 33654074; PubMed Central PMCID: 655 PMCPMC7925594. 656 28. Gitlin AD, Heger K, Schubert AF, Reja R, Yan D, Pham VC, et al. Integration of innate 657 immune signalling by caspase-8 cleavage of N4BP1. Nature. 2020;587(7833):275-80. Epub 658 2020/09/25. doi: 10.1038/s41586-020-2796-5. PubMed PMID: 32971525. 659 29. Cui X, Li Y, Xu H, Sun Y, Jiang S, Li W. Characteristics of Hepatitis B virus integration 660 and mechanism of inducing chromosome translocation. NPJ Genom Med. 2023;8(1):11. Epub 661 2023/06/03. doi: 10.1038/s41525-023-00355-y. PubMed PMID: 37268616; PubMed Central PMCID: 662 PMCPMC10238498. 663 30. Gu H, Zheng S, Han G, Yang H, Deng Z, Liu Z, et al. Porcine Reproductive and Respiratory 664 Syndrome Virus Adapts Antiviral Innate Immunity via Manipulating MALT1. mBio. 665 2022;13(3):e0066422. Epub 2022/04/26. doi: 10.1128/mbio.00664-22. PubMed PMID: 35467421; 666 PubMed Central PMCID: PMCPMC9239189. 667 31. Mino T, Takeuchi O. Regnase-1-related endoribonucleases in health and immunological 652 27. Shi H, Sun L, Wang Y, Liu A, Zhan X, Li X, et al. N4BP1 negatively regulates NF-kappaB 653 by binding and inhibiting NEMO oligomerization. Nat Commun. 2021;12(1):1379. Epub 2021/03/04. 654 doi: 10.1038/s41467-021-21711-5. PubMed PMID: 33654074; PubMed Central PMCID: 655 PMCPMC7925594. 656 28. Gitlin AD, Heger K, Schubert AF, Reja R, Yan D, Pham VC, et al. Integration of innate 657 immune signalling by caspase-8 cleavage of N4BP1. Nature. 2020;587(7833):275-80. Epub 656 28. Gitlin AD, Heger K, Schubert AF, Reja R, Yan D, Pham VC, et al. Integration of innate 657 immune signalling by caspase-8 cleavage of N4BP1. Nature. 2020;587(7833):275-80. Epub 658 2020/09/25. doi: 10.1038/s41586-020-2796-5. PubMed PMID: 32971525. 659 29. Cui X, Li Y, Xu H, Sun Y, Jiang S, Li W. Characteristics of Hepatitis B virus integration 660 and mechanism of inducing chromosome translocation. NPJ Genom Med. 2023;8(1):11. Epub 661 2023/06/03. doi: 10.1038/s41525-023-00355-y. PubMed PMID: 37268616; PubMed Central PMCID: 662 PMCPMC10238498. 659 29. Cui X, Li Y, Xu H, Sun Y, Jiang S, Li W. Characteristics of Hepatitis B virus integration 660 and mechanism of inducing chromosome translocation. NPJ Genom Med. 2023;8(1):11. Epub 661 2023/06/03. doi: 10.1038/s41525-023-00355-y. PubMed PMID: 37268616; PubMed Central PMCID: 662 PMCPMC10238498. 659 29. Cui X, Li Y, Xu H, Sun Y, Jiang S, Li W. Characteristics of Hepatitis B virus integration 660 and mechanism of inducing chromosome translocation. NPJ Genom Med. 2023;8(1):11. Epub 661 2023/06/03. doi: 10.1038/s41525-023-00355-y. PubMed PMID: 37268616; PubMed Central PMCID: 662 PMCPMC10238498. 663 30. 650 domain-like fold. RNA Biol. 2006;3(1):18-27. Epub 2006/11/23. doi: 10.4161/rna.3.1.2548. PubMed 327 Generation of RBP knockdown cells Gu H, Zheng S, Han G, Yang H, Deng Z, Liu Z, et al. Porcine Reproductive and Respiratory 664 Syndrome Virus Adapts Antiviral Innate Immunity via Manipulating MALT1. mBio. 665 2022;13(3):e0066422. Epub 2022/04/26. doi: 10.1128/mbio.00664-22. PubMed PMID: 35467421; 666 PubMed Central PMCID: PMCPMC9239189. 667 31. Mino T, Takeuchi O. Regnase-1-related endoribonucleases in health and immunological 667 31. Mino T, Takeuchi O. Regnase-1-related endoribonucleases in health and immunological . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 38 669 PMID: 34514623. 670 32. Melén K, Keskinen P, Lehtonen A, Julkunen I. Interferon-induced gene expression and 671 signaling in human hepatoma cell lines. J Hepatol. 2000;33(5):764-72. Epub 2000/11/30. doi: 672 10.1016/s0168-8278(00)80308-6. PubMed PMID: 11097485. 673 33. Marozin S, Altomonte J, Stadler F, Thasler WE, Schmid RM, Ebert O. Inhibition of the IFN- 674 β Response in Hepatocellular Carcinoma by Alternative Spliced Isoform of IFN Regulatory Factor-3. 675 Molecular Therapy. 2008;16(11):1789-97. doi: 10.1038/mt.2008.201. 676 34. Speer SD, Li Z, Buta S, Payelle-Brogard B, Qian L, Vigant F, et al. ISG15 deficiency and 677 increased viral resistance in humans but not mice. Nat Commun. 2016;7:11496. Epub 2016/05/20. 678 doi: 10.1038/ncomms11496. PubMed PMID: 27193971; PubMed Central PMCID: 679 PMCPMC4873964. 680 35. Dickensheets H, Sheikh F, Park O, Gao B, Donnelly RP. Interferon-lambda (IFN-λ) induces 681 signal transduction and gene expression in human hepatocytes, but not in lymphocytes or monocytes. 682 J Leukoc Biol. 2013;93(3):377-85. Epub 2012/12/22. doi: 10.1189/jlb.0812395. PubMed PMID: 683 23258595; PubMed Central PMCID: PMCPMC3579021. 684 36. Nakai M, Komiya K, Murata M, Kimura T, Kanaoka M, Kanegae Y, et al. Expression of 685 pIX gene induced by transgene promoter: possible cause of host immune response in first-generation 670 32. Melén K, Keskinen P, Lehtonen A, Julkunen I. Interferon-induced gene expression and 671 signaling in human hepatoma cell lines. J Hepatol. 2000;33(5):764-72. Epub 2000/11/30. doi: 672 10.1016/s0168-8278(00)80308-6. PubMed PMID: 11097485. 673 33. Marozin S, Altomonte J, Stadler F, Thasler WE, Schmid RM, Ebert O. Inhibition of the IFN- 674 β Response in Hepatocellular Carcinoma by Alternative Spliced Isoform of IFN Regulatory Factor-3. 675 Molecular Therapy. 2008;16(11):1789-97. doi: 10.1038/mt.2008.201. 676 34. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 327 Generation of RBP knockdown cells Speer SD, Li Z, Buta S, Payelle-Brogard B, Qian L, Vigant F, et al. ISG15 deficiency and 677 increased viral resistance in humans but not mice. Nat Commun. 2016;7:11496. Epub 2016/05/20. 678 doi: 10.1038/ncomms11496. PubMed PMID: 27193971; PubMed Central PMCID: 679 PMCPMC4873964. 684 36. Nakai M, Komiya K, Murata M, Kimura T, Kanaoka M, Kanegae Y, et al. Expression of 685 pIX gene induced by transgene promoter: possible cause of host immune response in first-generation 684 36. Nakai M, Komiya K, Murata M, Kimura T, Kanaoka M, Kanegae Y, et al. Expression of 685 pIX gene induced by transgene promoter: possible cause of host immune response in first-generation . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint 39 686 adenoviral vectors. Hum Gene Ther. 2007;18(10):925-36. Epub 2007/10/03. doi: 687 10.1089/hum.2007.085. PubMed PMID: 17907966. 688 37. Suzuki M, Kondo S, Pei Z, Maekawa A, Saito I, Kanegae Y. Preferable sites and orientations 689 of transgene inserted in the adenovirus vector genome: The E3 site may be unfavorable for transgene 690 position. Gene Ther. 2015;22(5):421-9. Epub 2015/01/16. doi: 10.1038/gt.2014.124. PubMed PMID: 691 25588742; PubMed Central PMCID: PMCPMC4424821. 692 38. Pei Z, Kondo S, Kanegae Y, Saito I. Copy number of adenoviral vector genome transduced 693 into target cells can be measured using quantitative PCR: application to vector titration. Biochem 694 Biophys Res Commun. 2012;417(3):945-50. Epub 2011/12/29. doi: 10.1016/j.bbrc.2011.12.016. 695 PubMed PMID: 22202173. 696 39. Watashi K, Liang G, Iwamoto M, Marusawa H, Uchida N, Daito T, et al. Interleukin-1 and 697 tumor necrosis factor-alpha trigger restriction of hepatitis B virus infection via a cytidine deaminase 698 activation-induced cytidine deaminase (AID). J Biol Chem. 2013;288(44):31715-27. Epub 2013/09/13. 699 doi: 10.1074/jbc.M113.501122. PubMed PMID: 24025329; PubMed Central PMCID: 700 PMCPMC3814766. 701 40. iDEP.951. Available from: http://bioinformatics.sdstate.edu/idep/. 702 686 adenoviral vectors. Hum Gene Ther. 2007;18(10):925-36. Epub 2007/10/03. doi: 687 10.1089/hum.2007.085. PubMed PMID: 17907966. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . 327 Generation of RBP knockdown cells CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint (w bio . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint CC BY 4 0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license available under a author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is . CC-BY 4.0 International license made available under a ( y p ) , g p y p p p p y . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint CC-BY 4.0 International licens made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a licens this version posted October 9, ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: oRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: ioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted October 9, 2023. ; https://doi.org/10.1101/2023.10.06.561254 doi: bioRxiv preprint
https://openalex.org/W2045051799	https://europepmc.org/articles/pmc4107277?pdf=render	English	null	Centromere identity from the DNA point of view	Chromosoma	2,014	cc-by	11,429	Miroslav Plohl & Nevenka Meštrović & Brankica Mravinac Received: 27 November 2013 /Revised: 28 March 2014 /Accepted: 1 April 2014 /Published online: 25 April 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com Received: 27 November 2013 /Revised: 28 March 2014 /Accepted: 1 April 2014 /Published online: 25 April 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com Abstract The centromere is a chromosomal locus responsi- ble for the faithful segregation of genetic material during cell division. It has become evident that centromeres can be established literally on any DNA sequence, and the possible synergy between DNA sequences and the most prominent centromere identifiers, protein components, and epigenetic marks remains uncertain. However, some evolutionary pref- erences seem to exist, and long-term established centromeres are frequently formed on long arrays of satellite DNAs and/or transposable elements. Recent progress in understanding functional centromere sequences is based largely on the high-resolution DNA mapping of sequences that interact with the centromere-specific histone H3 variant, the most reliable marker of active centromeres. In addition, sequence assembly and mapping of large repetitive centromeric regions, as well as comparative genome analyses offer insight into their complex organization and evolution. The rapidly advancing field of transcription in centromere regions highlights the functional importance of centromeric transcripts. Here, we comprehen- sively review the current state of knowledge on the composi- tion and functionality of DNA sequences underlying active centromeres and discuss their contribution to the functioning of different centromere types in higher eukaryotes. determined by the centromere. The centromere includes the core or functional centromere domain, a specialized locus at which microtubules attach to the complex multiprotein struc- ture of the kinetochore in order to segregate chromosomes in mitosis and meiosis. The core centromere domain is surrounded by large blocks of pericentromeric heterochroma- tin (also called the pericentromere), primary sites of sister chromatid cohesion. Centromere functionality is vital for all eukaryotic organisms. In addition to understanding its role as a biological structure, studying the centromere is also highly relevant from a biomedical point of view, because abnormal- ities in centromeric function are often lethal or associated with various congenital and acquired diseases, such as cancer, infertility, and birth disorders (reviewed in Thompson et al. 2010). ) Centromeres are considered to be shaped by both geno- mic and epigenetic mechanisms, but the synergy between DNA sequences, protein components, and epigenetic marks is still not well understood. M. Plohl (*): N. Meštrović: B. Mravinac Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10002 Zagreb, Croatia e-mail: plohl@irb.hr Miroslav Plohl & Nevenka Meštrović & Brankica Mravinac In the absence of a universal DNA sequence, species-specific histone H3 var- iant CENH3 (CENP-A in mammals, CID in Drosophila melanogaster, Cse4 in Saccharomyces cerevisiae) is the most prominent protein identifier of centromere function. Related forms of this protein have been detected in all studied active centromeres of single-cell and multicellular eukaryotes (Black and Bassett 2008; Malik and Henikoff 2009). CENH3 replaces the canonical histone H3 in such a way that arrays of CENH3-based nucleosomes alternate with those containing canonical H3 (Blower et al. 2002; Sullivan and Karpen 2004). In humans and flies, canonical H3 is in turn epigenetically modified in the centromere, by dimethylation at lysine 4 (H3K4me2), and thus distinctive from the histone H3 in adjacent pericentromeric hetero- chromatin, which is marked by methylation at lysine 9 (H3K9me). These differences qualify centromeric chroma- tin as a unique chromatin type centrochromatin (Sullivan and Karpen 2004). Keywords Centromere . Satellite DNA . Transposable elements . Transcription Keywords Centromere . Satellite DNA . Transposable elements . Transcription Chromosoma (2014) 123:313–325 DOI 10.1007/s00412-014-0462-0 Chromosoma (2014) 123:313–325 DOI 10.1007/s00412-014-0462-0 REVIEW REVIEW REVIEW Centromere identity from the DNA point of view Miroslav Plohl & Nevenka Meštrović & Brankica Mravinac Introduction An essential function of genetic material in any living organ- ism is its faithful segregation, the role which is in eukaryotes 314 Chromosoma (2014) 123:313–325 In the budding yeast S. cerevisiae, centromere function depends on a short, about 100 bp long DNA sequence motif. These centromeres are referred to as simple or point centro- meres (Hyman and Sorger 1995). In all other eukaryotes, centromeres are founded on repetitive DNA arrays of several hundred kilobase, commonly known as complex or regional centromeres (Pluta et al. 1995). A single centromere is nor- mally formed on each chromosome in a locus which is on the cytogenetical level recognized as a primary constriction of the monocentric chromosome. However, there are exceptions, and some organisms have holocentric chromosomes that lack a primary constriction and comprise of a centromere dispersed in many subdomains along the entire chromosome length (Dernburg 2001). humans (Ohzeki et al. 2002), as well as in various subclasses of alphoid repeats in mammalian species (Alkan et al. 2011). This motif is a binding site for the protein CENP-B, which probably facilitates kinetochore formation (Masumoto et al. 2004), but might also play a role in rearrangements of satDNA sequences (Kipling and Warburton 1997). The presence of CENP-B box-like motifs in unrelated satDNAs of some dis- tant invertebrates and plants suggests its potential functional relevance in non-mammalian organisms (Mravinac et al. 2005; Canapa et al. 2000; Meštrović et al. 2013; Gindullis et al. 2001). SatDNAs evolve according to the principles of concerted evolution. Within the genome, mutations are homogenized among repeats of the satDNA by the mechanisms of non- reciprocal sequence transfer, such as unequal crossover, gene conversion, rolling circle replication, and transposition-related mechanisms (Dover 1986). Although the centromere was traditionally treated as a region of suppressed recombination, unequal crossing-over and gene conversion have been identi- fied as the most widespread mechanism involved in satDNA dynamics (Mahtani and Willard 1998; Smith 1976; Talbert and Henikoff 2010). Nevertheless, recent studies on primates and plants postulated mechanism of segmental duplication as an important evolutionary force in the massive amplifications of satDNA arrays and long range rearrangements of (peri)centromere regions (Horvath et al. 2005; Ma and Jackson 2006). At the population level, satDNAs become fixed as a result of random assortment of genetic material in meiosis. Introduction As species diverge, satDNAs accumulate changes as a consequence of mutations and turnover mechanisms in separate lineages generating species-specific satDNA arrays (Dover 1986). However, rapidly accumulating differences in species-specific satDNA profiles can also be accomplished by amplifications/contractions of repeats existing in a so-called library of satDNAs common to related genomes. The hypoth- esis was originally proposed by Fry and Salser (1977) and experimentally proved by Meštrović et al. (1998). As predict- ed by the theory of concerted evolution, a small bias in favor of homogenization of a particular set of repeat variants would lead to extreme conservation of satDNAs (Ohta and Dover 1984; Strachan et al. 1985), observed in various organisms, for example, in sturgeons (De la Herran et al. 2001) and beetles (Mravinac et al. 2002). Because of the above men- tioned specificities, the scenario of satDNA evolution unifies array homogeneity and long-term sequence stability together with the ability of the satDNA library to act as a reservoir of sequences that allow rapid changes through expansions and contractions of arrays (Plohl et al. 2008). Mostly, due to limitations in sequencing and assembly of long arrays of nearly-identical repeats, our knowledge on the long-range functional organization of centromeric DNA is rather limited, and centromeres still represent the last frontiers in genome assemblies and sequence annotations (Hayden and Willard 2012). Here, we review the rapidly progressing field of functional centromere genomics. We present data relating DNA sequences and their functional interactions in different centromere types of higher eukaryotes, and point to the sig- nificance of transcriptional potential of centromeric sequences. Repetitive DNA sequences are the most common centromere components 2006 Arabidopsis thaliana/ thale cress pAL1 satDNA [180 bp]: homogenous family in all centromeres LTR-retrotransposon (Athila) in centromere core, multiple families of LTR retrotransposons and 5SrRNA in pericentromeric regions Nagaki et al. 2003; Kumekawa et al. 2000 Pisum sativum/pea 13 distinct satDNAs families [50–2,094 bp] localized in various combinations in different centromeres satDNAs: TR2, TR3, and TR5 in pericentromeric regions Neumann et al. 2012 Satellite DNAs and retrotransposons Oryza sativa/rice CentO satDNA [155 bp] and CRR retrotransposon in all centromeres Different retrotransposon families belonging to Ty3/ gypsy–class Dong et al. 1998; Cheng et al. 2002 Zea mays/maize Retrotransposons CRM1 and CRM2 and CenC satDNA [156 bp] in all centromeres Retrotransposons CRM3 and CRM4 Zhong et al. 2002; Wolfgruber et al. 2009 Retrotransposons Triticum spp./wheat CRW, Quinta and Weg retrotransposon families Different CRW retrotransposons families (Ty3/gypsy–class) Li et al. 2013 Repeats and non-repeats Equus caballus/horse satDNAs: different ECA families [221–475 bp], 37cen [221 bp], and 2PI [23 bp] localized in various combinations; repeat-free ch11 centromere NA Piras et al. 2010; Alkan et al. 2011 Gallus gallus/chicken Chromosome specific satDNAs [1.8–3.2 kb] in centromeres of eight macrochromosomes, CNM satDNA [42 bp] in some microchromosomes and in ch6 and ch9; repeat-free ch5, ch27, chZ centromeres NA Shang et al. 2010 Solanum tuberosum/ potato Six chromosome specific satDNAs [979 bp to 5.4 kb]; repeat-free ch4, ch6, ch10, ch11, and ch12 centromeres NA Gong et al. 2012 Other abundant DNAs contributing to centromere region References contributing to centromere region Monomeric forms of alpha- satDNA, diverse non-alphoid satDNAs (gamma, beta, Sat I, II, III) and LINE elements in pericentromeric regions Willard and Waye 1987; Waye and Willard 1989; Rudd and Willard 2004; Rudd et al. 2006; Sullivan et al. 2011; Schueler et al. 2001 Dp1187 centromere: LTR retrotransposons (HMS Beagle, 412, and Bel), non- LTR (LINE-like) retroposon (F) and 359-bp satDNA Sun et al. 1997, 2003 satDNAs: MS3 [150 bp] in centromeric core, Major satDNA [234 bp] and MS4 [300 bp] in pericentromeric regions Guenatri et al. 2004; Kuznetsova et al. 2006 LTR-retrotransposon (Athila) in centromere core, multiple families of LTR retrotransposons and 5SrRNA in pericentromeric regions Nagaki et al. 2003; Kumekawa et al. 2000 satDNAs: TR2, TR3, and TR5 in pericentromeric regions Neumann et al. 2012 Different retrotransposon families belonging to Ty3/ gypsy–class Dong et al. 1998; Cheng et al. 2002 Retrotransposons CRM3 and CRM4 Zhong et al. 2002; Wolfgruber et al. Repetitive DNA sequences are the most common centromere components The phenomenon of rapid evolution of centromeric DNA and protein 315 Chromosoma (2014) 123:313–325 Table 1 Centromere DNA features in higher eukaryote model organisms Centromere type according to functional DNA sequence Species/common name Characteristics of functional DNA sequence(s) Satellite DNA Homo sapiens/human alpha-satDNA [171 bp] in all centromeres; chromosome- specific subfamilies; higher- order organization Drosophila melanogaster/fruit fly Dp1187 centromere: AATAT and AAGAG satDNAs Mus musculus/house mouse Minor satDNA [120 bp]: homogenous family in all centromeres Arabidopsis thaliana/ thale cress pAL1 satDNA [180 bp]: homogenous family in all centromeres Pisum sativum/pea 13 distinct satDNAs families [50–2,094 bp] localized in various combinations in different centromeres Satellite DNAs and retrotransposons Oryza sativa/rice CentO satDNA [155 bp] and CRR retrotransposon in all centromeres Zea mays/maize Retrotransposons CRM1 and CRM2 and CenC satDNA [156 bp] in all centromeres Retrotransposons Triticum spp./wheat CRW, Quinta and Weg retrotransposon families Repeats and non-repeats Equus caballus/horse satDNAs: different ECA families [221–475 bp], 37cen [221 bp], and 2PI [23 bp] localized in various combinations; repeat-free ch11 centromere Gallus gallus/chicken Chromosome specific satDNAs [1.8–3.2 kb] in centromeres of eight macrochromosomes, CNM satDNA [42 bp] in some microchromosomes and in ch6 and ch9; repeat-free ch5 ch27, chZ centromeres Solanum tuberosum/ potato Six chromosome specific satDNAs [979 bp to 5.4 kb]; repeat-free ch4, ch6, ch10, ch11, and ch12 centromeres ( ) Table 1 Centromere DNA features in higher eukaryote model organisms Centromere type according to functional DNA sequence Species/common name Characteristics of functional DNA sequence(s) Other abundant DNAs contributing to centromere region References Satellite DNA Homo sapiens/human alpha-satDNA [171 bp] in all centromeres; chromosome- specific subfamilies; higher- order organization Monomeric forms of alpha- satDNA, diverse non-alphoid satDNAs (gamma, beta, Sat I, II, III) and LINE elements in pericentromeric regions Willard and Waye 1987; Waye and Willard 1989; Rudd and Willard 2004; Rudd et al. 2006; Sullivan et al. 2011; Schueler et al. 2001 Drosophila melanogaster/fruit fly Dp1187 centromere: AATAT and AAGAG satDNAs Dp1187 centromere: LTR retrotransposons (HMS Beagle, 412, and Bel), non- LTR (LINE-like) retroposon (F) and 359-bp satDNA Sun et al. 1997, 2003 Mus musculus/house mouse Minor satDNA [120 bp]: homogenous family in all centromeres satDNAs: MS3 [150 bp] in centromeric core, Major satDNA [234 bp] and MS4 [300 bp] in pericentromeric regions Guenatri et al. 2004; Kuznetsova et al. Repetitive DNA sequences are the most common centromere components Two classes of highly abundant repetitive sequences, satellite DNAs (satDNAs) and transposable elements (TEs), represent major DNA components of many centromeric regions. Both groups of sequences are extremely divergent, and understand- ing the mechanisms of their accumulation, diversification, protein-binding capacity, and linear distribution is essential for a complete picture of centromere genomics, both from a structural and functional perspective. Characteristics of func- tional DNA sequences and other abundant DNAs contributing to centromere region of the most common model organisms of higher eukaryotes are presented in Table 1. SatDNAs are a class of diverse tandemly repeated DNA sequences that comprise long arrays localized in a tightly packed heterochromatin. Features of satDNA sequences in centromeric regions have already been reviewed in detail (Plohl et al. 2008, 2012). A recent comprehensive bioinfor- matic analysis of centromeric satDNAs in a number of animal and plant species confirmed the rapid evolution of DNA sequences in these areas (Melters et al. 2013). Despite the extreme diversity of satDNA sequences, some sequence seg- ments can be shared among heterologous repeats. The best known example is the conserved 17 bp long sequence motif, the CENP-B box, which is specific for alpha-satDNA in Nevertheless, it is difficult to understand the rapid evolu- tion of satDNAs in a centromere solely by sequence dynamics of tandem repeats, especially in the light of the centromere structure-function paradox (Eichler 1999). Repeat-based centromeres Another repetitive component of importance for centro- meric regions are transposable elements (TEs), DNA se- quences which can move to new genomic locations and form interspersed repeats if replicated in the process of movement (Kazazian 2004; Tollis and Boissinot 2012). According to the mechanisms of transposition, TEs are categorized as RNA- mediated (retroelements such as long terminal repeat (LTR) and non-LTR-retrotransposons) or DNA-mediated (DNA transposons). In addition to sequence segments coding for their own enzymes and thus being self-sufficient in the pro- cess of mobility, enzymes of autonomous elements can trail a large number of various non-autonomous copies. The majority of eukaryotes studied in terms of centromeric DNA have monocentric chromosomes with large regional centromeres. Functional centromeric domains of these chro- mosomes are usually inserted into blocks of pericentromeric heterochromatin, a compartment composed of Mb-sized ar- rays of satDNAs. Arrays are in general much longer than necessary for centromeric function. For instance, functional centromere domains in Drosophila comprise only of 15– 40 kb, which is comparable to the minimum length of 30– 70 kb of alpha-satDNA in a functional centromere of human artificial chromosomes (Okamoto et al. 2007). Among TEs, LTR-retrotransposons in particular accumu- late frequently in centromeres and pericentromeres of both plants and animals (e.g., Pimpinelli et al. 1995; Copenhaver et al. 1999; Schueler et al. 2001; Cheng et al. 2002). TEs belonging to the chromovirus clade of Ty3/gypsy LTR- retrotransposons are widely distributed in centromeres of an- giosperms. It has been proposed that they are targeted to centromeres by a specific motif located at the C-terminus of their integrase (Neumann et al. 2011). Molecular determinants that need to be recognized by this motif in order to trigger specific integration are probably sequence-independent het- erochromatin marks, although their exact nature has not yet been unambiguously identified (Neumann et al. 2011; Tsukahara et al. 2012). In addition to active transposition, centromere-specific retrotransposons can become significant- ly enriched in centromeric regions as a consequence of mul- tiple rounds of segmental duplication, a process which can also be responsible for massive amplifications of satDNA arrays (Ma and Jackson 2006). Details on the complexity of organizational patterns and contribution of particular sequence types to repeat-based cen- tromeres differ significantly among species (Fig. 1). Repetitive DNA sequences are the most common centromere components 2009 Different CRW retrotransposons families (Ty3/gypsy–class) Li et al. 2013 NA Piras et al. 2010; Alkan et al. 2011 , NA Shang et al. 2010 NA Gong et al. 2012 Monomeric forms of alpha- satDNA, diverse non-alphoid satDNAs (gamma, beta, Sat I, II, III) and LINE elements in pericentromeric regions Gong et al. 2012 Gong et al. 2012 DNA components. Centromeres are thus not defined only by epigenetic factors but also through interactions between repet- itive DNA and protein components, mediated by meiotic drive (Dawe and Henikoff 2006). In other words, rapid evolution of centromere satDNA sequences is possible only assuming coevolution with CENH3 and other DNA-binding proteins. DNA components. Centromeres are thus not defined only by epigenetic factors but also through interactions between repet- itive DNA and protein components, mediated by meiotic drive (Dawe and Henikoff 2006). In other words, rapid evolution of centromere satDNA sequences is possible only assuming coevolution with CENH3 and other DNA-binding proteins. components in spite of conserved centromere function has been referred to as the centromere paradox (Henikoff et al. 2001). In this regard, evolution of CENH3 is subject to positive selection in Drosophila (Malik and Henikoff 2001) and Arabidopsis (Talbert et al. 2002), and probably in general (Talbert et al. 2004) because of its interactions with changing 316 Chromosoma (2014) 123:313–325 (MITE) from the cupped oyster Crassostrea virginica re- semble satDNAs in several other mollusks (Gaffney et al. 2003). In plants, a hypervariable region of one LTR- retrotransposon was found expanded into tandem repeats of a satDNA in the pea (Pisum sativum) genome (Macas et al. 2009). Similarly, Zea mays centromeres became enriched in tandem repeats derived from LTRs and un- translated regions of two unrelated centromere-specific retrotransposons, what probably happened in two indepen- dent evolutionary events (Sharma et al. 2013). Because satDNAs are the major DNA components of heterochromatin, differences in their composition can be linked with reproductive isolation and speciation (Bachmann et al. 1989). Differences among individuals in the centromere region accumulate as a consequence of centromere drive, leading to reduced compatibility of homologous chromo- somes in hybrids and ultimately to postzygotic isolation, thus triggering speciation (Henikoff et al. 2001). The role of satDNA in reproductive isolation caused by rapid centromere evolution has been recently studied in detail in monkey- flowers (Fishman and Saunders 2008) and Drosophila (Ferree and Barbash 2009). Repeat-based centromeres For ex- ample, global sequence characterization of rice centromeric satDNA CentO by next generation high-throughput sequenc- ing and ChIP experiments with CENH3 could not reveal any particular differences between monomers included in the functional centromere and pericentromeric arrays (Macas et al. 2010). A comparable uniform distribution of nearly- identical repeats of species-specific highly-abundant satDNAs (up to 50 % of the genome) in centromeric and pericentromeric heterochromatin of all chromosomes can be anticipated in some beetle species of the order Coleoptera (Palomeque and Lorite 2008). It has been proposed that the lack of chromosome-specific satDNA variants (Fig. 1a) indi- cates high efficiency of sequence homogenization in the bou- quet stage of meiotic prophase, in which all chromosomes of the complement align together (Durajlija Žinić et al. 2000; Mravinac and Plohl 2010). In contrast, well-known examples of satDNAs localizing to pericentromeric and centromeric regions are the mouse major and minor satDNA, respectively, (Guenatri et al. 2004; Kuznetsova et al. 2006). Despite differences in the structure, organization, dy- namics, and mechanisms of spread, a growing number of reports link TEs and satDNAs. A whole unit or a segment of a TE can be amplified in tandem, although the direction of transition between the two types of repetitive sequences is not always clear (Macas et al. 2009). For example, a part of the mammalian retrotransposon L1 shares similarity with a segment of the satDNA repeat in whales (Kapitonov et al. 1998). Internal tandem repeats of non- autonomous miniature inverted repeat transposable element The distribution of centromeric satDNAs can also be chro- mosome specific (Fig. 1b). The best studied example is the complex organizational pattern of centromeric sequences in human chromosomes. Two basic types of alpha-satDNA, monomeric and higher-order repeat (HOR), characterize hu- man centromeric regions (Willard and Waye 1987; Rudd and 317 Chromosoma (2014) 123:313–325 Fig. 1 Schematic presentation of functional DNA sequences in different centromere types Fig. 1 Schematic presentation of functional DNA sequences in different centromere types Fig. 1 Schematic presentation of functional DNA sequences in different centromere types The diversity of DNA sequences localized in functional centromeres and/or pericentromeres has been evidenced not only in terms of different satDNAs and their organizational forms, but also in terms of other sequences’ contribution. Different interspersion patterns of tandemly repeated DNA and TEs are found in many species (Fig. 1c). Repeat-based centromeres The centro- meric fraction of human HORs is mostly devoid of inserted TEs or other sequences, while pericentromeres are frequent- ly interrupted by unrelated satDNAs (e.g., gamma-satellite and SatIII) and LINE elements (Schueler et al. 2001). Different plants such as maize, rice, and wheat turned out to be valuable models for studying the specificities of centromere DNA sequence organization, particularly be- cause of the presence of substantial portions of centromere-specific retrotransposons. Retrotransposons are extensively intermingled with satDNAs and both sequence types mark functional parts of some plant centromeres (Ma Willard 2004). All regular human centromeres are formed on tandemly repeated HOR units composed of 2 to over 20 diverged 171-bp-long monomers, and HORs are usually chro- mosome specific (Rudd et al. 2006). However, only a fraction of HOR arrays of human alpha-satDNA underlies active cen- tromeres, while the rest, flanked by monomeric repeats, con- tributes to pericentromeric heterochromatin (Spence et al. 2002; Lam et al. 2006; Mravinac et al. 2009; Sullivan et al. 2011). Comparably, in the domestic dog, CENP-A chromatin immunoprecipitation (ChIP) experiments suggested monomer sequence subtypes of two related satDNAs as functional cen- tromere sequences (Hayden and Willard 2012). Recent efforts combining genomic and ChIP-obtained data on human alpha- satDNA allowed the possibility for comprehensive functional mapping of centromeric areas and led to a model in which the centromere is defined by sequence features and context- dependent epigenetic interactions (Hayden et al. 2013). 318 Chromosoma (2014) 123:313–325 et al. 2007). For instance, functional rice centromeres are characterized by CentO satDNA and the centromere-specific retrotransposon CRR (Cheng et al. 2002). A recent study in the wild rice Oryza brachyantha showed that CentO satDNA repeats as well as CRR retrotransposons have completely disappeared and are replaced by a new functional centromeric CentF satDNA in a short evolutionary time (Lee et al. 2005). the functional centromere of chromosome 11 lacks any tan- dem repeats (Piras et al. 2010). The extended cytogenetic analysis of congeneric species revealed that donkey and two zebra species contain several pairs of chromosomes with satellite-less centromeres (Piras et al. 2010). The chicken genome with 10 pairs of macrochromosomes, 28 pairs of microchromosomes, and Z/W sex chromosomes represents the first avian karyotype with molecular cytogenetic charac- terization of each chromosome (Masabanda et al. 2004), and thus has been a powerful resource for studying the genetic makeup. Neocentromeres and evolutionary new centromeres (ENCs) Neocentromeres are fully functional centromeres that arise at ectopic DNA loci not previously associated with kinetochore proteins (Fig. 1f). In humans, the majority of neocentromeres evidenced in clinical phenotypes rescue acentric chromosome fragments in cells with severe chromosomal rearrangements (Marshall et al. 2008). As the neocentromeres described to date show notable divergence of underlying DNA sequences and chromosome positions, the sequence attributes that might be favorable to their formation have not yet been established. Most of them are located in gene-poor regions with no appar- ent association with heterochromatin (Alonso et al. 2010), and although some of them form on repetitive DNA (Hasson et al. 2011), none of them are associated with alpha-satellite DNA. In addition to human cells, neocentromere formation and function have also been studied in different model organisms such as D. melanogaster, Schizosaccharomyces pombe, Can- dida albicans, and several plant species (reviewed in Burrack and Berman 2012). Complex organization of centromeric regions is further supported by the presence of protein coding genes or gene candidates in centromeric chromatin of D. melanogaster (Smith et al. 2007), rice (Wu et al. 2004; Nagaki et al. 2004), and wheat (Li et al. 2013), although the insertions of this type were not observed in Arabidopsis (Hosouchi et al. 2002) and human (Schueler et al. 2001). Repeat-based centromeres Thorough identification of centromeric DNA showed that the majority of chicken centromeres are founded on chromosome-specific satDNA spanning several hundred kilobase of homogeneous repetitive arrays, while centromeres of chromosomes 5, 27, and Z, spanning only ~30 kb, are devoid of tandem repeats (Shang et al. 2010). The presence of the two distinct types of centromeres has also been evi- denced in plants. In the potato, Solanum tuberosum, no satel- lite repeats were discovered in centromeres of five pairs of chromosomes, whereas six potato centromeres harbor megabase-sized chromosome-specific satellite repeat arrays (Gong et al. 2012). Similar to chicken, centromeric satellites in potato share partial sequence similarity to different retrotransposon sequences (Gong et al. 2012). Detailed mapping of the repeat content and arrays of com- plete centromeres in some chromosomes of maize (Wolfgruber et al. 2009) and wheat (Li et al. 2013) revealed species-specific centromeric retrotransposons as predominant CENH3-associated DNA sequences (Fig. 1d). Maize centro- meres still contain small amounts of CentC satDNAs, detected as functional centromeric sequences in other maize inbreds (Kato et al. 2004; Wolfgruber et al. 2009) and related to the CentO satDNA in rice (Cheng et al. 2002). Similar replace- ments of functional centromeric satDNA with retrotransposons occurred in wheat, followed by consecutive introduction of new functional retrotransposons. All these replacements occurred in a very short evolutionary time, <0.5 MY (Li et al. 2013). In principle, older retrotransposons typically lie outside of the functional centromere (Wolfgruber et al. 2009; Li et al. 2013) and can be compared with the distribution of LINE and other TEs in pericentromeres of human chromosomes (Schueler et al. 2001). It has been hy- pothesized that retrotransposons may accumulate in active centromeres because of favored integration into an epigenet- ically modified centromere environment, and not because of preferred association with CENH3 nucleosomes (Lamb et al. 2007; Wolfgruber et al. 2009). Organisms with both repeat-based and repeat-free centromeres From the methodological standpoint, due to the abundance of satellite repeats in eukaryotic species, it is understandable that the literature to date mostly describes the cases of centromeric regions rich in repetitive sequences. However, the develop- ment of chromatin immunoprecipitation and usage of CENH3 variants as the most reliable markers of active centromeres enabled high-resolution DNA mapping of interacting se- quences. Consequently, there are an increasing number of reports documenting the organisms that possess both repeat- based and repeat-free centromeres (Fig. 1e). Horse Equus caballus centromeres are enriched for satellite sequences but Evolutionary new centromeres (ENCs), also known as repositioned centromeres, are centromeres that moved to a new position along a single chromosome without any observ- able chromosomal rearrangements or phenotypic conse- quences. Once repositioned, ENCs are transmitted through generations and become fixed in the population. Since they 319 Chromosoma (2014) 123:313–325 dicentric B chromosomes. Without changing the sequence of underlying DNA, one of the B chromosome centromeres becomes nonfunctional by histone CENH3 depletion (Han et al. 2006) and increasing methylation of the underlying DNA (Koo et al. 2011). A structural tricentric chromosome in wheat acts like a functional monocentric by keeping active the large centromere, while at the same time both of the small centromeres, enriched for heterochromatic histone modifica- tions H3K27me2 and H3K27me3, are inactivated (Zhang et al. 2010). Dicentric chromosomes in humans can be quite stable, and it has been known for two decades that some human dicentric chromosomes also stay functional dicentrics through multiple cell divisions (Sullivan and Willard 1998). Stimpson et al. (2010) recently showed that the human dicen- trics, being functionally monocentric, undergo centromere inactivation through different processes: (1) by epigenetic mechanisms or (2) by size reduction of the alpha-satDNA array associated with CENP-A. Human chromosome HSA17, characterized by the two alpha-satellite arrays D17Z1 and D17Z1-B, is an example of a regular human chromosome structurally arranged as a dicentric that behaves as a functional monocentric. Its functional centromere is pre- dominantly linked to the D17Z1 array (Maloney et al. 2012). However, in vitro and in vivo studies proved that the HSA17 functional centromere can also assemble at D17Z1-B, and its location is inherited through multigenerational families. The structural differences in the D17Z1 and D17Z1-B HOR arrays imply genomic factors that, together with epigenetic mecha- nisms, influence centromere specification in humans (Maloney et al. 2012). Organisms with both repeat-based and repeat-free centromeres In other words, the analyses of natural and engineered dicentric chromosomes indicate that epigenet- ic plasticity, but also subtle genetic features of centromere- competent DNA sequences, plays an important role in defin- ing centromere identity. can be identified exclusively by comparing the ancestral and derived position of a specific centromere, systematic karyo- type analyses of related organisms are crucial. So far, the best studied model group is primates and it has been proved that nine macaque chromosomes possess ENCs (Ventura et al. 2007), whilst six human centromeres are evolutionarily new (reviewed in Rocchi et al. 2012). ENCs have also been re- vealed in other mammals (e.g., Carbone et al. 2006; Rocchi et al. 2012), birds (Kasai et al. 2003), and plants (Han et al. 2009). Although they arise in anonymous sequences, ENCs gradually incorporate repetitive arrays. In macaque, all the nine ENCs over time accumulated large arrays of alpha- satDNA becoming indistinguishable from other macaque cen- tromeres. At the same time, the inactivated centromeres completely lost their satellite arrays (Ventura et al. 2007). Similarly, centromere repositioning in cucurbit species was accompanied by the gain of centromeric satDNA repeats in ENCs and the loss of pericentromeric heterochromatin in inactivated centromeres (Han et al. 2009). What can be learned from neocentromere and ENC phe- nomena is that a centromere potentially can be seeded in any unique sequence, albeit the repetitive DNA setup provides a preferred chromatin environment for centromere mainte- nance. The hypothesis that repeat-free centromeres represent a primordial form is in accordance with the occurrence of neocentromeres and their maturation into repeat-based centro- meres by the accumulation of satellites and retrotransposons (Kalitsis and Choo 2012). Dicentric chromosomes Each chromosome normally possesses a single centromere, though genome rearrangements can generate chromosomes with two centromeres (Fig. 1g). In general, dicentric chromo- somes are inherently very unstable because of anaphase bridge formation resulting in broken or rearranged chromo- somes. Nevertheless, in some cases, dicentric chromosomes are stabilized due to inactivation of one of the two centro- meres, which allows the structural dicentric to act as a func- tional monocentric during cell divisions. The exact mecha- nism of centromere inactivation has not been completely elucidated; however, studies of naturally occurring and engineered dicentrics in different organisms predominantly indicate epigenetic changes. In the fission yeast, S. pombe, 99 % of the cells harboring an artificial dicentric chromosome died, but in 70 % of the survivors, one of the centromeres was functionally silenced by the loss of Cnp1 (the yeast CENH3 homolog), depletion of euchromatic histone modifications H3K9ac and H3K14ac, and by becoming enriched for the heterochromatic H3K9me2 mark without associated alter- ations in the DNA sequence (Sato et al. 2012). Epigenetic centromere inactivation has also been documented in maize Holocentric centromeres sativum, which represents the first example of an intermediate between monocentric and holocentric centro- meres, demonstrates that all functional centromere domains in the pea are tightly associated with clusters of 13 distinct satDNA families and with one family of retrotransposons (Neumann et al. 2012). The pea centromeres have from three to five explicit CENH3-containing regions composed of dif- ferent families of satDNAs (Fig. 1i). CENH3 distribution during interphase and prophase. In both species, diffuse centromeres are distributed along each chro- matid except in the telomeric regions (Heckmann et al. 2011). Data on the DNA sequences underlying holocentric centro- meres are generally lacking. Nevertheless, a recent study of animal and plant species shows that the genomic content of tandem repeats in holocentric species differs greatly (Melters et al. 2013). The C. elegans genome contains only a few tandem repeats (Hillier et al. 2007). ChIP analysis shows that even ~50 % of this genome is associated with CENH3, but association loci are not correlated with repeat density (Gassmann et al. 2012). In contrast, comprehensive character- ization of holocentric Luzula elegans shows that 61 % of its genome is built of highly repetitive DNAs, including over 30 highly divergent satellite families, while 33 % of the genome comprises Ty1/copia LTR retrotransposons of the Angela clade (Heckmann et al. 2013). Although retrotransposons in L. elegans are uniformly distributed along the chromosomes, they are not centromere-associated. Similarly, different satDNAs are present as blocks preferentially accumulated on chromosome ends which are declared as non-centromeric regions. However, a portion of centromere domains in the related holocentric species Luzula nivea is composed of scattered clusters of satellite LCS1 which display significant similarity to the major centromeric satellite of monocentric chromosomes of some Oryza species (Haizel et al. 2005). These data suggest that satDNA can be an important centro- mere determinant in this holocentric species. In support of this, a study of novel meta-polycentric chromosomes in the pea P. sativum, which represents the first example of an intermediate between monocentric and holocentric centro- meres, demonstrates that all functional centromere domains in the pea are tightly associated with clusters of 13 distinct satDNA families and with one family of retrotransposons (Neumann et al. 2012). The pea centromeres have from three to five explicit CENH3-containing regions composed of dif- ferent families of satDNAs (Fig. 1i). Holocentric centromeres studies on higher eukaryotic species showed a link between the RNAi machinery and heterochromatin-mediated transcrip- tional silencing in plants (Zilberman et al. 2003), flies (Drosophila; Pal-Bhadra et al. 2004), worms (C. elegans; Grishok et al. 2000), and mammals (Fukagawa et al. 2004). However, the ultimate impact of RNAi on heterochromatin assembly and chromosome segregation is less straightforward suggesting different mechanisms of the RNAi pathway in complex genomes (Chan and Wong 2012). In hybrid chicken cells carrying a human chromosome, loss of Dicer led to defects in centromere heterochromatin and chromosome seg- regation, pointing out the importance of siRNA for hetero- chromatin assembly (Fukagawa et al. 2004). Similarly to chicken cells, Dicer deficiency in mouse embryonic stem (ES) cells caused accumulation of pericentric satellite tran- scripts, but there are still controversies related to the impact of the RNAi machinery on mammalian centromere assembly (Kanellopoulou et al. 2005; Murchison et al. 2005). Kanellopoulou et al. (2005) reported loss of DNA methylation and of histone H3 modification H3K9me3 at the pericentromeric regions of Dicer-deficient ES cells and sug- gested that Dicer participates in the maintenance of centro- meric heterochromatin structure. In contrast, Murchison et al. (2005) concluded that the RNAi pathway is not essential for the regulation of heterochromatin assembly in mouse ES cells because in their experimental system Dicer loss had no sig- nificant effect on cytosine methylation nor changed H3K9me3 status at the centromere. More recent work on S. pombe suggests that the observed defects may be indirectly related to exosome RNA machinery (a multiprotein complex capable of degrading various RNA types), which acts in parallel with RNAi and promotes heterochromatin formation (Reyes-Turcu et al. 2011). In addition, a great progress has also been made in deter- mining non-siRNAs transcripts in the centromere of higher eukaryotes. The data suggest transcriptional competence of the entire centromere (both the centromere core and the pericentromere) and heterogenous transcripts appear to be variable in size and structure (Gent and Dawe 2012). They can be transcribed from both strands or display strand-specific characteristics (Topp et al. 2004; May et al. 2005). Some of them are exclusively nuclear while the other form cytoplasmatic polyadenylated RNA (Vourc’h and Biamonti 2011). Increasingly, evidence suggests an impact of centro- meric transcripts on development, cell differentiation, and response to environmental stimuli. Holocentric centromeres In contrast to monocentric, holocentric chromosomes have a long kinetochore plate with spindle fibers attached along the entire chromosome length (Dernburg 2001) (Fig. 1h). Based on cytological studies, it has been shown that holocentric chromosomes are scattered among plant and animal kingdoms arising at least 13 independent times during evolution (Mola and Papeschi 2006). A more precise understanding of centro- meric function in holocentric species, based on immunodetection of CENH3 homologs, has been intensively analyzed only in the nematode, Caenorhabditis elegans, and a few other species. In spite of polyphyletic origin, immunodetection of the corresponding CENH3 proteins in mitotic chromosomes of C. elegans (Buchwitz et al. 1999) and the plant Luzula (Nagaki et al. 2005; Heckmann et al. 2011) shows common structural features in the form of dispersed 320 Chromosoma (2014) 123:313–325 CENH3 distribution during interphase and prophase. In both species, diffuse centromeres are distributed along each chro- matid except in the telomeric regions (Heckmann et al. 2011). Data on the DNA sequences underlying holocentric centro- meres are generally lacking. Nevertheless, a recent study of animal and plant species shows that the genomic content of tandem repeats in holocentric species differs greatly (Melters et al. 2013). The C. elegans genome contains only a few tandem repeats (Hillier et al. 2007). ChIP analysis shows that even ~50 % of this genome is associated with CENH3, but association loci are not correlated with repeat density (Gassmann et al. 2012). In contrast, comprehensive character- ization of holocentric Luzula elegans shows that 61 % of its genome is built of highly repetitive DNAs, including over 30 highly divergent satellite families, while 33 % of the genome comprises Ty1/copia LTR retrotransposons of the Angela clade (Heckmann et al. 2013). Although retrotransposons in L. elegans are uniformly distributed along the chromosomes, they are not centromere-associated. Similarly, different satDNAs are present as blocks preferentially accumulated on chromosome ends which are declared as non-centromeric regions. However, a portion of centromere domains in the related holocentric species Luzula nivea is composed of scattered clusters of satellite LCS1 which display significant similarity to the major centromeric satellite of monocentric chromosomes of some Oryza species (Haizel et al. 2005). These data suggest that satDNA can be an important centro- mere determinant in this holocentric species. In support of this, a study of novel meta-polycentric chromosomes in the pea P. Conclusions Although being essential for the proper distribution of genetic material in eukaryotic cells, the centromere still continues to intrigue in the complexity of its structure and rapid evolution of its building components. Advances in methodological ap- proaches and high-throughput analyses in the last two decades fostered the rapid accumulation of centromere-related datasets in different model organisms, giving access to information about DNA, RNA, proteins, and their epigenetic modifica- tions. However, the complex networks of interactions among them as well as the details of functional features and roles of particular components are still far from being well understood. Epigenetic determinants are recognized as major identifiers of centromeres in higher eukaryotes, while the functional contri- bution of DNA remains obscure and seriously questioned because of the ability of the centromere to be formed and to persist on extremely diverse sequences. Recent studies of genomic and functional datasets based on combined sequenc- ing data and established CENH3-associated DNA sequences revealed a more detailed insight into genomic architecture of centromeres. In spite of the diversity of DNA sequences, the preferred forms populating functional centromeres appear to be tandem repetitions of satDNAs and/or mobile elements. Only a subset of centromere-located DNA sequences or their variants is predominantly CENH3-associated, indicating the importance of their linear composition. An increasing number of reports that evidence organisms with dually organized centromeres (repeat-rich and repeat-free) opens up the possi- bility that the dynamics of centromere formation is much higher than previously thought, and also highlights stable functioning of centromeres established on different sequence types within a single organism. It can be hypothesized that the repetitive DNA environment has the potential to preserve stability of the functional centromere, and at the same time, to provide a reservoir of new functional sequences. This creates a platform which allows rapid changes in centromere identity and as a consequence can directly stimulate reproduc- tive isolation. Several reasons for this continuous rapid change can be considered, such as specificities of evolution of satDNAs, targeted integration of TEs into the epigenetically marked centromeric environment, and coevolution of DNA In addition to the analysis of pericentromeric regions, an ever-growing number of studies on the centromere core do- main demonstrates the transcription of repetitive sequences from this region and suggests a contribution of these tran- scripts to centromere/kinetochore assembly and maintenance (Gent and Dawe 2012). Transcription of centromeric sequences So, it has been proposed that the differential transcription of human pericentromeric satellite III in response to heat-shock stress might be a consequence of inhibition or saturation of the RNAi machinery in the pericentromeric region (Jolly et al. 2004). BRCA1-deficient tumor cells show defective pericentromeric heterochromatin formation which leads to the disruption of gene silencing and activation of the pericentromeric alpha-satDNA transcription (Zhu et al. 2011). Derepression of satDNA transcription has also been detected in many human epithelial tumors, but it is not clear whether satDNA transcription causes or is a consequence of genomic instability and tumorigenesis (Ting et al. 2011). a critical epigenetic determinant of the neocentromere. A novel class of small RNAs encompassing contiguous satellites and retroviruses located at the centromere core and likely produced through the activity of retroviral LTR promoters was discovered in a marsupial (Carone et al. 2009). In-depth analysis discovered that hypermorphic expression of these retroelement-encoded small RNAs is critical for the mainte- nance and assembly of CENP-A in the marsupial centromere (Carone et al. 2013). a critical epigenetic determinant of the neocentromere. A novel class of small RNAs encompassing contiguous satellites and retroviruses located at the centromere core and likely produced through the activity of retroviral LTR promoters was discovered in a marsupial (Carone et al. 2009). In-depth analysis discovered that hypermorphic expression of these retroelement-encoded small RNAs is critical for the mainte- nance and assembly of CENP-A in the marsupial centromere (Carone et al. 2013). Transcription of centromeric sequences The non-coding nature of repetitive sequences in centromeres and pericentromeres led to the opinion that centromeres are transcriptionally inactive. However, new evidences show that small-interfering RNAs (siRNAs) transcribed from pericentromeric tandem repeats in S. pombe modify the het- erochromatin. In brief, transcription of pericentromeric se- quences in the form of double stranded RNAs and their processing into siRNAs by the ribonuclease Dicer proved to be crucial in heterochromatin assembly and transcriptional silencing (Volpe et al. 2002). Impairment of the RNA inter- ference (RNAi) pathway resulted in severe chromosome seg- regation defects in S. pombe (Hall et al. 2003). Subsequent Pericentromeric major satDNA in mice is highly tran- scribed during embryogenesis, and transcripts are responsible for reorganization of pericentromeric satDNA into chromo- centers. Disruption of these transcripts led to developmental arrest indicating their role in de novo heterochromatin forma- tion and proper developmental progression (Probst et al. 2010). In humans, polyadenylated RNA transcripts from the Chromosoma (2014) 123:313–325 321 pericentromeric region of the Y chromosome are involved in trans-splicing in the CDC2L2 kinase mRNA generating a testis-specific isoform (Jehan et al. 2007). This example illus- trates specific regulation of euchromatic gene expression by pericentromeric transcripts and provides a link between satDNA transcription and cell differentiation. The overexpres- sion of centromeric RNA transcripts may be the result of derepression of heterochromatic regions under disease or stress conditions. So, it has been proposed that the differential transcription of human pericentromeric satellite III in response to heat-shock stress might be a consequence of inhibition or saturation of the RNAi machinery in the pericentromeric region (Jolly et al. 2004). BRCA1-deficient tumor cells show defective pericentromeric heterochromatin formation which leads to the disruption of gene silencing and activation of the pericentromeric alpha-satDNA transcription (Zhu et al. 2011). Derepression of satDNA transcription has also been detected in many human epithelial tumors, but it is not clear whether satDNA transcription causes or is a consequence of genomic instability and tumorigenesis (Ting et al. 2011). pericentromeric region of the Y chromosome are involved in trans-splicing in the CDC2L2 kinase mRNA generating a testis-specific isoform (Jehan et al. 2007). This example illus- trates specific regulation of euchromatic gene expression by pericentromeric transcripts and provides a link between satDNA transcription and cell differentiation. The overexpres- sion of centromeric RNA transcripts may be the result of derepression of heterochromatic regions under disease or stress conditions. Conclusions The single-stranded centromeric alpha-satellite RNA and the centromere protein CENP-C as- sociate and facilitate nucleoprotein assembly (CENP-C, innercentromere protein INCENP, and INCENP-interacting protein survivin) at the human mitotic centromere (Wong et al. 2007). Inhibition of RNA polymerase II activity, which results in depletion of alpha-satellite RNA in mitotic human cells, reduces CENP-C binding at the kinetochore and leads to chromosome missegregation (Chan et al. 2012). Similarly, Minor satDNA transcripts from the mouse centromere are integral components of the CENP-A chromatin fraction and associate with proteins of the chromosomal passenger com- plex Aurora B, survivin, and INCENP. In addition to a role in mediating interactions between protein components in the centromere/kinetochore complex, it has also been evidenced that Minor satellite RNA controls the enzymatic function of the Aurora A kinase (Ferri et al. 2009). In addition to centro- meric satDNA transcripts, transcripts derived from retrotransposons were also shown to be essential components of the centromere core. For example, in maize, single-stranded non-siRNAs (40–200 nt) transcribed from centromeric CentC satDNA and CRM retrotransposon are tightly bound to CENH3 (Topp et al. 2004). Similarly, RNA transcripts of the LINE-1 retrotransposon were found to bind CENP-A chro- matin in Mardel (10) 10q25 neocentromere (Chueh et al. 2009). RNAi-mediated knockdown of the LINE transcripts led to a significant reduction in the mitotic stability of the neocentromere suggesting that retrotransposable elements are 322 Chromosoma (2014) 123:313–325 Cheng Z, Dong F, Langdon T, Ouyang S, Buell CR, Gu M, Blattner FR, Jiang J (2002) Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon. Plant Cell 14: 1691–1704 sequences and CENH3 proteins. The complexity of the DNA sequence and functional relationships in centromeres becomes even more perplexing as a growing number of recent reports indicate roles for centromere DNA transcripts in centromere structure and function. Recent efforts have begun to decipher the rules in sequential patterns of centromeric DNA sequences and their functional interactions in different centromere types which will ultimately lead to a novel integrated view on the centromere genomics. Chueh AC, Northrop EL, Brettingham-Moore KH, Choo KH, Wong LH (2009) LINE retrotransposon RNA is an essential structural and functional epigenetic component of a core neocentromeric chroma- tin. References Eichler EE (1999) Repetitive conundrums of centromere structure and function. Hum Mol Genet 8:151–155 Alkan C, Cardone MF, Catacchio CR et al (2011) Genome-wide charac- terization of centromeric satellites from multiple mammalian ge- nomes. Genome Res 21:137–145 Ferree PM, Barbash DA (2009) Species-specific heterochromatin pre- vents mitotic chromosome segregation to cause hybrid lethality in Drosophila. PLoS Biol 7:e1000234 Alonso A, Hasson D, Cheung F, Warburton PE (2010) A paucity of heterochromatin at functional human neocentromeres. Epigenetics Chromatin 8:6 Ferri F, Bouzinba-Segard H, Velasco G, Hubé F, Francastel C (2009) Non-coding murine centromeric transcripts associate with and po- tentiate Aurora B kinase. Nucleic Acids Res 37:5071–5080 Bachmann L, Raab M, Sperlich D (1989) Satellite DNA and speciation— a species-specific satellite DNA of Drosophila guanche. J Zool Syst Evol Res 27:84–93 Fishman L, Saunders A (2008) Centromere-associated female meiotic drive entails male fitness costs in monkeyflowers. Science 322:1559–1562 Black BE, Bassett EA (2008) The histone variant CENP-A and centro- mere specification. Curr Opin Cell Biol 20:91–100 Fry K, Salser W (1977) Nucleotide sequences of HS- alpha satellite DNA from kangaroo rat Dipodomys ordii and characterization of similar sequences in other rodents. Cell 12:1069–1084 Blower MD, Sullivan BA, Karpen GH (2002) Conserved organization of centromeric chromatin in flies and humans. Dev Cell 2:319–330 Fukagawa T, Nogami M, Yoshikawa M, Ikeno M, Okazaki T, Takami Y, Nakayama T, Oshimura M (2004) Dicer is essential for formation of the heterochromatin structure in vertebrate cells. Nat Cell Biol 6:784–791 Buchwitz BJ, Ahmad K, Moore LL, Roth MB, Henikoff S (1999) A histone-H3-like protein in C. elegans. Nature 401:547–548 Gaffney PM, Pierce JC, Mackinley AG, Titchen DA, Glenn WK (2003) Pearl, a novel family of putative transposable elements in bivalve mollusks. J Mol Evol 56:308–316 Burrack LS, Berman J (2012) Neocentromeres and epigenetically inherited features of centromeres. Chromosome Res 20:607–619 Canapa A, Barucca M, Cerioni PN, Olmo E (2000) A satellite DNA containing CENP-B box-like motifs is present in the antarctic scal- lop Adamussium colbecki. Gene 247:175–180 Gassmann R, Rechtsteiner A, Yuen KW et al (2012) An inverse relation- ship to germline transcription defines centromeric chromatin in C. elegans. Nature 484:534–537 Carbone L, Nergadze SG, Magnani E et al (2006) Evolutionary move- ment of centromeres in horse, donkey, and zebra. Genomics 87:777– 782 Gent JI, Dawe RK (2012) RNA as a structural and regulatory component of the centromere. Conclusions PLoS Genet 5:e1000354 Copenhaver GP, Nickel K, Kuromori T, Benito MI, Kaul S, Lin X, Bevan M, Murphy G, Harris B, Parnell LD, McCombie WR, Martienssen RA, Marra M, Preuss D (1999) Genetic definition and sequence analysis of Arabidopsis centromeres. Science 286:2468–2474 nalysis of Arabidopsis centromeres. Science 286:2468–2474 Dawe RK, Henikoff S (2006) Centromeres put epigenetics in the driver's seat. Trends Biochem Sci 31:662–669 Acknowledgments We thank Mary Sopta for critical reading of the manuscript. This work was supported by Research Fund of Republic of Croatia, project no. 098-0982913-2756 and by funds received from the European Union’s Seventh Framework Programme for research, techno- logical development and demonstration under grant agreement No 316289 - InnoMol, FP7-REGPOT-2012-2013-1. De la Herran R, Fontana F, Lanfredi M, Congiu L, Leis M, Rossi R, Ruiz Rejón C, Ruiz Rejón M, Garrido-Ramos MA (2001) Slow rates of evolution and sequence homogenization in an ancient satellite DNA family of sturgeons. Mol Biol Evol 18:432–436 Dernburg AF (2001) Here, there, and everywhere: kinetochore function on holocentric chromosomes. J Cell Biol 153:F33–F38 Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Dong F, Miller JT, Jackson SA, Wang G-L, Ronald PC, Jiang J (1998) Rice (Oryza sativa) centromeric regions consist of complex DNA. Proc Natl Acad Sci U S A 95:8135–8140 Dover GA (1986) Molecular drive in multigene families: how biological novelties arise, spread and are assimilated. Trends Genet 2:159–165 Ž Durajlija-Žinić S, Ugarković Đ, Cornudella L, Plohl M (2000) A novel interspersed type of organization of satellite DNAs in Tribolium madens heterochromatin. Chromosome Res 8:201–212 References Science 293:1098–1102 Ma J, Wing RA, Bennetzen JL, Jackson SA (2007) Plant centromere organization: a dynamic structure with conserved functions. Trends Genet 23:134–139 Hillier LW, Miller RD, Baird SE, Chinwalla A, Fulton LA, Koboldt DC, Waterston RH (2007) Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny. PLoS Biol 5:e167 Macas J, Koblízková A, Navrátilová A, Neumann P (2009) Hypervariable 3' UTR region of plant LTR-retrotransposons as a source of novel satellite repeats. Gene 448:198–206 Horvath JE, Gulden CL, Vallente RU, Eichler MY, Ventura M, McPherson JD, Graves TA, Wilson RK, Schwartz S, Rocchi M, Eichler EE (2005) Punctuated duplication seeding events during the evolution of human chromosome 2p11. Genome Res 15:914–927 Macas J, Neumann P, Novák P, Jiang J (2010) Global sequence charac- terization of rice centromeric satellite based on oligomer frequency analysis in large-scale sequencing data. Bioinformatics 26:2101– 2108 Hosouchi T, Kumekawa N, Tsuruoka H, Kotani H (2002) Physical map- based sizes of the centromeric regions of Arabidopsis thaliana chromosomes 1, 2, and 3. DNA Res 9:117–121 Mahtani MM, Willard HF (1998) Physical and genetic mapping of the human X chromosome centromere: repression of recombination. Genome Res 8:100–110 Hyman AA, Sorger PK (1995) Structure and function of kinetochores in budding yeast. Annu Rev Cell Dev Biol 11:471–495 Malik HS, Henikoff S (2001) Adaptive evolution of Cid, a centromere- specific histone in Drosophila. Genetics 157:1293–1298 Jehan Z, Vallinayagam S, Tiwari S, Pradhan S, Singh L, Sure Jehan Z, Vallinayagam S, Tiwari S, Pradhan S, Singh L, Suresh A, Reddy HM, Ahuja YR, Jesudasan RA (2007) Novel noncoding RNA from human Y distal heterochromatic block (Yq12) generates testis- specific chimeric CDC2L2. Genome Res 17:433–440 specific histone in Drosophila. Genetics 157:1293–1298 Malik HS, Henikoff S (2009) Major evolutionary transitions in centro- mere complexity. Cell 138:1067–1082 Jolly C, Metz A, Govin J, Vigneron M, Turner BM, Khochbin S, Vourc'h C (2004) Stress-induced transcription of satellite III repeats. J Cell Biol 164:25–33 Maloney KA, Sullivan LL, Matheny JE, Strome ED, Merrett SL, Ferris A, Sullivan BA (2012) Functional epialleles at an endogenous human centromere. Proc Natl Acad Sci U S A 109:13704–13709 Kalitsis P, Choo KH (2012) The evolutionary life cycle of the resilient centromere. Chromosoma 121:327–340 Marshall OJ, Chueh AC, Wong LH, Choo KH (2008) Neocentromeres: new insights into centromere structure, disease development, and karyotype evolution. References Proc Natl Acad Sci U S A 106:14937–14941 Kumekawa N, Hosuchi T, Tsuruoka H, Kotani H (2000) The size and sequence organization of the centromeric region of Arabidopsis thaliana chromosome 5. DNA Res 7:315–321 Kuznetsova I, Podgornaya O, Ferguson-Smith MA (2006) High- resolution organization of mouse centromeric and pericentromeric DNA. Cytogenet Genome Res 112:248–255 Hasson D, Alonso A, Cheung F, Tepperberg JH, Papenhausen PR, Engelen JJ, Warburton PE (2011) Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres. Chromosoma 120:621–632 Lam AL, Boivin CD, Bonney CF, Rudd MK, Sullivan BA (2006) Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA. Proc Natl Acad Sci U S A 103: 4186–4191 Hayden KE, Willard HF (2012) Composition and organization of active centromere sequences in complex genomes. BMC Genomics 13:324 Hayden KE, Strome ED, Merrett SL, Lee HR, Rudd MK, Willard HF (2013) Sequences associated with centromere competency in the human genome. Mol Cell Biol 33:763–772 Lamb JC, Meyer JM, Birchler JA (2007) A hemicentric inversion in the maize line knobless Tama flint created two sites of centromeric elements and moved the kinetochore-forming region. Chromosoma 116:237–247 Heckmann S, Schroeder-Reiter E, Kumke K, Ma L, Nagaki K, Murata M, Wanner G, Houben A (2011) Holocentric chromosomes of Luzula elegans are characterized by a longitudinal centromere groove, chromosome bending, and a terminal nucleolus organizer region. Cytogenet Genome Res 134:220–222 Lee HR, Zhang W, Langdon T, Jin W, Yan H, Cheng Z, Jiang J (2005) Chromatin immunoprecipitation cloning reveals rapid evolutionary patterns of centromeric DNA in Oryza species. Proc Natl Acad Sci U S A 102:11793–11798 Li B, Choulet F, Heng Y, Hao W, Paux E, Liu Z, Yue W, Jin W, Feuillet C, Zhang X (2013) Wheat centromeric retrotransposons: the new ones take a major role in centromeric structure. Plant J 73:952–965 Heckmann S, Macas J, Kumke K, Fuchs J, Schubert V, Ma L, Novák P, Neumann P, Taudien S, Platzer M, Houben A (2013) The holocentric species Luzula elegans shows interplay between centro- mere and large-scale genome organization. Plant J 73:555–565 Ma J, Jackson SA (2006) Retrotransposon accumulation and satellite amplification mediated by segmental duplication facilitate centro- mere expansion in rice. Genome Res 16:251–259 Henikoff S, Ahmad K, Malik HS (2001) The centromere paradox: Stable inheritance with rapidly evolving DNA. References Annu Rev Genet 46:443–453 Gindullis F, Desel C, Galasso I, Schmidt T (2001) The large-scale organization of the centromeric region in Beta species. Genome Res 11:253–265 Carone DM, Longo MS, Ferreri GC, Hall L, Harris M, Shook N, Bulazel KV, Carone BR, Obergfell C, O'Neill MJ et al (2009) A new class of retroviral and satellite encoded small RNAs emanates from mam- malian centromeres. Chromosoma 118:113–125 Gong Z, Wu Y, Koblízková A et al (2012) Repeatless and repeat-based centromeres in potato: implications for centromere evolution. Plant Cell 24:3559–3574 Carone DM, Zhang C, Hall LE, Obergfell C, Carone BR, O’Neill MJ, O’Neill RJ (2013) Hypermorphic expression of centromeric retroelement-encoded small RNAs impairs CENP-A loading. Chromosome Res 21:49–62 Grishok A, Tabara H, Mello CC (2000) Genetic requirements for inher- itance of RNAi in C. elegans. Science 287:2494–2497 Guenatri M, Bailly D, Maison C, Almouzni G (2004) Mouse centric and pericentric satellite repeats form distinct functional heterochromatin. J Cell Biol 166:493–505 Chan FL, Wong LH (2012) Transcription in the maintenance of centro- mere chromatin identity. Nucleic Acids Res 40:11178–11188 Chan FL, Marshall OJ, Saffery R, Kim BW, Earle E, Choo KH, Wong LH (2012) Active transcription and essential role of RNA polymerase II at the centromere during mitosis. Proc Natl Acad Sci U S A 109: 1979–1984 Haizel T, Lim YK, Leitch AR, Moore G (2005) Molecular analysis of holocentric centromeres of Luzula species. Cytogenet Genome Res 109:134–143 Chromosoma (2014) 123:313–325 323 Kazazian HH Jr (2004) Mobile elements: Drivers of genome evolution. Science 303:1626–1632 Hall IM, Noma K, Grewal SI (2003) RNA interference machinery regu- lates chromosome dynamics during mitosis and meiosis in fission yeast. Proc Natl Acad Sci U S A 100:193–198 Kipling D, Warburton PE (1997) Centromeres, CENP-B and Tigger too. Trends Genet 13:141–145 Han F, Lamb JC, Birchler JA (2006) High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize. Proc Natl Acad Sci U S A 103:3238–3243 Koo DH, Han F, Birchler JA, Jiang J (2011) Distinct DNA methylation patterns associated with active and inactive centromeres of the maize B chromosome. Genome Res 21:908–914 Han Y, Zhang Z, Liu C, Liu J, Huang S, Jiang J, Jin W (2009) Centromere repositioning in cucurbit species: implication of the genomic impact from centromere activation and inactivation. References Plant Cell 17:1886–1893 Smith GP (1976) Evolution of repeated DNA sequences by unequal crossover. Science 191:528–535 Neumann P, Navrátilová A, Koblížková A, Kejnovský E, Hřibová E, Hobza R, Widmer A, Doležel J, Macas J (2011) Plant centromeric retrotransposons: a structural and cytogenetic perspective. Mob DNA 2:4 Smith CD, Shu S, Mungall CJ, Karpen GH (2007) The Release 5.1 annotation of Drosophila melanogaster heterochromatin. Science 316:1586–1591 Spence JM, Critcher R, Ebersole TA, Valdivia MM, Earnshaw WC, Fukagawa T, Farr CJ (2002) Co-localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X alpha-satellite array. EMBO J 21:5269–5280 Neumann P, Navrátilová A, Schroeder-Reiter E, Koblížková A, Steinbauerová V, Chocholová E, Novák P, Wanner G, Macas J (2012) Stretching the rules: monocentric chromosomes with multi- ple centromere domains. PLoS Genet 8:e1002777 ple centromere domains. PLoS Genet 8:e1002777 Stimpson KM, Song IY, Jauch A, Holtgreve-Grez H, Hayden KE, Bridger JM, Sullivan BA (2010) Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes. PLoS Genet 6:e1001061 Ohta T, Dover GA (1984) The cohesive population genetics of molecular drive. Genetics 108:501–521 Ohzeki J, Nakano M, Okada T, Masumoto H (2002) CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA. J Cell Biol 159:765–775 Strachan T, Webb D, Dover GA (1985) Transition stages of molecular drive in multiple-copy DNA families in Drosophila. EMBO J 4: 1701–1708 alphoid DNA. J Cell Biol 159:765–775 Okamoto Y, Nakano M, Ohzeki J, Larionov V, Masumoto H (2007) A minimal CENP-A core is required for nucleation and maintenance of a functional human centromere. EMBO J 26:1279–1291 Sullivan BA, Willard HF (1998) Stable dicentric X chromosomes with two functional centromeres. Nat Genet 20:227–228 Pal-Bhadra M, Leibovitch BA, Gandhi SG, Rao M, Bhadra U, Birchler JA, Elgin SC (2004) Heterochromatic silencing and HP1 localiza- tion in Drosophila are dependent on the RNAi machinery. Science 303:669–672 Sullivan BA, Karpen GH (2004) Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchro- matin and heterochromatin. Nat Struct Mol Biol 11:1076– 1083 Palomeque T, Lorite P (2008) Satellite DNA in insects: a review. Heredity 100:564–573 Sullivan LL, Boivin CD, Mravinac B, Song IY, Sullivan BA (2011) Genomic size of CENP-A domain is proportional to total alpha satellite array size at human centromeres and expands in cancer cells. References Am J Hum Genet 82:261–282 Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, Jenuwein T, Livingston DM, Rajewsky K (2005) Dicer-deficient mouse em- bryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev 19:489–501 Masabanda JS, Burt DW, O'Brien PC et al (2004) Molecular cytogenetic definition of the chicken genome: the first complete avian karyo- type. Genetics 166:1367–1373 g Kapitonov VV, Holmquist GP, Jurka J (1998) L1 repeat is a basic unit of heterochromatin satellites in cetaceans. Mol Biol Evol 15:611–612 Masumoto H, Nakano M, Ohzeki J (2004) The role of CENP-B and alpha-satellite DNA: de novo assembly and epigenetic maintenance of human centromeres. Chromosome Res 12:543–556 Kasai F, Garcia C, Arruga MV, Ferguson-Smith MA (2003) Chromosome homology between chicken (Gallus gallus domesticus) and the red- legged partridge (Alectoris rufa); evidence of the occurrence of a neocentromere during evolution. Cytogenet Genome Res 102:326–330 May BP, Lippman ZB, Fang Y, Spector DL, Martienssen RA (2005) Differential regulation of strand-specific transcripts from Arabidopsis centromeric satellite repeats. PLoS Genet 1:e79 Kato A, Lamb JC, Birchler JA (2004) Chromosome painting using repetitive DNA sequences as probes for somatic chromosome iden- tification in maize. Proc Natl Acad Sci U S A 101:13554–13559 Melters DP, Bradnam KR, Young HA et al (2013) Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution. Genome Biol 14:R10 324 Chromosoma (2014) 123:313–325 Meštrović N, Plohl M, Mravinac B, Ugarković Đ (1998) Evolution of satellite DNAs from the genus Palorus—Experimental evidence for the "library" hypothesis. Mol Biol Evol 15:1062–1068 Plohl M, Meštrović N, Mravinac B (2012) Satellite DNA evolution. In: Garrido-Ramos MA (ed) Repetitive DNA, Genome dynamics, 7th edn. Karger, Basel, pp 126–152 Pluta AF, Mackay AM, Ainsztein AM, Goldberg IG, Earnshaw WC (1995) The centromere: hub of chromosomal activities. Science 270:1591–1594 Meštrović N, Pavlek M, Car A, Castagnone-Sereno P, Abad P, Plohl M (2013) Conserved DNA motifs, including the CENP-B box-like, are possible promoters of satellite DNA array rearrangements in nema- todes. PLoS One 8:e6732 Probst AV, Okamoto I, Casanova M, El Marjou F, Le Baccon P, Almouzni G (2010) A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development. Dev Cell 19:625–638 Mola LM, Papeschi AG (2006) Holokinetic chromosomes at a glance. References BAG J Basic Appl Genet 17:17–33 Mravinac B, Plohl M (2010) Parallelism in evolution of highly repetitive DNAs in sibling species. Mol Biol Evol 27:1857–1867 Reyes-Turcu FE, Zhang K, Zofall M, Chen E, Grewal SIS (2011) Defects in RNA quality control factors reveal RNAi-independent nucleation of heterochromatin. Nat Struct Mol Biol 18:1132–1138 Mravinac B, Plohl M, Meštrović N, Ugarković Đ (2002) Sequence of PRAT satellite DNA "frozen" in some Coleopteran species. J Mol Evol 54:774–783 Rocchi M, Archidiacono N, Schempp W, Capozzi O, Stanyon R (2012) Centromere repositioning in mammals. Heredity 108:59–67 Mravinac B, Ugarković Đ, Franjević D, Plohl M (2005) Long inversely oriented subunits form a complex monomer of Tribolium brevicornis satellite DNA. J Mol Evol 60:513–525 Rudd MK, Willard H (2004) Analysis of the centromeric regions of the human genome assembly. Trends Genet 20:529–533 Rudd MK, Wray GA, Willard HF (2006) The evolutionary dynamics of alpha-satellite. Genome Res 16:88–96 Mravinac B, Sullivan LL, Reeves JW, Yan CM, Kopf KS, Farr CJ, Schueler MG, Sullivan BA (2009) Histone modifications within the human X centromere region. PLoS One 4:e6602 Sato H, Masuda F, Takayama Y, Takahashi K, Saitoh S (2012) Epigenetic inactivation and subsequent heterochromatinization of a centromere stabilize dicentric chromosomes. Curr Biol 22:658–667 Murchison EP, Partridge JF, Tam OH, Cheloufi S, Hannon GJ (2005) Characterization of Dicer-deficient murine embryonic stem cells. Proc Natl Acad Sci U S A 102:12135–12140 Schueler MG, Higgins AW, Rudd MK, Gustashaw K, Willard HF (2001) Genomic and genetic definition of a functional human centromere. Science 294:109–115 Nagaki K, Talbert PB, Zhong CX, Dawe RK, Henikoff S, Jiang J (2003) Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres. Genetics 163:1221–1225 Shang WH, Hori T, Toyoda A, Kato J, Popendorf K, Sakakibara Y, Fujiyama A, Fukagawa T (2010) Chickens possess centromeres with both extended tandem repeats and short non-tandem- repetitive sequences. Genome Res 20:1219–1228 Nagaki K, Cheng Z, Ouyang S, Talbert PB, Kim M, Jones KM, Henikoff S, Buell CR, Jiang J (2004) Sequencing of a rice centromere un- covers active genes. Nat Genet 36:138–145 Sharma A, Wolfgruber TK, Presting GG (2013) Tandem repeats derived from centromeric retrotransposons. BMC Genomics 14:142 Nagaki K, Kashihara K, Murata M (2005) Visualization of diffuse cen- tromeres with centromere-specific histone H3 in the holocentric plant Luzula nivea. References Chromosome Res 19:457–470 Pimpinelli S, Berloco M, Fanti L, Dimitri P, Bonaccorsi S, Marchetti E, Caizzi R, Caggese C, Gatti M (1995) Transposable elements are stable structural components of Drosophila melanogaster hetero- chromatin. Proc Natl Acad Sci U S A 92:3804–3808 Sun X, Wahlstrom J, Karpen G (1997) Molecular structure of a functional Drosophila centromere. Cell 91:1007–1019 p Sun X, Le HD, Wahlstrom JM, Karpen GH (2003) Sequence analysis of a functional Drosophila centromere. Genome Res 13:182–194 Piras FM, Nergadze SG, Magnani E, Bertoni L, Attolini C, Khoriauli L, Raimondi E, Giulotto E (2010) Uncoupling of satellite DNA and centromeric function in the genus Equus. PLoS Genet 6:e1000845 Talbert PB, Henikoff S (2010) Centromeres convert but don’t cross. PLoS Biol 8:e1000326 Plohl M, Luchetti A, Meštrović N, Mantovani B (2008) Satellite DNAs between selfishness and functionality: structure, genomics and evo- lution of tandem repeats in centromeric (hetero)chromatin. Gene 409:72–82 Talbert PB, Masuelli R, Tyagi AP, Comai L, Henikoff S (2002) Centromeric localization and adaptive evolution of an Arabidopsis histone H3 variant. Plant Cell 14:1053–1066 Chromosoma (2014) 123:313–325 325 repetitive tandem DNA. Proc Natl Acad Sci U S A 86: 6250–6254 Talbert PB, Bryson TD, Henikoff S (2004) Adaptive evolution of cen- tromere proteins in plants and animals. J Biol 3:18 Willard HF, Waye JS (1987) Chromosome-specific subsets of human alpha satellite DNA: analysis of sequence divergence within and between chromosomal subsets and evidence for an ancestral pentameric repeat. J Mol Evol 25:207–214 Thompson SL, Bakhoum SF, Compton DA (2010) Mechanisms of chro- mosomal instability. Curr Biol 20:R285–95 Ting DT, Lipson D, Paul S et al (2011) Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers. Science 331:593–596 Wolfgruber TK, Sharma A, Schneider KL et al (2009) Maize centromere structure and evolution: sequence analysis of centromeres 2 and 5 reveals dynamic Loci shaped primarily by retrotransposons. PLoS Genet 5:e1000743 Tollis M, Boissinot S (2012) The evolutionary dynamics of transposable elements in eukaryote genomes. In: Garrido-Ramos MA (ed) Repetitive DNA, Genome dynamics, 7th edn. Karger, Basel, pp 68–91 Wong LH, Brettingham-Moore KH, Chan L et al (2007) Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere. Genome Res 17:1146–1160 Topp CN, Zhong CX, Dawe RK (2004) Centromere-encoded RNAs are integral components of the maize kinetochore. References Proc Natl Acad Sci U S A 101:15986–15991 Wu J, Yamagata H, Hayashi-Tsugane M et al (2004) Composition and structure of the centromeric region of rice chromosome 8. Plant Cell 16:967–976 Tsukahara S, Kawabe A, Kobayashi A, Ito T, Aizu T, Shin-i T, Toyoda A, Fujiyama A, Tarutani Y, Kakutani T (2012) Centromere-targeted de novo integrations of an LTR retrotransposon of Arabidopsis lyrata. Genes Dev 26:705–713 Zhang W, Friebe B, Gill BS, Jiang J (2010) Centromere inactivation and epigenetic modifications of a plant chromosome with three func- tional centromeres. Chromosoma 119:553–563 Ventura M, Antonacci F, Cardone MF, Stanyon R, D'Addabbo P, Cellamare A, Sprague LJ, Eichler EE, Archidiacono N, Rocchi M (2007) Evolutionary formation of new centromeres in macaque. Science 316:243–246 Zhong CX, Marshall JB, Topp C, Mroczek R, Kato A, Nagaki K, Birchler JA, Jiang JM, Dawe RK (2002) Centromeric retroelements and satellites interact with maize kinetochore protein CENH3. Plant Cell 14:2825–2836 Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297:1833–1837 Zhu Q, Pao GM, Huynh AM, Suh H, Tonnu N, Nederlof PM, Gage FH, Verma IM (2011) BRCA1 tumour suppression occurs via heterochromatin-mediated silencing. Nature 477:179–184 Vourc'h C, Biamonti G (2011) Transcription of Satellite DNAs in Mammals. Prog Mol Subcell Biol 51:95–118 Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methyla- tion. Science 299:716–719 Waye JS, Willard HF (1989) Human beta satellite DNA: genomic organization and sequence definition of a class of highly
W2021598974.txt	https://zenodo.org/record/1556038/files/article.pdf	de		Über die Ziele der Forstlehranstalt zu Stargard in Mecklenburg	Forstwissenschaftliches Centralblatt	1,910	public-domain	1,298	Über die Ziele der Forstlehranstalt zn Stargard in Medlenöurg. 287 unternehmen es and) die GeseHfchasten, aus privatem Wege ihre Er¬ sahrungen und Versuche ständiger Prüfung zu unterziehen und darf man aus die endlichen Nesnltate, welche zurzeit noch nicht vorliegen, gespannt sein. JedensaUs sind sie ein weiterer Beitrag zu der wichtigen Frage der Konservierungssähigkeit des Holzes. Der praktische Sinn der Amerikaner treibt sie, auch scheinbar neben¬ sächliche Dinge in den Bereich ihrer Untersuchungen zu ziehen, und so beschäftigt sich Eircular 46 des Forest Service mit der „Haltekraft" der Eisenbahnschienennägel in hölzernen Schwellen. Hiervon verwendeten sie die gebräuchlichen 4 Formen und kommen dabei zu folgenden wefentlichen Nefultaten: Schraubennägel eignen sich vornehmlich für Weichholzfchwetten. Kannelierte Schienennägel haben nur etwa 60% der $raft zum Festhalten wie Schraubenbolzen. Getrodnete Schwellen halten die Schrauben und Nägel besser als mit Dampf behandelte. Die Fähigkeit, die Schrauben und Nägel festzuhalten ist unter einem Drud von 30 Pfund = 13V2 kg gleich bei einer grünen und bei einer bampfgetrodneten Schwelle. Dämpfung durch vier Stunden und mit einem geringeren Drud als 30 Pfund scheint die Haltekraft zu erhöhen, während Dämpfung länger als 4 Stunden mit 20 Pfund Drud sie vermindert. Gedämpfte und kreosotierte oder gedämpfte und mit Zinkchlorid behandelte Schwellen fcheinen geringere Kraft zum Festhalten der Schienennägel zn haben als lediglich gedämpfte. — rto— Über die \|)iele der ^orstlehranstalt zu Stargard in inedPlenburg. Von Oberförster D. TI)i)en. Wer eine Sache beurteilen will, der muß sie wenigstens kennen, um in den Kreisen darüber Auskunst geben zn können, denen sie nü\|en soll. Wer sich damit nicht befassen will, der hat kein Necht zn einer Kritik: er kann eine Sache, die ihm ans irgend einem Grunde unsympathisch ist, höchstens ignorieren. Abgesehen von einigen staatlichen Forstlehrlings., schulen existierten bisher überhaupt noch keine derartigen Anstalten. Die Forsilehrlingsschule in Templin genügt den Anforderungen, nach einer Nichtung hin nicht: in ihr können nur Lehrlinge ausgebildet werden. Der Verein der Privatforstbeamten Deutschlands hat für feine Mitglieder wohl Prüfungsftellen geschaffen, die Hauptfache aber hat er bisher außer 19* 288 Shhen: acht gelassen: eine Stelle, an der unsere älteren Grünrode ihren praktischen Kenntnissen einen Abschluß geben konnten. Es fehlte ihnen die Unter¬ Weisung, ohne die jede Prüfung undenkbar ist. Der Selbstunterricht ist ein schweres Ding! Und der ost schwerfällige Geist, der wohl gewohnt war, die praktischen Waldbitder zu erfassen, ist nicht immer imstande, die Lehren Westermener's, so klar sie auch gegeben sein mögen, ohne Erklärung und Erläuterung in auszuehmen; — die schwere Hand, die früher nur die Büchfe und die Kluppe führte, kann ohne fachgemäße Leitung den Zeichenstift oder den Pinfel nicht führen. J n richtiger Erfenntnis diefer Verhältnisse wurde dem Technikum sür Landeskultur zu Stargard i n Medtenburg, — das früher lediglich für Gärtner in Betracht kam, vor etwa Jahresfrist eine Forstabteilung angegliedert. Neben zwei akademisch gebildeten Oberförstern und einem tüchtigen Ober¬ förster-Kandidaten unterrichten an ihr ein Lehrer für Naturwissenschaft, ein Kultur-Jngenieur und ein Lehrer für Stenographie. Da der eine forstliche Lehrer in den Kolonien tätig war, kann auch auf die Kultur von Tropenpflanzen Nüdficht genommen werden, was befonders deshalb vorteilhaft ist, als die Besser der großen Plantagen von West- und Oft¬ afrika, in Hamburg und Bremen, mit Vorliebe junge Forstleute auf ihren Pflanzungen beschästigen. Dem individuellen Anschauungsvermögen der Schüler wird ebenso Nechnung getragen wie dem praktischen Können. Danach sind die Kurse eingerichtet. S i e dauern mindestens 6 Monate. Ost aber auch, wenn bei den Schlußprüsungen ein negatives Nesultat ^crausfteHt^ 12 Monate. Damit die Schlußprüsungen nicht zu umfangreich werden, finden am Schlüsse eines jeden Quartals Prüfungen statt, bie dfjentKd) fhtfe. Befonders sind Forstleute stets willkommen. Die Vorträge sind für jeden praktischen Forstmann verständlich. S i e werden an Waldbitbern erläutert, was um so leichter ist, als das Städtchen Stargard mitten im Kessel eines kleinen Gebirges liegt, das von den denkbar instruktivsten Forsten bestanden ist. Aus die Vermittelung geeigneter Stellen wirb besonderes Gewicht gelegt. Bisher ist sast kein Schüler abgegangen, dem nicht eine paffende Stelle nachgewiesen werden konnte. Manche kehrten allerdings auch in ihre alten Verwaltungen zurüd. Und da die Lehrer bie Schüler kennen, wird aus die Leistungen besondere Nüdficht genommen, was in erster Linie den Herren Waldbesijäern zugute kommt! Früher gingen bei einer SteHen-Neubese\|ung etwa 120 Bewerbungen ein und wenn ein Beamter mit den besten Zeugnissen richtig gewählt war, paßte er zu allem anderen besser, als zu der betreffenden Stelle! — Jn diefe Kurse werden nur Beamte ausgenommen, die nachweisen können, daß sie sci h sich Über die ölele der Forstlehranstalt ju Stargard in Mecklenburg. 289 jahrelang in der Praxis tätig gewesen sind. Außerdem werben aber auch Lehrlinge ausgebildet. D i e s e müssen, saHs sie eine entsprechende Schulbildung nicht durch ein Zeugnis belegen können, sich einer Ausnahmeprüsung unterwerfen. Am Unterricht nehmen diese Anfänger zunächst nur in den Vorbereitungssachern (Deutfch, Nechnen u s w . ) teil und natürlich auch an ben Exkursionen. Jm übrigen beteiligen sie sich an allen praktischen Arbeiten in der Stadtforst. Erst nach gründlicher, praktischer Unterweisung erfolgt auch die theoretische Ausbildung. Sehen wir uns nun den Stundenplan näher a n , so werden wir erkennen, daß nichts zu wünschen übrig bleibt. Diese Fächer muß ein moderner Förster hente beherrschen! $tunt>mvlan SKontag ©runbtiffe Der gorsteinriäjtung 8—10 10«/,-12V f 6-7 ftorft- unb gelbntessen 8ltttt!S= u n d mtäborstetyer<§ef<Pfte (bei einem Ober* sörjier, ber lange 3ab.re a!8 ämt6» unb Gutäöorpe^er tatig tt>ar) Sonnentag lurflonsbertdjt (5Rad& laus. @jtat(ion.) tylan- panzefiijnftt gitöetlierett 2-4 ©ienätag Seidjnett gorftöotanif (3oologie, gifdjerei im SBatbe Vienenju<St) '»tenoßrnptjie SomfponDenj Vortrag, Siftat ©üitnnbcnb gorftbtnuljuitg SDeutffi, abfassen teon ©teilengejudjen gorftfdjuft, Snseftenfunbe nst». SBatDtmu ^tanjeidjnen »vtantf ä5alDt)üm= blasen ©eilljreiöenbe Slufsäfce, SnODfuit&e 8W)t0itDcr (au« bem £ier unb Pftanjen(eben), Vorfüljrung 101.?= nialer Silber (ein Dberfürfter tuar in ben Kolonien tätig!) blasen 290 Literarische Berichte. Die Aufficht über Schulbesuch und ein gesittetes Leben wird zwar streng gehandhabt, aber das Lehrerkollegium ist der Ansicht, daß das Leben der Grüntöcke ein freies sein muß. Wie das Milieu, das der Wald bietet, der auch keine Treibhauskultur verträgt, ein sreies ist, steht jeder militärische Drill in Stargard in Mißkredit. Die Schüler haben Vertrauen zu ihren Lehrern und das hat die heften Erfolge gezeitigt. Die Schüler können sich in allen Lebenslagen so benehmen, wie es von einem gebildeten Förster oerlangt werden muß und sie erhalten auch nach ihrem Abgange von den Lehrern stets Nat und Hilse. Stargard ist idyllisch gelegen und das Leben ist billig dort. Volle, gute Pension ift für 50—60 Ji zu bekommen. Das Schulgeld beträgt für einen Kursus von 6 Monaten 150 Ji. Dazu kommen noch die Auslagen sür Bücher und Taschengeld. Es ist hier nicht angebracht, über die verschiedenen Ausbildungsarten unserer Forstschufcbeamlen zu urteilen. Der eine wünscht Schulen, der andere militärische Ausbildung. Jrgend eine Ausbildung aber verlangt jeder! Und jeder Staat erteilt sie! Wer gibt sie aber den Privat¬ grünröcken! Wenn sie das Glück haben, bei einem Jäger-Bataillon ihrer Militär-Dienstpflicht genügen zu können, dürfen fie noch nicht einmal am forstlichen Unterricht teilnehmen! Diesem mit der Zeit schreiend ge wordenen Bedürsnis will Stargard abhelsen. Und wie sehr die Grün¬ tacke danach streben, ihren praktischen Kenntnissen hier einen Abschluß zu geben, — das beweisen am besten die zahlreichen Anfragen und An¬ meldungen, die hier aus aller Herren Länder einlaufen! III. literarische Berichte. Nr. 33. Itttfeve ^ o v f f t t i i t t f ö a f t i m 3 0 . %oS)K\)\\vfotxi. I L Betrachtungen über den forftlichen Unterricht und das Einrichtungswefen, von Professor E. W a g n e r in Tübingen. Tübingen, Verlag der Laupp'schen Vuchhandtung, 1910. 65 S . Preis brosch. 1,20 Ji. Der unter gleichem Titel im Jahre 1909 erschienenen Broschüre ) hat W a g n e r nun eine zweite folgen lassen, die gleichsam eine Fortse\|ung der ersten bildet; in ihr saßt er drei Auffä\|e zufammen, die er in württembergischen Tageszeitungen erfcheinen ließ und nun in erweiterter Form auch weiteren Kreifen zugänglich macht. 1 Vergt. gorßö). (Eentrbt: 1909. @. 442.
https://openalex.org/W2897958053	https://europepmc.org/articles/pmc5896993?pdf=render	English	null	A case-control study of lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer using PCBaSe 3.0	PloS one	2,018	cc-by	5,304	RESEARCH ARTICLE Objectives To investigate the association between lower urinary-tract infections, their associated antibi- otics and the subsequent risk of developing PCa. Results It was found that lower urinary-tract infections did not affect PCa risk, however, having a lower urinary-tract infection or a first antibiotic prescription 6–12 months before PCa were both associated with an increased risk of PCa (OR: 1.50, 95% CI: 1.23–1.82 and 1.96, 1.71–2.25, respectively), as compared to men without lower urinary-tract infections. Com- pared to men with no prescriptions for antibiotics, men who were prescribed 10 antibiotics, were 15% less likely to develop PCa (OR: 0.85, 95% CI: 0.78–0.91). Data Availability Statement: The data used in this study comes from the Swedish prostate cancer database PCBaSe. We can confirm that the authors did not have any special access privileges and the data was made available following a formal application. The steering groups of the NPCR and PCBaSe welcome external collaborations. For more information please see [www.npcr.se/in-english], where registration forms, manuals and annual reports from the NPCR are found as well as a full list of publications from PCBaSe. Beth Russell1, Hans Garmo1,2, Kerri Beckmann1,3, Pa¨r Stattin4, Jan Adolfsson5, Mieke Van Hemelrijck1 1 King’s College London, Division of Cancer Studies, Translational Oncology & Urology Research (TOUR), London, United Kingdom, 2 Regional Cancer Centre, Uppsala, Sweden, 3 University of South Australia, Centre for Population Health Research, Adelaide, Australia, 4 Department Surgical Sciences, Uppsala University, Uppsala, Sweden, 5 CLINTEC department, Karolinska Institutet, Stockholm, Sweden a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 beth.russell@kcl.ac.uk OPEN ACCESS Citation: Russell B, Garmo H, Beckmann K, Stattin P, Adolfsson J, Van Hemelrijck M (2018) A case- control study of lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer using PCBaSe 3.0. PLoS ONE 13 (4): e0195690. https://doi.org/10.1371/journal. pone.0195690 Published: April 12, 2018 Copyright: © 2018 Russell 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. * beth.russell@kcl.ac.uk A case-control study of lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer using PCBaSe 3.0 Beth Russell1*, Hans Garmo1,2, Kerri Beckmann1,3, Pa¨r Stattin4, Jan Adolfsson5, Mieke Van Hemelrijck1 Subjects/Patients (or materials) and methods Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: September 6, 2017 Accepted: March 27, 2018 Published: April 12, 2018 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: September 6 2017 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: September 6, 2017 Accepted: March 27, 2018 Published: April 12, 2018 Copyright: © 2018 Russell 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 Using data from the Swedish PCBaSe 3.0, we performed a matched case-control study (8762 cases and 43806 controls). Conditional logistic regression analysis was used to assess the association between lower urinary-tract infections, related antibiotics and PCa, whilst adjusting for civil status, education, Charlson Comorbidity Index and time between lower urinary-tract infection and PCa diagnosis. Introduction Inflammation caused by infectious agents or environmental conditions has now been put forward as a possible cause for an increased prostate cancer (PCa) risk. Lower urinary tract infections are one example of such infections that are known to cause inflammation [1]. In addition, certain microorganisms have been implicated in the role of carcinogenesis [2]. The microorganism Escherichia coli has been identified as being the major pathogen involved in lower urinary-tract infection and is believed to account for around 80% of all urinary tract infections [3,4]. It has however not yet been proven to be carcinogenic. Competing interests: The authors have declared that no competing interests exist. Inflammation of the prostate gland (prostatitis) is also known to be attributed by infection of the microorganisms that are causative of lower urinary tract infections (LUTIs). With a link between the infectious agents, inflammation and the location of the prostate gland, a potential association between lower urinary-tract infections and PCa may be conceived [5]. Further links between infections and PCa are found with the gram-positive bacillus, Propio- nibacterium acnes as well as the most common non-viral sexually transmitted infection Tricho- monas vaginalis. It has been proposed that the inflammatory response and prostatitis that occurs from these pathogens may play an important role in the development of PCa [6–8]. Conversely, sexually transmitted infections caused by pathogens such as Chlamydia trachoma- tis and human papillomavirus (HPV) have been found to have little or no association with PCa risk [9,10]. Further links between infections and PCa are found with the gram-positive bacillus, Propio- nibacterium acnes as well as the most common non-viral sexually transmitted infection Tricho- monas vaginalis. It has been proposed that the inflammatory response and prostatitis that occurs from these pathogens may play an important role in the development of PCa [6–8]. occurs from these pathogens may play an important role in the development of PCa [6 8]. Conversely, sexually transmitted infections caused by pathogens such as Chlamydia trachoma- tis and human papillomavirus (HPV) have been found to have little or no association with PCa risk [9,10]. The few studies on the link between lower urinary-tract infections and PCa are inconclu- sive. One study by Pelucchi and colleagues found that having cystitis increased the men’s risk of having PCa by 76% [11]. Data source All data was obtained from PCBaSe Sweden 3.0 in which data from >98% of PCa cases from the National Prostate Cancer Register (NPCR) of Sweden are recorded [13,14]. This database started in 1998 and encompasses data from around 11 national registers such as the Prescribed Drug Register and the National Patient Register up until 2012. It is comprised of information from 137985 registered PCa men including information based on their records of various demographic and clinical aspects as well as the men’s socio-economic status. Introduction On the contrary, a similar study which focused on urethritis, con- cluded that this particular lower urinary-tract infection was not associated with PCa risk [12]. y sive. One study by Pelucchi and colleagues found that having cystitis increased the men’s risk of having PCa by 76% [11]. On the contrary, a similar study which focused on urethritis, con- cluded that this particular lower urinary-tract infection was not associated with PCa risk [12]. Using data from the Prostate Cancer Database Sweden (PCBaSe) on diagnosis and prescrip- tions of antibiotics for lower urinary-tract infections, the current study aims to further disen- tangle their potential role in the development of PCa. Using data from the Prostate Cancer Database Sweden (PCBaSe) on diagnosis and prescrip- tions of antibiotics for lower urinary-tract infections, the current study aims to further disen- tangle their potential role in the development of PCa. Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer detection bias, which highlights the importance of data on the diagnostic work-up when studying potential risk factors for PCa. detection bias, which highlights the importance of data on the diagnostic work-up when studying potential risk factors for PCa. Funding: This work was supported by the Swedish Research Council for Working Life, Health, and Welfare (825-2012-5047), the Swedish Cancer Society (14- 0570), the Cancer Society in Stockholm, and Uppsala County council. The research was also supported by the National Institute for Health Research (NIHR) BRC based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. Conclusion PCa was not found to be associated with diagnosis of a urinary-tract infection or frequency, but was positively associated with short time since diagnoses of lower urinary-tract infection or receiving prescriptions for antibiotics. These observations can likely be explained by 1 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer according to the National Drug Registry. Matching on county was ignored for those over the age of 90, due to a smaller sample size. The lower urinary-tract infection diagnoses were defined according to their ICD-10 codes. N30 referred to all diagnoses of cystitis; N34 to urethritis; and N39 to disorder of urinary sys- tem caused by infection with unspecified location. For each lower urinary-tract infection diag- nosis, the time between first diagnosis and date of being diagnosed with PCa or becoming a control (i.e. date that corresponding case was diagnosed with PCa) was calculated and catego- rized as 6–12 months, 2–4 years, 5–9 years and 10 years. Those lower urinary-tract infections diagnosed within six months prior to PCa diagnosis were not incorporated into the above- described exposure variables, as to avoid reverse causation. For those men who had more than one lower urinary-tract infection, a minimum of 30 days between diagnoses was required. A total of nine antibiotics were included in the study, all of which have been indicated to be used for lower urinary-tract infections specifically: Trimethoprim/sulfamethoxazole (ATC: J01XX01), Trimethoprim (J01EA01), Ciprofloxacin (J01MA02), Levofloxacin (J01MA12), Cephalexin (J01DB01), Doxycycline (J01AA02), Nitrofurantoin (J01XE01), Amoxicillin/Cla- vulanate (J01CR02) and Norfloxacin (J01MA06). The number of antibiotic prescriptions, as well as the time between first prescription and event, were also calculated and categorized as: 6–12 months, 1–2 years, 3–4 years and 5 years. Prescriptions within 6 months of diagnosis of prostate cancer were not considered as exposed, again to avoid reverse causation. For those men who had more than one prescription of antibiotics, a minimum of 7 days between pre- scriptions was required. The new prescription could have been for either a different antibiotic or the same. Furthermore, we collected information on Charlson Comorbidity Index (CCI). The latter assigns weights to a number of medical conditions, including diabetes and hypertension, based on discharge diagnoses prior to the date of diagnosis in the Patient Register [15]. Educa- tion was divided into three categories; Low referred to those who spent 10 years in the Swed- ish education system; Medium included the men with 10–12 years’ experience in Swedish schooling; High included those who went to university. Statistical analysis Odds ratios (ORs) were calculated using conditional multivariate logistic regression models. The various exposure variables describing the diagnosis of lower urinary-tract infections or use of antibiotics were studied in relation to risk of being diagnosed with PCa. All ORs were adjusted for civil status, education and CCI. The ORs, with respect to lower urinary-tract infec- tions, were additionally adjusted for ‘time between first lower urinary-tract infection diagnosis and event’, whilst the ORs for exposure variables solely relating to antibiotics were instead additionally adjusted for the ‘time between first antibiotic and event’. All data management was performed on SAS version 9.3 (SAS Institute, Cary, NC, USA), whilst all statistical analysis was performed on STATA/IC 12.1 (Texas, USA). Study population and study design From PCBaSe 3.0, we identified 8,762 men who were diagnosed with PCa in 2012 (i.e. cases) and matched them with five controls based on age and county (n = 43,806). Within this cohort, we then identified those who had a diagnosis of a lower urinary-tract infection in the patient register, plus those who had received a lower urinary-tract infection-specific antibiotic 2 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 Results Cases and controls showed similar distributions for age, education level, civil status and CCI (Table 1). Table 2 displays the PCa specific variables for the cases. In total, 9% of men were found to have distant metastatic PCa, while 3% of cases were recorded as having metastasis to their lymph nodes local to the prostate. The mean PSA at time of diagnosis for all men with PCa was 8.6 μg/ml. The frequency, type, and time since lower urinary-tract infection diagnoses are displayed in Table 3. The frequency of lower urinary-tract infections was similar across both cases and 3 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer Table 1. Characteristics of cases and controls selected from PCBaSe 3.0. Variable Cases n (%) Controls n (%) Age 40–49 81 (0.92) 431 (0.98) 50–59 1072 (12.23) 5341 (12.19) 60–69 3577 (40.82) 17816 (40.67) 70–79 2785 (31.78) 13997 (31.95) 80+ 1247 (14.23) 6221 (14.20) Mean (SD) 69.84 (8.98) 69.83 (8.99) Educational Level High 2254 (25.72) 10374 (23.68) Low 2956 (33.74) 15535 (35.46) Middle 3491 (39.84) 17285 (39.46) Missing 61 (0.70) 612 (1.40) Civil Status Married 5832 (66.56) 27201 (62.09) Not married 1052 (12.00) 6140 (14.02) Divorced 1280 (14.61) 5832 (13.31) Widower 596 (6.80) 3258 (7.44) Missing data 2 (0.02) 52 (0.12) CCI 0 6062 (69.19) 29226 (66.72) 1 1304 (14.88) 6887 (15.72) 2 817 (9.32) 4101 (9.36) 3+ 579 (6.61) 3592 (8.20) CCI = Charlson comorbidity index https://doi.org/10.1371/journal.pone.0195690.t001 controls, with 5% of cases and controls having had 1–2. More men were diagnosed with a lower urinary-tract infection of unspecified location than any other type (4% of cases and con- trols). The adjusted ORs displayed in Table 3 only indicate a statistically significant association with PCa for the period of time between initial lower urinary-tract infection and PCa diagno- sis: OR for lower urinary-tract infection 6–12 months prior to PCa diagnosis: 1.50 (95% CI: 1.23–1.82). Table 4 shows the distribution of specific antibiotic use among cases and controls, and their corresponding crude and adjusted ORs. Of the men who had been prescribed antibiotics, most had received between 1 and 3 prescriptions (34% cases and 30% controls), whilst considerably fewer men received over 10 (1% cases and controls). PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 Results The multivariate logistic regression analy- sis revealed that men who had received any antibiotic were at a 19% increased risk of PCa (OR: 1.19, 95% CI: 1.12–1.27). In addition, trimethoprim/sulfamethoxazole, ciprofloxacin, doxycycline and norfloxacin were all associated with an increased risk of PCa (OR: 1.33, 95% CI: 1.10–1.62; OR: 1.39, 95% CI: 1.28–1.52; OR = 1.10, 95% CI: 1.02–1.18 and OR: 1.25, 95% CI: 1.08–1.46 respectively). Conversely, men who had 10 or more antibiotic prescriptions were 15% less likely to develop PCa (OR: 0.85, 95% CI: 0.79–0.91). With respect to time between the first antibiotic prescription and event, it was found that the majority of cases and controls (60% and 64% respectively), had at least 5 years between these two dates. Time since antibiotic prescription was positively associated with PCa diagnosis, but the strongest association was PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 4 / 10 Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer Table 2. PCa specific characteristics of cases selected from PCBaSe 3.0. Variable Cases n (%) Controls n (%) Gleason Score <7 3263 (37.24) - 7 3285 (37.49) - >7 1982 (22.62) - Missing data 232 (2.65) - T Stage T0 31 (0.35) - T1a 263 (3.00) - T1b 157 (1.79) - T1c 4083 (46.60) - T2 2421 (27.63) - T3 1350 (15.41) - T4 280 (3.20) - TX 171 (1.95) - Missing data 6 (0.07) - N-Stage N0 1960 (22.37) - N1 220 (2.51) - NX 6567 (74.95) - Missing data 15 (0.17) - M-Stage M0 7957 (90.81) - M1 788 (8.99) - MX 2 (0.02) - Missing data 15 (0.17) - Number of biopsy cores taken <10 2265 (25.85) - 10–19 5723 (65.32) - 20–29 45 (0.51) - 30+ 4 (0.05) - None 725 (8.27) - Number of biopsy cores with cancer 0 1 (0.01) - 1–4 5012 (57.20) - 5–9 2313 (26.40) - 10–14 605 (6.90) - 15+ 1 (0.01) - None 830 (9.47) - PSA at time of diagnosis (μg/ml) Median (IQR) 8.6 (15.7) - PSA = prostate specific antigen. https://doi.org/10.1371/journal.pone.0195690.t002 seen for those men who had 6–12 months between the first antibiotic and PCa diagnosis (OR: 1.96, 95% CI: 1.71–2.25), as compared to men with no prescriptions. seen for those men who had 6–12 months between the first antibiotic and PCa diagnosis (OR: 1.96, 95% CI: 1.71–2.25), as compared to men with no prescriptions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer Table 3. Odds ratios (OR) and 95% confidence intervals (CI) for PCa based on history of lower urinary-tract infection. Cases N (%) Controls N (%) Crude 0R 95% CI Adjusted OR 95% CI Number of lower urinary tract infections 0 8288 (94.59) 41457 (94.64) 1.00 Ref. 1.001 Ref. 1–2 454 (5.18) 2224 (5.08) 1.02 (0.92–1.13) 0.96 (0.72–1.30) 3–4 15 (0.17) 102 (0.23) 0.74 (0.43–1.27) 0.75 (0.40–1.40) 5+ 5 (0.06) 23 (0.05) 1.09 (0.41–2.86) 1.13 (0.41–3.11) Type of lower urinary-tract infections No LUTI 8288 (94.59) 41457 (94.64) 1.00 Ref. 1.001 Ref. Any 474 (5.41) 2349 (5.36) 1.01 (0.91–1.12) 0.96 (0.71–1.28) Cystitis N30 77 (0.88) 408 (0.93) 0.94 (0.74–1.21) 0.91 (0.64–1.31) Urethritis N34 10 (0.11) 54 (0.12) 0.93 (0.47–1.82) 0.90 (0.43–1.87) LUTI of unspecified location N39 356 (4.06) 1694 (3.87) 1.05 (0.93–1.18) 1.00 (0.73–1.36) Multiple types 31 (0.35) 193 (0.44) 0.8 (0.55–1.18) 0.84 (0.53–1.33) Time between first lower urinary-tract infections diagnosis and event (yrs) No LUTI diagnosed 8288 (94.59) 41457 (94.64) 1.00 Ref. 1.002 Ref. 6–12 months 265 (3.02) 1229 (2.81) 1.41 (1.16–1.70) 1.50 (1.23–1.82) 2–4 156 (1.78) 827 (1.89) 0.87 (0.72–1.05) 0.93 (0.77–1.12) 5–9 53 (0.60) 282 (0.64) 0.94 (0.79–1.22) 0.97 (0.81–1.16) 10+ 0 (0) 11 (0.03) 0.91 (0.67–1.21) 0.96 (0.71–1.28) 1Adjusted for civil status, education, CCI and time between first UTI diagnosis and event. 2Adjusted for civil status, education and CCI. Date for controls in ‘Time between first LUTI diagnosis and event’ have been taken as the date of the man becoming a control. Multiple types of UTI means any combination of the three types of UTI. j , Date for controls in ‘Time between first LUTI diagnosis and event’ have been taken as the date of the man becoming a control. Multiple types of UTI means any combination of the three types of UTI. https://doi.org/10.1371/journal.pone.0195690.t003 Results seen for those men who had 6–12 months between the first antibiotic and PCa diagnosis (OR: 1.96, 95% CI: 1.71–2.25), as compared to men with no prescriptions. Finally, results were stratified by PCa risk categories and comparable ORs were produced between low risk and high risk groups for time since UTI diagnosis and antibiotic prescription. Finally, results were stratified by PCa risk categories and comparable ORs were produced between low risk and high risk groups for time since UTI diagnosis and antibiotic prescription. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 5 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 https://doi.org/10.1371/journal.pone.0195690.t003 Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer Table 4. Odds ratios (OR) and 95% confidence intervals (CI) for PCa based on prescriptions of lower urinary-tract infection-related antibiotics. Cases N (%) Controls N (%) Crude 0R 95% CI Adjusted OR 95% CI Number of antibiotic prescriptions None 5237 (59.77) 28199 (64.37) 1.00 Ref. 1.001 Ref. 1–3 2961 (33.79) 13030 (29.74) 0.96 (0.85–1.08) 1.02 (0.90–1.15) 4–6 376 (4.29) 1736 (3.96) 1.01 (0.82–1.26) 1.06 (0.85–1.33) 7–9 106 (1.21) 462 (1.05) 0.95 (0.75–1.21) 1.05 (0.82–1.34) 10+ 82 (0.94) 379 (0.87) 0.82 (0.76–0.86) 0.85 (0.79–0.91) Antibiotic received None 1.00 Ref. 1.001 Ref. Trimethoprim/sulfamethoxazole 309 (3.53) 1398 (3.19) 1.31 (1.09–1.59) 1.33 (1.10–1.62) Trimethoprim 313 (3.57) 1475 (3.37) 1.07 (0.89–1.29) 1.10 (0.91–1.33) Ciprofloxacin 1785 (20.37) 6905 (15.76) 1.41 (1.31–1.51) 1.39 (1.28–1.52) Levofloxacin 13 (0.15) 65 (0.15) 0.64 (0.25–1.63) 0.65 (0.26–1.66) Cephalexin 11 (0.13) 31 (0.07) 1.43 (0.53–3.82) 1.49 (0.55–4.00) Doxycycline 1949 (22.24) 9175 (20.94) 1.12 (1.05–1.19) 1.10 (1.02–1.18) Nitrofurantoin 156 (1.78) 749 (1.71) 1.14 (0.81–1.61) 1.12 (0.78–1.60) Amoxicillin & Clavulanate 185 (2.11) 799 (1.82) 1.13 (0.91–1.41) 1.12 (0.89–1.40) Norfloxacin 314 (3.58) 1348 (3.08) 1.26 (1.09–1.46) 1.25 (1.08–1.46) Any - - 1.22 (1.16–1.28) 1.19 (1.12–1.27) Time between first antibiotic and event (yrs) No antibiotic given 291 (3.32) 812 (1.85) 1.00 Ref. 1.002 Ref. 6–12 months 780 (8.90) 3473 (7.93) 1.93 (1.68–2.22) 1.96 (1.71–2.25) 1–2 949 (10.83) 4426 (10.10) 1.21 (1.12–1.32) 1.23 (1.13–1.33) 3–4 1505 (17.18) 6896 (15.74) 1.16 (1.07–1.25) 1.17 (1.08–1.26) 5+ 5237 (59.77) 28199 (64.37) 1.18 (1.11–1.26) 1.20 (1.12–1.27) Table 4. Odds ratios (OR) and 95% confidence intervals (CI) for PCa based on prescriptions of lower urinary-tract infec Adjusted for civil status, education and CCI. Type of antibiotic is the first antibiotic received if more than one has been prescribed. Date for controls in ‘Time between first antibiotic and event have been taken as the date of the man becoming a control i.e. date of event for corresponding case. E.coli therefore these antibodies may not provide the same carcinogenic protection as those of P.acnes [17]. E.coli therefore these antibodies may not provide the same carcinogenic protection as those of P.acnes [17]. E.coli therefore these antibodies may not provide the same carcinogenic protection as those of P.acnes [17]. Discussion This population-based case-control study did not find any association between lower urinary- tract infections and their frequency with PCa risk. Only those who had a lower urinary-tract infection within the past year were found to have an increased risk of PCa diagnosis. Taking lower urinary-tract infection related antibiotics and the most recent prescriptions or diagnoses were found to be linked with increased odds of PCa diagnosis. However, it needs to be noted that men being prescribed many antibiotics (10) were discovered to have a lower risk of PCa. Our null results for lower urinary-tract infections in relation to PCa risk were somewhat unexpected and are inconsistent with the results reported by Fan et al who found a possible eti- ological role for urethritis and cystitis in the development of PCa [5]. In particular, they found that urethritis had the most significant increase in PCa risk with an OR of 1.72 (95% CI: 1.26– 2.34). This study however, only looked at a cohort of Taiwanese men and hence the findings may not be generalizable to a European population. Conversely, the null finding for lower uri- nary-tract infections in our study was also established in a similar case control study in which UTIs were studied in relation to bladder cancer [2]. Initially, this investigation looked at all diagnosis of cystitis in relation to bladder cancer and found an OR of 1.52 (95% CI: 1.12–2.06). However, upon excluding any cases of cystitis that occurred within one year prior to diagnosis of bladder cancer, the association disappeared. In a paper by Severi et al, they reported that an increase in antibodies against the pathogen P.acnes provided protection against PCa [16]. Therefore, our result that an increased frequency of lower urinary-tract infections did not increase the OR for risk in this present study was sur- prising. Nonetheless, the majority of lower urinary-tract infections are caused by the pathogen PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 6 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 Our observation that having an lower urinary-tract infection 6–12 months before PCa diag- nosis increased the risk of PCa by 49% is somewhat in line with a study by Pelucchi et al [11], who found an OR of 1.76 when the lower urinary-tract infection occurred less than 5 years before PCa diagnosis. These observations are likely to be explained by detection bias as we did not see any association with frequency or type of lower urinary-tract infection. Men who have lower urinary-tract infections are likely to undergo a work-up and hence have an increased risk of detecting PCa. Furthermore, lower urinary-tract infections can also be caused by a blad- der outlet obstruction (BOO) as a result of benign prostate hyperplasia (BPH). The lower uri- nary tract symptoms (LUTS) caused by BPH may then result in a visit to the doctors and thus increased risk of PCa diagnosis [18]. Our observations for time since receiving antibiotics showed a similar trend. Men who had received any of the lower urinary-tract infection associated antibiotics were at a 19% increased risk of PCa diagnosis. Interestingly, however, men who received 10 or more prescriptions for antibiotics had a 15% lowered risk of developing PCa. No other study to date PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 7 / 10 Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer has researched the use of lower urinary-tract infection-specific antibiotics and PCa, therefore these conflicting results cannot be compared with previous data. However, it can be hypothe- sized that detection bias is also an issue here. Those who received antibiotics would have been to see a medical practitioner and are subsequently more likely to have any PCa detected. Con- versely, the decrease in risk for men who received 10 of more antibiotics may be explained by a decrease in inflammation. One study revealed a chemopreventive role of the antibiotic eryth- romycin in mice [19]. Hamoya et al hypothesised that the decrease in cancer development was in part caused by the attenuation of local inflammation [19]. However, in our study, it is not possible to tease out this potential chemopreventive role and one could also hypothesize that this decrease in PCa risk is simply due to the fact that these men are longitudinally followed and truly have no PCa. Conclusion We did not find an increased risk of PCa following the diagnosis of a lower urinary-tract infec- tion. However, shortly after the filling of a prescription for an antibiotic or the diagnosis of a lower urinary-tract infection, there was an increased risk of PCa, which suggests detection bias. This thus highlights the importance of data on the diagnostic work-up when studying potential risk factors for PCa. Furthermore, since our results clearly point towards detection bias rather than causality, we feel that this paper means that the existing literature should be assessed carefully. Strengths of this study include its large sample size, adjustment for a variety of confounders including CCI, and a combination of data on both lower urinary-tract infection diagnosis and antibiotics. However, many antibiotics are commonly prescribed to treat several types of infec- tions and not just lower urinary-tract infections [20]. Penicillins for example (such as amoxi- cillin) can be used to treat anything from skin infections to chest infections as well as urinary tract infections [20]. Consequently, some prescriptions included in this present study might have been used to treat other indications than lower urinary-tract infections—however this potential misclassification would have been the same for both cases and controls. Another pos- sible limitation to this study was the identification of men with lower urinary-tract infections. PCBaSe does not have detailed information on indication for diagnosis (e.g. confirmation of culture)–again this misclassification bias would have been applicable to both cases and con- trols. Furthermore, all cases were selected based on their hospital admission. Therefore, it is reasonable to presume that these cases would have encompassed the more severe lower uri- nary-tract infection diagnoses and so may not be representative of all lower urinary-tract infections. Acknowledgments This project was made possible by the continuous work of the National PCa Register of Swe- den (NPCR) steering group: Pa¨r Stattin (chairman), Anders Widmark, Camilla Thellenberg Karlsson, Ove Andre´n, Anna Bill Axelson, Ann-Sofi Fransson, Magnus To¨rnblom, Stefan Carlsson, Marie Hja¨lm Eriksson, David Robinson, Mats Ande´n, Jonas Hugosson, Ingela Franck Lissbrant, Maria Nyberg, Go¨ran Ahlgren, Rene´ Blom, Lars Egevad, Calle Waller, Olof Akre, Per Fransson, Eva Johansson, Fredrik Sandin, Karin Hellstro¨m. References 1. Masajtis-Zagajewska A, Nowicki M. New markers of urinary tract infection. Clin Chim Acta. 2017; 2. Jhamb M, Jie A, Ae L, Ballow R, Kamat AM, Barton AH, et al. Urinary tract diseases and bladder cancer risk: a case–control study. Cancer Causes Control. 2007; 18:839–45. https://doi.org/10.1007/s10552- 007-9028-2 PMID: 17593531 3. Stamm WE. Scientific and clinical challenges in the management of urinary tract infections. Am J Med. 2002 Jul; 113(1):1–4. 4. Sfanos KS, De Marzo AM. Prostate cancer and inflammation: the evidence. Histopathology. 2012; 60 (1):199–215. https://doi.org/10.1111/j.1365-2559.2011.04033.x PMID: 22212087 5. Fan C-Y, Huang W-Y, Lin K-T, Lin C-S, Chao H-L, Yang J-F, et al. Lower Urinary Tract Infection and Subsequent Risk of Prostate Cancer: A Nationwide Population-Based Cohort Study. PLoS One. 2017; 12(1). 6. Cohen RJ, Shannon BA, Mcneal JE, Shannon T, Garrett KL. Infection/Inflammation Propionibacterium acnes associated with inflammation in radical prostatectomy specimens: A possible link to cancer evo- lution? J Urol. 2005; 173:1969–74. 7. Davidsson S, Mo¨lling P, Rider JR, Unemo M, Karlsson MG, Carlsson J, et al. Frequency and typing of Propionibacterium acnes in prostate tissue obtained from men with and without prostate cancer. Infect Agent Cancer. 2016; 11(26). 8. Sutcliffe S, Giovannucci E, Alderete JF, Chang T-H, Gaydos CA, Zenilman JM, et al. Plasma Antibodies against Trichomonas vaginalis and Subsequent Risk of Prostate Cancer. Cancer Epidemiol Biomarkers Prev. 2006; 15(5):939–45. https://doi.org/10.1158/1055-9965.EPI-05-0781 PMID: 16702374 9. Anttila T, Tenkanen L, Lumme S, Leinonen M, Gislefoss RE, Hallmans G, et al. Chlamydial Antibodies and Risk of Prostate Cancer. Cancer Epidemiol Prev Biomarkers. 2005; 14(2):385–9. 10. Korodi Z, Dillner J, Jellum E, Lumme S, Hallmans G, Thoresen S, et al. Human Papillomavirus 16, 18, and 33 Infections and Risk of Prostate Cancer: A Nordic Nested Case-Control Study. Cancer Epidemiol Prev Biomarkers. 2005; 14(12):2952–5. 11. Pelucchi C, Talamini R, Negri E, Franceschi S, La Vecchia C. Genital and urinary tract diseases and prostate cancer. Eur J Cancer Prev. 2006; 15:254–7. https://doi.org/10.1097/01.cej.0000198897. 40303.d4 PMID: 16679869 12. Boehm K, Valdivieso R, Meskawi M, Larcher A, Schiffman J, Sun M, et al. Prostatitis, other genitouri- nary infections and prostate cancer: results from a population-based case–control study. World J Urol. 2016; 34:425–30. https://doi.org/10.1007/s00345-015-1625-1 PMID: 26108732 13. Van Hemelrijck M, Wigertz A, Sandin F, Garmo H, Hellstro¨m K, Fransson P, et al. Cohort profile: The National Prostate Register of Sweden and Prostate Cancer Data Base Sweden 2.0. Int J Epidemiol. 2013; 42:956–67. https://doi.org/10.1093/ije/dys068 PMID: 22561842 14. Author Contributions Conceptualization: Kerri Beckmann, Mieke Van Hemelrijck. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 8 / 10 Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer Formal analysis: Beth Russell, Hans Garmo. Methodology: Beth Russell, Hans Garmo. Supervision: Kerri Beckmann, Mieke Van Hemelrijck. Writing – original draft: Beth Russell. Writing – review & editing: Beth Russell, Hans Garmo, Kerri Beckmann, Pa¨r Stattin, Jan Adolfsson, Mieke Van Hemelrijck. Supervision: Kerri Beckmann, Mieke Van Hemelrijck. Writing – original draft: Beth Russell. Writing – review & editing: Beth Russell, Hans Garmo, Kerri Beckmann, Pa¨r Stattin, Jan Adolfsson, Mieke Van Hemelrijck. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 18. Sundaram D, Sankaran PK, Raghunath G, Vijayalakshmi S, Vivayakumar J, Yuvarj MF, et al. Correla- tion of Prostate Gland Size and Uroflowmetry in Patients with Lower Urinary Tract Symptoms MATERI- ALS AND METHODS. J Clin Diagnostic Res. 2017; 11(5):1–4. 19. Hamoya T, Miyamoto S, Tomono S, Fujii G, Nakanishi R, Komiya M, et al. Chemopreventive effects of a lowwsideeeffect antibiotic drug, erythromycin, on mouse intestinal tumors. J Clin Biochem Nutr. 2017; 60(3):199–207. https://doi.org/10.3164/jcbn.16-107 PMID: 28584401 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 20. NHS. Antibiotics—NHS Choices [Internet]. Department of Health; 2017 [cited 2017 Jun 7]. http://www. nhs.uk/conditions/Antibiotics-penicillins/Pages/Introduction.aspx Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer References Van Hemelrijck M, Garmo H, Wigertz A, Nilsson P, Stattin PR. Cohort Profile Update: The National Prostate Cancer Register of Sweden and Prostate Cancer data Base—a refined prostate cancer trajec- tory. Int J Epidemiol. 2016; 45(1):73–82. https://doi.org/10.1093/ije/dyv305 PMID: 26659904 15. Charlson ME, Pompei P, Ales KL, Mackenzie CR. A new method of classifying prognostic comorbidity in longitudinal studies: Development and Validation. J Chron Dis. 1987; 40(5):373–83. PMID: 3558716 16. Severi G, Shannon B, Hoang H, Baglietto L, English D, Hopper J, et al. Plasma concentration of Propio- nibacterium acnes antibodies and prostate cancer risk: results from an Australian population-based case–control study. Br J Cancer. 2010; 103:411–5. https://doi.org/10.1038/sj.bjc.6605757 PMID: 20606679 17. Longhi C, Comanducci A, Riccioli A, Ziparo E, Marazzato M, Aleandri M, et al. Features of uropatho- genic Escherichia coli able to invade a prostate cell line. Short Commun New Microbiol. 2016; 39 (2):146–9. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195690 April 12, 2018 9 / 10 Lower urinary-tract infections, associated antibiotics and the risk of developing prostate cancer 10 / 10
https://openalex.org/W3004109122	https://europepmc.org/articles/pmc6086003?pdf=render	English	null	Viral tumor antigen expression is no longer required in radiation-resistant subpopulation of JCV induced mouse medulloblastoma cells	Genes & cancer	2,018	cc-by	8,745	Department of Neuroscience and Center for Neurovirology, Temple University Lewis Katz School o PA, USA Correspondence to: Ilker Kudret Sariyer, email: isariyer@temple.edu Keywords: JC virus; PML; cancer; medulloblastoma; viral oncogene Received: June 22, 2018 Accepted: July 16, 2018 Correspondence to: Ilker Kudret Sariyer, email: isariyer@temple.edu Keywords: JC virus; PML; cancer; medulloblastoma; viral oncogene Received: June 22, 2018 Accepted: July 16, 2018 Published: July 25, 2018 Published: July 25, 2018 Copyright: Donadoni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: Donadoni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT The human neurotropic polyomavirus JC, JC virus (JCV), infects the majority of human population during early childhood and establishes a latent/persistent infection for the rest of the life. JCV is the etiologic agent of the fatal demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML) that is seen primarily in immunocompromised individuals. In addition to the PML, JCV has also been shown to transform cells in culture systems and cause a variety of tumors in experimental animals. Moreover, JCV genomic DNA and tumor antigen expression have been shown in a variety of human tumors with CNS origin. Similar to all polyomaviruses, JCV encodes for several tumor antigens from a single transcript of early coding region via alternative splicing. There is little known regarding the characteristics of JCV induced tumors and impact of DNA damage induced by radiation on viral tumor antigen expression and growth of these cells. Here we analyzed the possible impact of ionizing radiation on transformed phenotype and tumor antigen expression by utilizing a mouse medulloblastoma cell line (BSB8) obtained from a mouse transgenic for JCV tumor antigens. Our results suggest that a small subset of BSB8 cells survives and shows radiation resistance. Further analysis of the transformed phenotype of radiation resistant BSB8 cells (BSB8-RR) have revealed that they are capable of forming significantly higher numbers and sizes of colonies under anchorage dependent and independent conditions with reduced viral tumor antigen expression. Moreover, BSB8-RR cells show an increased rate of double-strand DNA break repair by homologous recombination (HR). More interestingly, knockout studies of JCV tumor antigens by utilizing CRISPR/Cas9 gene editing reveal that unlike parental BSB8 cells, BSB8-RR cells are no longer required the expression of viral tumor antigens in order to maintain transformed phenotype. Genes & Cancer, Vol. 9 (3-4), March 2018 Genes & Cancer, Vol. 9 (3-4), March 2018 www.Genes&Cancer.com Viral tumor antigen expression is no longer required in radiation- resistant subpopulation of JCV induced mouse medulloblastoma cells Effect of radiation on mouse medulloblastoma cells induced by JCV tumor antigens JC virus can transform cells in culture systems and cause a variety of tumors in experimental animals. Cell lines from a medulloblastoma induced in a mouse model transgenic for JCV tumor antigens were generated previously [11]. Among these cell lines, we utilized BSB8 cells, which express viral tumor antigens and shows transformed phenotype and BS1A cells, which were immortal but did not express viral tumor antigens with no obvious transformed phenotype. BSB8 and BS1A cells were first grown to full confluency in tissue culture dishes. Cells were exposed to various dosages of ionizing radiation (3-6-12 Gy) and analyzed by MTT cell viability assay at 72h post-radiation. As shown in Figure 1A, radiation had no impact on BS1A cell viability due to the contact inhibition of their growth at full confluency. On the other hand, radiation caused a significant reduction in cellular viability of BSB8 cells at 3-Gy and did not further alter cellular viability loss at 6 and 12-Gy dosages. In parallel, whole cell protein extracts were also obtained from BSB8 and BS1A cells exposed to ionizing radiation at 6 and 12-Gy dosages and analyzed by Western blotting at 72hrs post-radiation. As shown in Figure 1B, radiation caused a significant decrease in JCV large T-ag expression at 6-Gy with no further change at 12-Gy. Interestingly, both apoptosis and autophagy were induced in BSB8 cells as evidenced by cleaved caspase-3 activation and LC3- II conversion, respectively (Figure 1B, compare lane 1 The oncogenic potential of JCV is strongly associated with the expression of viral tumor antigens. Several line of evidence suggests that JCV-mediated cellular transformation relies on the sequestration and suppression of the tumor suppressor proteins, p53 and the pRb family, by the viral large T antigen [21, 22, 23]. JCV large T antigen can also interact with other cellular proteins such as insulin receptor substrate 1 (IRS-1), β-catenin, neurofibromatosis type 2 gene product, and antiapoptotic protein survivin which are also implicated in pathways associated with cellular transformation, [24, 25, 26, 27, 28]. We have previously showed that downregulation of JCV tumor antigen expression in BSB8 cells, a cell line that originated from a medulloblastoma developed in a transgenic mouse expressing the JCV- early region, and in HJC-2 cells, a cell line obtained from a glioblastoma induced by intracranial injection of JCV in newborn hamsters, results in growth arrest and induction of apoptosis [6]. INTRODUCTION virus genomic DNA can be detected in serum and urine of immunocompetent individuals that suggests the presence of a low level viral replication leading to viral persistency in healthy subjects [1, 2, 3]. Beside its role in the development of PML, JC virus has also been associated with various tumors in laboratory animals and humans. Similar to the simian polyomavirus 40 (SV40), JC virus also shows ability to transform primary cells in vitro JC virus (JCV) is a human polyomavirus, which infects the majority of human population during early childhood, forms a latent/persistent infection for the rest of the life, and reactivates in individuals mostly under immunosuppressive conditions leading to the development of progressive multifocal leukoencephalopathy (PML). JC www.Genes&Cancer.com Genes & Cancer 130 impact of secondary insults, such as ionizing radiation, on viral gene expression and growth of tumor cells transformed by JCV has not been reported. [4]. JCV-transformed primary human cells express viral transforming antigens and exhibit transformed phenotype [5, 6]. Here we investigated the effects of ionizing radiation on JCV tumor antigen expression and viability of BSB8 cells. Interestingly, although radiation induced apoptosis in the majority of BSB8 cells, a small subset of cells showed radiation resistance. Further analysis of radiation resistant subpopulation of BSB8 cells (BSB8- RR) revealed that these cells expressed reduced levels of viral tumor antigens, were no longer sensitive to repeated insults of radiation, had an increase in cell-cycle, and were able to repair double-strand breaks with a better efficiency than parental cells. Moreover, knockout of JCV tumor antigens from BSB8-RR cells by excision mediated by CRISPR/Cas9 gene editing suggest that viral tumor antigen expression was no longer required for the growth and colonization of the BSB8-RR cells. These results may have implications for the involvement of JCV tumor antigens in early stages of human cancers and suggest a possible mechanism of radiation resistance. On the other hand, inoculation of JCV into experimental animals, including mice, hamster, and primates results in tumor development rather than lytic viral replication. Intracerebral inoculation of JCV PML strain into Syrian hamsters leads to the development of glial and neuronal origin tumors including glioblastomas, neuroblastomas, and medulloblastomas [7, 8]. JCV has also been shown to be tumorigenic in nonhuman primates [9, 10]. Mice lines transgenic for JCV early coding region encoding for viral tumor antigens under the control of viral promoter were also created. INTRODUCTION Interestingly, viral promoter activity was attributed to the neuronal cells with the formation of different tumors that derived from neural origin in these transgenic mice models [11, 12, 13]. JCV genomic sequences and viral proteins have also been detected and reported in variety of human tumors. Sporadic development of human tumors with CNS origin, such as oligodendroglioma, astrocytomas, and neuroblastomas were reported in PML patients [14, 15, 16]. Expression of viral tumor antigens was observed in the absence of productive lytic infection in PML patients. Expression of the JCV large T antigen and presence of JCV genome have also been detected in human brain tumors in the absence of PML lesions [17, 18, 19, 20]. Such findings provided evidence for a possible association of JCV for the formation of human tumors with CNS origin. In fact, according to Del Valle et al, 2001 and 2002 [19, 20] , JCV early gene sequences were detected in 62.5% of oligoastrocytomas, 83.3% of ependymomas, 80% of pilocytic astrocytomas, 57.1% of oligodendrogliomas, 76.9% of astrocytomas, and 66% of anaplastic oligodendrogliomas. Effect of radiation on mouse medulloblastoma cells induced by JCV tumor antigens These observations suggest that the fate of cells transformed by JCV might be indeed depended on viral tumor antigen expression. However, the possible www.Genes&Cancer.com Genes & Cancer 131 cells than BSB8 and BSB8-RR cells with no sign of JCV tumor antigen expression as expected. with lanes 2 and 3). On the other hand, BS1A cells did not show any large T-ag expression or cleaved caspase-3 activation although LC3-II conversion was induced by radiation (Figure 1B, compare lane 4 with lanes 5 and 6). These results suggest that ionizing radiation induces autophagy in both BSB8 and BS1A cells but apoptosis is only induced in BSB8 cells. The observed impact of radiation on BSB8 cell viability and large T-ag expression were not altered by increased radiation dosages suggesting that a subset of these cells were possibly resistant to the apoptosis induced by radiation. In order to test this, BSB8 cells were irradiated at 6-Gy and nearly 80% of cells were lost due to the extensive apoptosis by 72h post radiation. The remaining radiation resistant sub-population of BSB8 cells was further cultured for two weeks and called “BSB8-RR”. As shown in Figure 1C-1D, BSB8-RR cells retained the low levels of large T-ag and small-t-ag expressions with no significant difference in p53 levels as compared to original BSB8 cells. On the other hand, basal p53 expression levels were significantly lower in BS1A BSB8-RR cells are resistant to repeated radiation insults To further investigate the role of JCV tumor antigen expression in resistance to the radiation, BSB8 and BSB8- RR cells were irradiated at 6-Gy and cellular viability was analyzed by MTT assay at 48h and 72h post-radiation. While cellular viabilities of both BSB8 and BSB8-RR cells were not altered at 48h post-radiation, there was a significant decrease in BSB8 viability at 72h (Figure 2A and B). Interestingly, unlike BSB8 cells, BSB8-RR cells did not show any significant alteration in cellular viabilities even after 72h post-radiation (Figure 2B, compare lane 4 with 2) suggesting that they were resistant to cell death induced by secondary radiation. In parallel, whole cell extracts were also prepared and processed by Western blotting for the detection and analysis of viral Figure 1: Effect of radiation on apoptosis and autophagy. A. MTT assay of BS1A and BSB8 cells after treatment with ionizing radiation. B. Expression of proteins involved in apoptosis and autophagy in BS1A and BSB8 cells after radiation treatment. C. Western blot analysis for Large T-Ag, small-t , and p53 in BSB8, BSB8-RR, and BS1A cells. D. Band intensities of Large T antigen, small t antigen, and p53 from Western blot analysis in panel C were normalized to tubulin and shown as bar graph. Figure 1: Effect of radiation on apoptosis and autophagy. A. MTT assay of BS1A and BSB8 cells after treatment with ionizing radiation. B. Expression of proteins involved in apoptosis and autophagy in BS1A and BSB8 cells after radiation treatment. C. Western blot analysis for Large T-Ag, small-t , and p53 in BSB8, BSB8-RR, and BS1A cells. D. Band intensities of Large T antigen, small t antigen, and p53 from Western blot analysis in panel C were normalized to tubulin and shown as bar graph Figure 1: Effect of radiation on apoptosis and autophagy. A. MTT assay of BS1A and BSB8 cells after treatment with ionizing radiation. B. Expression of proteins involved in apoptosis and autophagy in BS1A and BSB8 cells after radiation treatment. C. Western blot analysis for Large T-Ag, small-t , and p53 in BSB8, BSB8-RR, and BS1A cells. D. Band intensities of Large T antigen, small t antigen, and p53 from Western blot analysis in panel C were normalized to tubulin and shown as bar graph. www.Genes&Cancer.com Genes & Cancer 132 the secondary radiation (Figures 1 and 2). BSB8-RR cells are resistant to repeated radiation insults Next we asked if there were also any alterations in their growth characteristics and tumorigenic capacity compared to original BSB8 cells. In order to investigate and compare growth rates, BSB8 and BSB8-RR cells were first serum- starved for 24 hours. Starvation was released by adding 10% fetal bovine serum and cells were harvested at 0h-12h-24h post-release from starvation. Cells were treated with propidium iodide and cell-cycle analyses were performed using flow cytometry. As shown in Figure 3A, both BSB8 and BSB8-RR cells had a synchronized G1/G0 stage at 0h (24h post-starvation) as expected. Interestingly, BSB8-RR cells showed a significantly higher percentage of cells at G1/G0 and lower percentage of cells at G2/M after 24h post-starvation release suggesting that initiation and completion of cell cycle of BSB8-RR cell were most likely faster than the original BSB8 cells. Following cell cycle analysis, growth characteristics and tumorigenesis of BSB8 and BSB8-RR cells were also analyzed by colony formation assay and soft agar growth analyses under anchorage dependent and independent conditions, respectively. As shown in Figure 3B, BSB8-RR cells form tumor antigen expression from the same set of studies. As shown in Figure 2C and 2D, viral large T-ag and small t-ag expressions were dramatically reduced in BSB8 cells at 48 and 72h post-radiation. On the other hand, while there was no significant alteration at 48h, large T-ag expression was induced by secondary radiation in BSB8-RR cells at 72h post-radiation (Figure 2C and 2D, compare lane 3 with 4 and 7 with 8). Interestingly, unlike large T-ag expression, small t-ag levels were not altered by secondary radiation in BSB8-RR cells. These data suggest that the alterations in the expression levels of tumor antigens before and after radiations in original BSB8 cells and BSB8-RR cells may have a role in the resistance to the radiation. BSB8-RR cells show increased cell-cycle rates and grow more aggressively than original BSB8 cells under anchorage dependent and independent conditions As shown above, BSB8-RR cells show alterations in viral tumor antigen expression and are resistant to Figure 2: BSB8-RR cells show resistance to secondary radiation. A. MTT assay on BSB8 and BSB8-RR control or irradiated with dose of 6-Gy at forty-eight hours after radiation. B. MTT assay on BSB8 and BSB8-RR control or irradiated with dose of 6-Gy at seventy-two hours after radiation. C. Western blot analysis of Large T-Ag and small-t in control and irradiated BSB8 and BSB8-RR cells at 48 and 72hrs post-radiation. D. Band intensities of Large T antigen and small t antigen from Western blot analysis in panel C were normalized to tubulin and shown as bar graph. Figure 2: BSB8-RR cells show resistance to secondary radiation. A. MTT assay on BSB8 and BSB8-RR control or irradiated with dose of 6-Gy at forty-eight hours after radiation. B. MTT assay on BSB8 and BSB8-RR control or irradiated with dose of 6-Gy at seventy-two hours after radiation. C. Western blot analysis of Large T-Ag and small-t in control and irradiated BSB8 and BSB8-RR cells at 48 and 72hrs post-radiation. D. Band intensities of Large T antigen and small t antigen from Western blot analysis in panel C were Figure 2: BSB8-RR cells show resistance to secondary radiation. A. MTT assay on BSB8 and BSB8-RR control or irradiated with dose of 6-Gy at forty-eight hours after radiation. B. MTT assay on BSB8 and BSB8-RR control or irradiated with dose of 6-Gy at seventy-two hours after radiation. C. Western blot analysis of Large T-Ag and small-t in control and irradiated BSB8 and BSB8-RR cells at 48 and 72hrs post-radiation. D. Band intensities of Large T antigen and small t antigen from Western blot analysis in panel C were normalized to tubulin and shown as bar graph. Genes & Cancer www.Genes&Cancer.com 133 how fast and efficient they can repair the DNA damage induced by radiation. As shown above, BSB8-RR cells were originated from the subpopulation of BSB8 cells after radiation and they were resistant to the secondary radiation. Moreover, they showed increased cell-cycle rates and grew more aggressively than original BSB8 cells. We next analyzed and evaluated double-strand break DNA repair efficiencies of BSB8 and BSB8-RR cells. Double- strand DNA breaks can be repaired by non-homologous DNA end joining (NHEJ) or homologous recombination (HR). BSB8-RR cells show increased cell-cycle rates and grow more aggressively than original BSB8 cells under anchorage dependent and independent conditions Regarding the NHEJ assay, nuclear extracts were prepared from BSB8 and BSB8-RR cells and analyzed for NHEJ activity as described in materials and methods. As shown in Figure 4A, nuclear extracts from BSB8-RR cells had a lower NHEJ activity than the nuclear extracts from the original BSB8 cells (compare lane 4 with 3) as significantly higher number of colonies under anchorage- dependent conditions than BSB8 cells as evident by the increased intensities of the colonies formed. Furthermore, BSB8-RR cells also formed significantly higher number of colonies in soft agar under anchorage-independent conditions (Figure 3C) suggesting that they acquire a greater tumorigenicity. BSB8-RR cells show reduced NHEJ but increased homologous recombination DNA repair Ionizing radiation holds its application by causing extensive double-strand DNA breaks in cancer cells leading to the activation of apoptosis and cell death. Upon radiation the fate of cancer cells is truly depended on Genes & Cance 134 www.Genes&Cancer.com Figure 3: Growth characteristics of BSB8 and BSB8-RR cells. A. Cell cycle analysis of BSB8 and BSB8-RR cells at three time points after FBS deprivation. B. BSB8-RR cells form increased number of colonies under anchorage-dependent conditions. Colony formation assay after transfection with pcDNA3.1 and selection with G418 treatments. C. BSB8-RR cells form increased number and size of colonies under anchorage independent conditions. Soft agar growth after transfection with pcDNA3.1 and selection with G418. Figure 3: Growth characteristics of BSB8 and BSB8-RR cells. A. Cell cycle analysis of BSB8 and BSB8-RR cells at three time points after FBS deprivation. B. BSB8-RR cells form increased number of colonies under anchorage-dependent conditions. Colony formation assay after transfection with pcDNA3.1 and selection with G418 treatments. C. BSB8-RR cells form increased number and size of colonies under anchorage independent conditions. Soft agar growth after transfection with pcDNA3.1 and selection with G418. Genes & Cancer www.Genes&Cancer.com 134 evident from the formation of dimer, trimer, and tetramer forms of the plasmid DNA. In addition, DNA repair activity of BSB8 and BSB8-RR cells was also analyzed by homologous recombination assay as described previously [29]. Cells were transiently transfected with pCBASceI, pDsRedl-Mito, and increasing amounts of pDRGFP plasmids by electroporation. Cells were harvested and analyzed by flow cytometry for the percentages of green and red fluorescein cells at 72h post-transfections. The efficiency in repairing double-strand DNA breaks using homologous recombination was established as the number of cells positive for both green and red fluorescence normalized to the cells positive only for red fluorescence. As shown in Figure 4B, number of GFP and Red-mito expressing cells were significantly higher in BSB8-RR cells than BSB8 cells. Since these experiments required transient transfection of three plasmids with considerably low co-transfection efficiencies, BSB8 and BSB8-RR cells were also transfected with pDRGFP plasmid and stable single-cell clones were generated. To confirm transient transfection results, BSB8 and BSB8-RR cells with stable pDRGFP expression were transfected with pDsRedl-Mito alone or in combination with increasing concentrations of pCBASceI plasmid. Cells were harvested and analyzed by flow cytometry for the number of green and red fluorescein cells at 96h post-transfections. BSB8-RR cells show reduced NHEJ but increased homologous recombination DNA repair www.Genes&Cancer.com Genes & Cancer 135 BSB8-RR cells than original BSB8 cells suggesting that BSB8-RR cells possess an increased rate of double-strand DNA repair by homologous recombination. editing strategy. Sub-cell lines of both BSB8 and BSB8- RR cells were first generated for doxycycline inducible expression of Cas9. Cells were then transduced with the combination of lentiviruses encoding three gRNAs targeting various regions within the viral early coding sequence (Figure 5A) as described previously [30]. Cells were either treated or untreated with doxycycline for the induction of Cas9 expression and cellular DNA were extracted for excision by PCR utilizing a pair of primers spanning the all gRNA target sites. As shown in Figure 5B, the excision products of 327bp and 824bp were amplified for gRNAs 1/3 and gRNAs 2/3, respectively, in both BSB8 and BSB8-RR cells. In parallel to the DNA samples, whole cell protein extracts were also collected from the same set of experiments. Expression of Cas9 and down-regulation of JCV tumor antigen expression were also analyzed and confirmed by Western blotting (Figure 5C). Finally, effect of tumor antigen knockout on growth and transformed BSB8-RR cells do no longer require JCV tumor antigen expression for the maintenance of the transformed phenotype The BSB8 cells were originally obtained from a mouse medulloblastoma developed in a mouse transgenic for JCV tumor antigens [11]. They show transformed phenotype and express viral early gene products (tumor antigens). Theoretically, their transformed phenotype and viabilities are depended on expression of viral tumor antigens. Next, we analyzed and compared the impact of JCV tumor antigen expression on transformed phenotype of BSB8 and BSB8-RR cells by CRISPR/Cas9 gene Figure 5: BSB8-RR cells do not require expression of viral tumor antigens for the maintenance of transformed phenotype. A. Schematic representation of JCV tumor antigen genes and position of gRNAs with expected size of amplification products upon cleavage by CRISPR/Cas9. B. PCR analysis of genomic DNA isolated from BSB8 and BSB8-RR cells stable for Cas9 expression upon induction with doxycycline and transduction with lentiviruses encoding gRNAs 1,2,3. C. Western blot analysis of whole cell protein lysates prepared from the same set of experiments presented in panel B for the detection of Cas9 and large T-ag. D. Colony formation assay and quantification of number of colonies in BSB8 and BSB8-RR cells after Cas9 induction with doxycycline. ure 5: BSB8-RR cells do not require expression of viral tumor antigens for the maintenan Figure 5: BSB8-RR cells do not require expression of viral tumor antigens for the maintenance of transformed phenotype. A. Schematic representation of JCV tumor antigen genes and position of gRNAs with expected size of amplification products upon cleavage by CRISPR/Cas9. B. PCR analysis of genomic DNA isolated from BSB8 and BSB8-RR cells stable for Cas9 expression upon induction with doxycycline and transduction with lentiviruses encoding gRNAs 1,2,3. C. Western blot analysis of whole cell protein lysates prepared from the same set of experiments presented in panel B for the detection of Cas9 and large T-ag. D. Colony formation assay and quantification of number of colonies in BSB8 and BSB8-RR cells after Cas9 induction with doxycycline. ells do not require expression of viral tumor antigens for the maintenance of transformed Figure 5: BSB8 RR cells do not require expression of viral tumor antigens for the maintenance of transformed phenotype. A. Schematic representation of JCV tumor antigen genes and position of gRNAs with expected size of amplification products upon cleavage by CRISPR/Cas9. B. PCR analysis of genomic DNA isolated from BSB8 and BSB8-RR cells stable for Cas9 expression upon induction with doxycycline and transduction with lentiviruses encoding gRNAs 1,2,3. C. BSB8-RR cells show reduced NHEJ but increased homologous recombination DNA repair As shown in Figure 4C, consistent with transient transfection studies, number of GFP and red positive cells were significantly higher in Genes & Cancer 135 www Genes&Cancer com Figure 4: DNA repair properties of BSB8 and BSB8-RR cells. A. Non-homologous end joining (NHEJ) activity in nuclear extracts from BSB8 and BSB8-RR cells. B. Representative scatter plots of untransfected cells, transfected BSB8 and BSB8-RR with pCBASceI and pDsRedl-Mito but not pDRGFP and BSB8 and BSB8-RR transfected with pCBASceI + pDsRedl-Mito + pDRGFP. Quantification of green positive cells after transfection with 3 ug and 6 ug of pDRGFP is represented as bar graph. C. Representative scatter plots of transfected BSB8 and BSB8-RR with pDsRedl-Mito and BSB8 and BSB8-RR transfected with pCBASceI + pDsRedl-Mito. Quantification of green positive cells after transfection with 3 ug and 6ug of pCBASceI is represented as bar graph. Figure 4: DNA repair properties of BSB8 and BSB8-RR cells. A. Non-homologous end joining Figure 4: DNA repair properties of BSB8 and BSB8-RR cells. A. Non-homologous end joining (NHEJ) activity in nuclear extracts from BSB8 and BSB8-RR cells. B. Representative scatter plots of untransfected cells, transfected BSB8 and BSB8-RR with pCBASceI and pDsRedl-Mito but not pDRGFP and BSB8 and BSB8-RR transfected with pCBASceI + pDsRedl-Mito + pDRGFP. Quantification of green positive cells after transfection with 3 ug and 6 ug of pDRGFP is represented as bar graph. C. Representative scatter plots of transfected BSB8 and BSB8-RR with pDsRedl-Mito and BSB8 and BSB8-RR transfected with pCBASceI + pDsRedl-Mito. Quantification of green positive cells after transfection with 3 ug and 6ug of pCBASceI is represented as bar graph. Figure 4: DNA repair properties of BSB8 and BSB8-RR cells. A. Non-homologous end joining (NHEJ) activity in nuclear extracts from BSB8 and BSB8-RR cells. B. Representative scatter plots of untransfected cells, transfected BSB8 and BSB8-RR with pCBASceI and pDsRedl-Mito but not pDRGFP and BSB8 and BSB8-RR transfected with pCBASceI + pDsRedl-Mito + pDRGFP. Quantification of green positive cells after transfection with 3 ug and 6 ug of pDRGFP is represented as bar graph. C. Representative scatter plots of transfected BSB8 and BSB8-RR with pDsRedl-Mito and BSB8 and BSB8-RR transfected with pCBASceI + pDsRedl-Mito. Quantification of green positive cells after transfection with 3 ug and 6ug of pCBASceI is represented as bar graph. BSB8-RR cells do no longer require JCV tumor antigen expression for the maintenance of the transformed phenotype Western blot analysis of whole cell protein lysates prepared from the same set of experiments presented in panel B for the detection of Cas9 and large T-ag. D. Colony formation assay and quantification of number of colonies in BSB8 and BSB8-RR cells after Cas9 induction with doxycycline. www.Genes&Cancer.com Genes & Cancer 136 phenotype of BSB8 and BSB8-RR cells were analyzed by colony formation assay. As shown in Figure 5D, as expected, BSB8-RR cells were able to form much higher numbers of colonies than BSB8 cells. Interestingly, BSB8 cells with doxycycline treatment leading tumor antigen knockout showed a dramatic reduction in the number of colonies compared to control BSB8 cells, suggesting that the growth and colony forming ability of BSB8 cells were truly depended on JCV tumor antigen expression. On the other hand, number of colonies formed by BSB8-RR cells with tumor antigen knockout was slightly reduced compared to the control cells (compare lane 3 with 4), but it was still significantly higher than original BSB8 cells with tumor antigen expression (compare lanes 4 and 1) suggesting that JCV tumor antigen expression was no longer needed for the maintenance of the transformed phenotype in BSB8-RR cells. dependent and -independent conditions suggesting that adaptation to the radiation led to the more aggressive tumor phenotype of these cells. Further analysis for the possible mechanism of this adaptation and change in growth phenotype of the BSB8-RR cells revealed that they possessed an increase rate of double-strand DNA break repair. These results are in agreement with previous observations indicating that radiation-resistant cancer cells can gain adaptation to repair double-strand breaks induced by radiation [34, 35, 36]. On the other hand, only a small subset of cells shows resistance to the radiation while the majority of the cells are highly responsive. Although cancers become more heterogeneous during the course of tumor development [37], factors contributing to the radiation resistance in a small subset of tumor cells and role of viral tumor antigens in this progress remains to be determined. Whether JCV is involved in tumors in human have been debated since the virus first isolated. It is without any concern that JCV can transform primary cells in vitro and lead to formation of various tumors in experimental animals [7, 8, 9, 10, 11, 12, 13]. BSB8-RR cells do no longer require JCV tumor antigen expression for the maintenance of the transformed phenotype Moreover, viral tumor antigens and genomic sequences were detected in human neuronal origin tumors, including glioblastoma, oligodendroglioma, and medulloblastoma by our group and various labs across the globe [14, 15, 16, 17, 18, 19, 20]. Among all polyomaviruses, merkel cell polyomavirus (MCPyV) is the only polyomavirus discovered as the underlying etiology of human merkel cell carcinoma, an aggressive form of skin cancer [38, 39]. MCPyV and JCV show very similar genomic organization with conserved homologies in viral tumor antigen sequences. Recent studies indicate that viral tumor antigen expression is required for the growth of merkel cell carcinoma cells [40, 41]. Unlike MCPvY, our results suggest for the first time that the growth of tumor cells induced by JCV does no longer required the expression of tumor antigens in order to sustain their transformed phenotype. This unique observation may have implications for possible involvement of JCV in the formation of human tumors with CNS origin. One possibility is that the expression of viral tumor antigens in the absence of viral replication may initiate the cellular transformation process in glial cells persistently infected with the virus. Due to the additional insults, such as UV irradiation and DNA damaging agents/ chemicals, tumor cells may gradually gain adaptation and tolerate the absence of viral tumor antigen expression for their growth. This type of phenomena of tumorigenesis can be explained by “hit-and-run” strategy as proposed previously for JCV [42]. Likewise, “hit-and-run” mechanism has also been proposed in the transformation initiated by the viral oncoproteins expressed by adenovirus [43], Hepatitis B virus [44], and more recently for the Beta HPV38 virus [45]. In line with these observations, our results support the concept that tumor antigens expressed by JCV act at the initial stages of the cellular DISCUSSION JCV infection in experimental animals, including hamsters, nonhuman primates, and mice results in cellular transformation and tumor development rather than lytic viral replication suggesting that viral early promoter is activated for the expression of tumor antigens leading to genome instability and inactivation of oncosuppressor proteins that eventually may lead to tumorigenesis [31]. Radiotherapy is commonly used as a part of cancer therapy in patients with many different forms of localized tumors before and after surgical operations. Regarding postsurgical application, radiation is commonly used to eliminate any remaining tumor cells at the side of surgery. Unfortunately, some small subset of tumor cells may become resistant to radiation and be responsible for the tumor recurrence. In order to gain insight into the effects of radiation on tumor cell growth, we utilized BSB8 cells derived from a medulloblastoma developed in a mouse transgenic for JCV [11]. As expected, radiation of BSB8 cells resulted in induction of autophagy along with apoptosis in the majority of the cells in the culture plate. Interestingly, a small subset of cells survived, become resistance to repeated dosages of radiation with reduced levels of viral tumor antigen expression. Since the JCV tumor antigens were the main driver of transformation in these cells, the reduction in its expression levels in BSB8-RR cells was not anticipated. JCV large tumor antigen is a phosphoprotein, which interacts with two key tumor suppressor proteins that regulate cell cycle progression, pRb and p53 [32, 33]. Possible alteration of its posttranslational modification, interaction with p53 and Rb proteins, and binding to viral promoter in BSB8-RR compared to original BSB8 cells remains to be further characterized. Our results have revealed several interesting features of BSB8-RR cells that include an increase in cell cycle rate and number of colonies formed under anchorage- www.Genes&Cancer.com Genes & Cancer 137 transformation and secondary insults, such as radiation, may relief the cells for the need of their expression. membranes were blocked for 30 min at room temperature with 10% non-fat dry milk in 1X phosphate-buffered saline containing 0.1% Tween-20 (PBST). Membranes were then incubated with primary antibodies overnight at 4°C. The membranes were subsequently washed three times with PBST and incubated with secondary antibodies at a 1:5000 dilution for 1 h at room temperature. After secondary antibodies incubation, membranes were visualized with an Odyssey CLx Imaging System (LI- COR). SDS-PAGE and western blotting Whole cell protein extracts were prepared from BSB8, BSB8-RR, and BS1A cells with TNN lysis buffer (40 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 1% NP40, and 1% protease inhibitors cocktail). Protein concentrations were quantified using Bradford reagent. Protein extracts were then heated to 95°C for 5 min and resolved through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then they were transferred to nitrocellulose membranes in a transfer buffer containing 25 mM Tris base (pH 7.4), 200 mM glycine, and 20% methanol. After transfer, MATERIAL AND METHODS BSB8 and BSB8-RR cells were harvested at three different time point: 0h, 12h, 24h. Cells were washed with PBS, re-suspended in 1 ml of PBS and added drop-wise to 4 ml of 88% ethanol for the fixation in a final concentration of 70% ethanol. Cells were then washed with PBS, re- suspended in 300 µl of PBS and incubated with 10 µg/ ml of propidium iodide and 100ug/ml solution of RNase A at 37°C for 30 minutes at dark. After incubation, cells were cooled at 4°C and data were acquired using a Guava EasyCyte Mini flow cytometer (Guava Technologies). MTT assay for cell viability To assess tumorigenesis, BSB8 and BSB8-RR cells either control or clones with inducible expression of Cas9 were plated in 100 mm tissue culture dishes. BSB8-Cas9 and BSB8-RR-Cas9 cells were transduced with lentiviral constructs encoding gRNAs 1, 2, and 3 for JCV early region and plated for colony formation assays in the presence or absence of doxycycline. Cells were kept in DMEM complete medium for two to three weeks till colonies were formed and visually observable. Colonies were fixed and stained with 0.1% crystal violet in 20% (vol/vol) aqueous methanol solution. BSB8, BSB8-RR or BS1A cells were plated in a concentration of 3 x 105 cells / well in 6-well tissue culture plates and irradiated with different amount of ionizing radiation (3-Gy, 6-Gy or 12-Gy). 48 hours after treatment, cells were incubated with 1 ml of MTT (3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide), working solution (0.5 mg/ml) for 2 hours at 37°C. The converted insoluble purple formazan was solubilized with 1 ml of acidic isopropanol (0.004 M HCl in isopropanol). Absorbance of the converted formazan was measured at a wavelength of 570 nm with a background subtraction at 650 nm. Cell lines and cultures The mouse medulloblastoma cell line expressing JCV tumor antigens (BSB8) and BS1A cells were previously described [11]. BSB8-radiation resistant (BSB8-RR) cells were obtained from radiation resistant subpopulation of BSB8 cells. BS1A, BSB8 and BSB8-RR cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5% heat-inactivated FBS and 100µg/ ml penicillin/streptomycin. All the cells were maintained at 37°C in a humidified atmosphere with 7% CO2. DISCUSSION In conclusion, our results suggest that radiation triggers a cascade of events in tumor cells induced by JCV leading to elimination of their dependency for the expression of viral tumor antigens. These observations may suggest possible involvement of JCV in human tumors and open a new avenue of research to better understand and characterize the role of JCV in human tumors with CNS origin. Soft agar growth BSB8 and BSB8-RR cells were transfected with pcDNA3.1 (+) and after 24 hours harvested and seeded in 60-mm dishes containing 2 mL of a 0.3% agarose suspension in DMEM complete medium with G418. Plates were then incubated at 37°C with for three to four weeks. Colonies were fixed and stained with 0.1% crystal violet in 20% (vol/vol) aqueous methanol solution and counted. Nonhomologous end joining assay for double strand DNA break repair Cells were harvested, washed with ice-cold PBS and re-suspended in ice-cold hypotonic lysis buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT and protease inhibitors) and left on ice for 15 minutes. Integrity of cellular membrane was disrupted by adding 50 µl 10% NP-40 solution per 500µl cell suspension and gently vortexing for 10 seconds. Cell nucleus was pelleted by centrifugation at 1,800 rpm for 5 minutes and the supernatants were aliquoted as cytoplasmic extracts. Nuclear pellets were re-suspended in ice-cold PBS and washed to remove any remaining cytoplasmic proteins. Nuclear pellets then were re- suspended in ice-cold hypertonic nuclear lysis buffer C (20 mM HEPES pH 7.9, 0.6 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM DTT, 0.5 mM PMSF and protease inhibitors), rotated 45 minutes at 4°C, and the nuclear lysates were obtained by centrifugation at 15k rpm for 15 minutes. Supernatants were aliquoted as nuclear extracts and dialyzed overnight against a dialyzing buffer consist of 25 mM Tris HCl (pH 7.5), l mM EDTA, 10% glycerol (vol/vol) and protease inhibitors.The NHEJ assay was performed as follows. pBlueScript(KS+) plasmid was linearized by restriction endonuclease digestion in order to obtain a four nucleotides 5′ overhang using Bam HI, and 3’- blunt-end using EcoR V restriction enzymes. The resulting 3 Kb linear DNA was used as DNA substrate for the end-joining reactions. Reactions were set up in 25 mM Tris OAc (pH 7.5), 100 mM KOAc, 10 mM MgOAc, 1 mM DTT, 2 mM ATP and 200 mM dNTPs. In all reaction, total of 50 μg nuclear extract from BSB8 or BSB8-RR cell were utilized. The reactions were incubated for 5 min at 37°C before addition of 400 ng of DNA substrate followed by one hour incubation at 37°C. DNA products were de- proteinized by treatment with proteinase K and analyzed by electrophoresis on a 0.7% agarose gel. detected and quantified using Guava EasyCyte Mini flow cytometer (Guava Technologies). Sub-cell lines of BSB8 and BSB8-RR cells were also obtained for stable expression of DRGFP as follow: BSB8 and BSB8-RR cells were electroporated with pDRGFP plasmid. Twenty- four hours after electroporation, cells were harvested and re-plated in 100-mm dishes with complete DMEM media and puromycin for selection. BSB8-DRGFP and BSB8- RR-DRGFP cells were then electroporated with increasing concentrations of pCBASceI and pDsRedl-Mito. CRISPR/Cas9 system BSB8 and BSB8-RR were plated in 6 well plates and transduced with a lentiviral vector encoding Cas9 in presence of polybrene (8 ug/ml) as previously described [30]. At twenty-four hours post-transduction, cells were plated in 100-mm dishes at low density and kept in DMEM complete medium with puromycin for selection. Cas9 expression was induced with doxycycline (2ug/ ml) and expression screened by Western blotting. Single cell line clones were selected and screened for Cas9 expression. Cas9 clones were then plated in 6-well tissue culture dishes and transduced with a combination of three lentiviral vectors encoding gRNAs (m1, m2 and m3). At twenty-four hour post-transductions, cells were treated with doxycycline for the induction of Cas9 expression. At 48hrs post-inductions, cells were either harvested for whole cell protein and genomic DNA extraction or re- plated for colony formation assay as described above. The excision of JCV tumor antigens were analyzed by PCR using primers that flank the JCV large T antigen coding region (5’- GCTTATGCCATGCCCTGAAGGT-3’ and 5’-ATGGACAAAGTGCTGAATAGGGA-3’). PCR products were separated on 2% agarose gel by electrophoresis and amplified products were visualized by ethidium bromide staining. Homologous recombination assay for double strand DNA break repair The homologous recombination (HR) events were analyzed as previously described (Pierce et al., 1999) with some modifications. For the transient transfection, BSB8 and BSB8-RR cells were electroporated with expression plasmids pDRGFP (recombination substrate), pCBASceI (encoding I-SceI endonuclease), and pDsRedl-Mito (Clontech, Paolo Alto, CA). The expression of I-SceI leads to a double strand break (DSB) in the specific restriction site within the pDRGFP cassette and HR events in the cells can restore the functional GFP expression. The co-transfection with pDsRedl-Mito, a plasmid that encodes for a red fluorescent protein with a mitochondrial localization, was used to calculate the efficiency of the transfection. Seventy-two hours after electroporation, cells were harvested and GFP and RFP expressions were Nonhomologous end joining assay for double strand DNA break repair The nonhomologous end joining (NHEJ) assay was performed by a modification of the method of Baumann and West, 1998 as previously described [46]. Nuclear www.Genes&Cancer.com Genes & Cancer 138 protein extracts from BSB8 and BSB8-RR cells were prepared as follows. Cells were harvested, washed with ice-cold PBS and re-suspended in ice-cold hypotonic lysis buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT and protease inhibitors) and left on ice for 15 minutes. Integrity of cellular membrane was disrupted by adding 50 µl 10% NP-40 solution per 500µl cell suspension and gently vortexing for 10 seconds. Cell nucleus was pelleted by centrifugation at 1,800 rpm for 5 minutes and the supernatants were aliquoted as cytoplasmic extracts. Nuclear pellets were re-suspended in ice-cold PBS and washed to remove any remaining cytoplasmic proteins. Nuclear pellets then were re- suspended in ice-cold hypertonic nuclear lysis buffer C (20 mM HEPES pH 7.9, 0.6 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM DTT, 0.5 mM PMSF and protease inhibitors), rotated 45 minutes at 4°C, and the nuclear lysates were obtained by centrifugation at 15k rpm for 15 minutes. Supernatants were aliquoted as nuclear extracts and dialyzed overnight against a dialyzing buffer consist of 25 mM Tris HCl (pH 7.5), l mM EDTA, 10% glycerol (vol/vol) and protease inhibitors.The NHEJ assay was performed as follows. pBlueScript(KS+) plasmid was linearized by restriction endonuclease digestion in order to obtain a four nucleotides 5′ overhang using Bam HI, and 3’- blunt-end using EcoR V restriction enzymes. The resulting 3 Kb linear DNA was used as DNA substrate for the end-joining reactions. Reactions were set up in 25 mM Tris OAc (pH 7.5), 100 mM KOAc, 10 mM MgOAc, 1 mM DTT, 2 mM ATP and 200 mM dNTPs. In all reaction, total of 50 μg nuclear extract from BSB8 or BSB8-RR cell were utilized. The reactions were incubated for 5 min at 37°C before addition of 400 ng of DNA substrate followed by one hour incubation at 37°C. DNA products were de- proteinized by treatment with proteinase K and analyzed by electrophoresis on a 0.7% agarose gel. protein extracts from BSB8 and BSB8-RR cells were prepared as follows. Nonhomologous end joining assay for double strand DNA break repair After five days, cells were harvested and GFP and RFP expressions were detected and quantified as described above. The HR efficiency was established as the number of GFP positive cells with red mitochondrial fluorescence co-localization. REFERENCES 15. Castaigne P, Rondot P, Escourolle R, Ribadeau dumas JL, Cathala F, Hauw JJ. Progressive multifocal leukoencephalopathy and multiple gliomas.Rev Neurol (Paris). 1974 Sep-Oct; 130:379-92. 1. Boukoum H, Nahdi I, Sahtout W, Skiri H, Segondy M, Aouni M. BK and JC virus infections in healthy patients compared to kidney transplant recipients in Tunisia. Microb Pathog. 2016 Aug; 97:204-8. 1. Boukoum H, Nahdi I, Sahtout W, Skiri H, Segondy M, Aouni M. BK and JC virus infections in healthy patients compared to kidney transplant recipients in Tunisia. Microb Pathog. 2016 Aug; 97:204-8. 16. Shintaku M, Matsumoto R, Sawa H, Nagashima K. Infection with JC virus and possible dysplastic ganglion- like transformation of the cerebral cortical neurons in a case of progressive multifocal leukoencephalopathy. J Neuropathol Exp Neurol. 2000 Oct; 59:921-9. 2. Chattaraj S, Bera NK, Dutta C, Bhattacharjee S. Quantification of human polyomavirus JC virus load in urine and blood samples of healthy tribal populations of North-Eastern part of West Bengal, India. Indian J Med Microbiol. 2015 Oct-Dec; 33:491-5. 17. Boldorini R, Caldarelli-Stefano R, Monga G, Zocchi M, Mediati M, Tosoni A, Ferrante P. PCR detection of JC virus DNA in the brain tissue of a 9-year-old child with pleomorphic xanthoastrocytoma. J Neurovirol. 1998 Apr; 4:242-5. 3. Van Loy T, Thys K, Tritsmans L, Stuyver LJ. Quasispecies analysis of JC virus DNA present in urine of healthy subjects. PLoS One. 2013 Aug 15; 8:e70950. 18. Rencic A, Gordon J, Otte J, Curtis M, Kovatich A, Zoltick P, Khalili K, Andrews D. Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma. Proc Natl Acad Sci U S A. 1996 Jul 9; 93:7352-7. 4. Mandl C, Walker DL, Frisque RJ. Derivation and characterization of POJ cells, transformed human fetal glial cells that retain their permissivity for JC virus. J Virol. 1987 Mar; 61:755-63. 5. Daniel AM, Frisque RJ. Transcription initiation sites of prototype and variant JC virus early and late messenger RNAs. Virology. 1993 May; 194:97-109. 19. Del Valle L, Gordon J, Assimakopoulou M, Enam S, Geddes JF, Varakis JN, Katsetos CD, Croul S, Khalili K. Detection of JC virus DNA sequences and expression of the viral regulatory protein T-antigen in tumors of the central nervous system. Cancer Res. 2001 May 15; 61:4287-93. 6. Uleri E, Beltrami S, Gordon J, Dolei A, Sariyer IK. ACKNOWLEDGMENTS The authors thank Dr. Kamel Khalili for sharing ideas and critical reading of the manuscript. We would like to thank Dr. Rafal Kaminski for sharing the plasmids for NHEJ and HR assays. We would like to also thank Dr. Jennifer Gordon for providing BSB8 cells. We also thank Dr. Nicoletta Basilico for helpful suggestions and comments. This study utilized services offered by core www.Genes&Cancer.com Genes & Cancer 139 ubiquitous JCV(CY) T-antigen gene induces brain tumors in experimental animals. Oncogene. 1999 Jan 7; 18:39-46. facilities of the Comprehensive NeuroAIDS Center (CNAC NIMH Grant Number P30MH092177) at Temple University Lewis Katz School of Medicine. This work was made possible by grants awarded by NIH to IKS (AI101192) 12. Franks RR, Rencic A, Gordon J, et al. Formation of undifferentiated mesenteric tumors in transgenic mice expressing human neurotropic polymavirus early protein. Oncogene. 1996; 12:2573-8. Author contributions 13. Gordon J, Del Valle L, Otte J, Khalili K. Pituitary neoplasia induced by expression of human neurotropic polyomavirus, JCV, early genome in transgenic mice. Oncogene. 2000; 19:4840-6. Conceived and designed the experiments: IKS. Performed the experiments: MD, RS, HW, and AB. Analyzed the data: IKS and MD. Contributed reagents/ materials/analysis tools: IKS. Wrote the paper: MD and IKS. 14. Sima AA, Finkelstein SD, McLachlan DR. Multiple malignant astrocytomas in a patient with spontaneous progressive multifocal leukoencephalopathy. Ann Neurol. 1983 Aug; 14:183-8. REFERENCES Gan DD, Khalili K. Interaction between JCV large T-antigen and beta-catenin. Oncogene. 2004 Jan 15; 23:483-90. 38. Feng H, Shuda M, Chang Y, Moore PS. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science. 2008; 319:1096-1100. 27. Shollar D, Del Valle L, Khalili K, Otte J, Gordon J. JCV T-antigen interacts with the neurofibromatosis type 2 gene product in a transgenic mouse model of malignant peripheral nerve sheath tumors. Oncogene. 2004 Jul 15; 23:5459-67. 39. Feng H, Taylor JL, Benos PV, Newton R, Waddell K, Lucas SB, Chang Y, Moore PS. Human transcriptome subtraction by using short sequence tags to search for tumor viruses in conjunctival carcinoma. J Virol. 2007; 81:11332-11340. 28. Piña-Oviedo S, Urbanska K, Radhakrishnan S, Sweet T, Reiss K, Khalili K, Del Valle L. Effects of JC virus infection on anti-apoptotic protein survivin in progressive multifocal leukoencephalopathy. Am J Pathol. 2007 Apr; 170:1291-304. 40. Houben R, Adam C, Baeurle A, Hesbacher S, Grimm J, Angermeyer S, Henzel K, Hauser S, Elling R, Bröcker EB, Gaubatz S, Becker JC, Schrama D. An intact retinoblastoma protein-binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cells. Int J Cancer. 2012 Feb 15; 130:847-56. 29. Pierce AJ, Johnson RD, Thompson LH, Jasin M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 1999 Oct 15; 13:2633-8. 41. Angermeyer S, Hesbacher S, Becker JC, Schrama D, Houben R. Merkel cell polyomavirus-positive Merkel cell carcinoma cells do not require expression of the viral small T antigen. J Invest Dermatol. 2013 Aug; 133:2059-64. 30. Wollebo HS, Bellizzi A, Kaminski R, Hu W, White MK, Khalili K. CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection. PLoS One. 2015 Sep 11; 10:e0136046. 42. Khalili K, Del Valle L, Otte J, Weaver M, Gordon J. Human neurotropic polyomavirus, JCV, and its role in carcinogenesis. Oncogene. 2003 Aug 11; 22:5181-91. 31. Delbue S, Comar M and Ferrantep. Review on the role of the human Polyomavirus JC in the development of tumors. Infect Agent Cancer, 2017. 12: p. 10. 43. Nevels M, Täuber B, Spruss T, Wolf H, Dobner T. “Hit-and- run” transformation by adenovirus oncogenes. J Virol. 2001 Apr; 75:3089-94. 32. Khalili K, Del Valle L, Otte J, Weaver M, Gordon J. Human neurotropic polyomavirus, JCV, and its role in carcinogenesis. Oncogene. 2003 Aug 11; 22:5181-91. 44. REFERENCES Extinction of Tumor Antigen Expression by SF2/ASF in JCV-Transformed Cells. Genes Cancer. 2011 Jul; 2:728-36. 20. Del Valle L, Enam S, Lara C, Ortiz-Hidalgo C, Katsetos CD, Khalili K. Detection of JC polyomavirus DNA sequences and cellular localization of T-antigen and agnoprotein in oligodendrogliomas. Clin Cancer Res. 2002 Nov; 8:3332- 40. 7. Walker DL, Padgett BL, ZuRhein GM, Albert AE, Marsh RF. uman papovavirus (JC): induction of brain tumors in hamsters. Science. 1973 Aug 17; 181:674-6. 8. Varakis J, ZuRhein GM, Padgett BL, Walker DL. Induction of peripheral neuroblastomas in Syrian hamsters after injection as neonates with JC virus, a human polyoma virus. Cancer Res. 1978 Jun; 38:1718-22. 21. Reiss K, Khalili K. Viruses and cancer: lessons from the human polyomavirus, JCV. Oncogene. 2003 Sep 29; 22:6517-23. 9. London WT, Houff SA, Madden DL, Fuccillo DA, Gravell M, Wallen WC, Palmer AE, Sever JL, Padgett BL, Walker DL, ZuRhein GM, Ohashi T. Brain tumors in owl monkeys inoculated with a human polyomavirus (JC virus). Science. 1978 Sep 29; 201:1246-9. 22. Bollag B, Prins C, Snyder EL, Frisque RJ. Purified JC virus T and T’ proteins differentially interact with the retinoblastoma family of tumor suppressor proteins. Virology. 2000 Aug 15; 274:165-78. 23. Felsani A, Mileo AM, Paggi MG. Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins. Oncogene. 2006 Aug 28; 25:5277-85. 10. London WT, Houff SA, McKeever PE, Wallen WC, Sever JL, Padgett BL, Walker DL. Viral-induced astrocytomas in squirrel monkeys. Prog Clin Biol Res. 1983; 105:227-37. 24. Enam S., Del Valle L., Lara C., Gan D.D., Ortiz-Hidalgo C., 11. Krynska B, Otte J, Franks R, Khalili K, Croul S. Human www.Genes&Cancer.com Genes & Cancer 140 Palazzo J.P., Khalili K. Association of human polyomavirus JCV with colon cancer: evidence for interaction of viral T-antigen and beta- catenin. Cancer Res. 2002; 62:7093- 7101 36. Nickson CM, Moori P, Carter RJ, Rubbi CP, Parsons JL. Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity. Oncotarget. 2017 May 2; 8:29963-29975. 25. Lassak A, Del Valle L, Peruzzi F, Wang JY, Enam S, Croul S, Khalili K, Reiss K. Insulin receptor substrate 1 translocation to the nucleus by the human JC virus T-antigen. J Biol Chem. 2002 May 10; 277:17231-8. 37. Dagogo-Jack I, Shaw AT. Tumor heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol. 2018 Feb; 15:81-94. 26. REFERENCES Hessein M, Saad el G, Mohamed AA, Kamel el AM, Abdel Hady AM, Amina M, Rogler CE. Hit-and-run mechanism of HBV-mediated progression to hepatocellular carcinoma. Tumori. 2005 May-Jun; 91:241-7. 33. White MK, Khalili K. Polyomaviruses and human cancer: Molecular mechanisms underlying patterns of tumorigenesis. Virology. 2004; 324:1-16. 45. Viarisio D, Müller-Decker K, Accardi R, Robitaille A, Dürst M, Beer K, Jansen L, Flechtenmacher C, Bozza M, Harbottle R, Voegele C, Ardin M, Zavadil J, Caldeira S, Gissmann L, Tommasino M. Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation- induced skin carcinogenesis in mice. PLoS Pathog. 2018 Jan 11; 14:e1006783. 34. Schwartz JL, Rotmensch J, Giovanazzi S, Cohen MB, Weichselbaum RR. Faster repair of DNA double-strand breaks in radioresistant human tumor cells. Int J Radiat Oncol Biol Phys. 1988 Oct; 15:907-12. 35. Eguchi-Kasai K, Kosaka T, Sato K, Kaneko I. Reparability of DNA double-strand breaks and radiation sensitivity in five mammalian cell lines. Int J Radiat Biol. 1991 Jan; 59:97-104. 46. Kaminski R, Darbinyan A, Merabova N, Deshmane SL, White MK, Khalili K. Protective role of Puralpha to cisplatin. Cancer Biol Ther. 2008 Dec; 7:1926-35. www.Genes&Cancer.com Genes & Cancer 141
https://openalex.org/W3037218930	https://www.biorxiv.org/content/biorxiv/early/2020/06/24/2020.06.23.167387.full.pdf	English	null	Genetic diversity increases food-web persistence in the face of climate warming	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	7,544	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint Genetic diversity increases food-web persistence in the face of climate warming Matthew A. Barbour,1⇤Daniel J. Kliebenstein,2 Jordi Bascompte1 1Department of Evolutionary Biology and Environmental Studies, University of Zurich, 8057 Zurich, Switzerland 2Department of Plant Science, University of California Davis, Davis 95616 CA, USA ⇤To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh.ch. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. Genetic diversity provides the raw material for species to adapt and persist in the face of climate change. Yet, the extent to which these genetic effects scale at the level of ecological communities remains unclear. Here we experimentally test the effect of plant genetic diversity on the persistence of an insect food web under a current and future warming scenario. We found that plant genetic di- versity increased food-web persistence by increasing the intrinsic growth rates of species across multiple trophic levels. This positive effect was robust to a 3◦C warming scenario and resulted from allelic variation at two genes that control the biosynthesis of chemical defenses. Our results suggest that the on- going loss of genetic diversity may undermine the persistence and functioning of ecosystems in a changing world. 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint One Sentence Summary: The loss of genetic diversity accelerates the extinction of inter- connected species from an experimental food web. Main Text: Gene-to-ecosystem processes sustain life on Earth. For example, genes encode information that determines an organism’s phenotype and ﬁtness in the environment (1), which 5 in turn plays a fundamental role in determining its trophic interactions with other species (2,3). Similarly, the strength and organization of trophic interactions in a food web play an impor- tant role in maintaining species diversity across multiple trophic levels (4, 5). Predicting how living systems will respond to ongoing climate change, therefore, requires a mechanistic un- derstanding of linkages across biological scales (6,7). Given that the current rate of population 10 extinction, and subsequent loss of genetic diversity, is orders of magnitude higher than the rate of species extinction (8), there is a pressing need to know to what degree this loss of genetic diversity will undermine the persistence of food webs in a changing world. To understand how gene-to-ecosystem processes will respond to climate change, we used an experimental food web consisting of a plant (Arabidopsis thaliana), two species of aphids (Bre- 15 vicoryne brassicae and Lipaphis erysimi), and a parasitoid wasp (Diaeretiella rapae) (Fig. 1A). To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. These species form a naturally occurring food web (10, 11), which contains three of the most common interaction structures found in natural food webs, including resource competition (aphid-plant-aphid), apparent competition (aphid-parasitoid-aphid), and a food chain (plant- To understand how gene-to-ecosystem processes will respond to climate change, we used an experimental food web consisting of a plant (Arabidopsis thaliana), two species of aphids (Bre- 15 vicoryne brassicae and Lipaphis erysimi), and a parasitoid wasp (Diaeretiella rapae) (Fig. 1A). These species form a naturally occurring food web (10, 11), which contains three of the most common interaction structures found in natural food webs, including resource competition (aphid-plant-aphid), apparent competition (aphid-parasitoid-aphid), and a food chain (plant- aphid-parasitoid) (12). Interactions in this food web are partly mediated by a group of spe- 20 cialized metabolites called aliphatic glucosinolates (10). Extensive knowledge of genotype-to- phenotype causality in Arabidopsis aliphatic glucosinolates (Fig. 1B) provides a system to test how genetic change inﬂuences ecological interactions in a food web. We used 3 transgenic lines that recreate natural null alleles in the aliphatic glucosinolate pathway (MAM1, AOP2, and GSOH), which determine natural variation in aliphatic glucosinolates across multiple Brassi- 25 2 2 MAM1 AOP2 GS-OH gsm1 AOP2 AOP2/gsoh Col-0 A B Fig. 1. Study system. (A) In our experimental food web, the parasitoid Diaeretiella rapae (top) parasitizes the aphids Brevicoryne brassicae (left) and Lipaphis erysimi (right), and these aphids compete for their shared resource Arabidopsis thaliana (bottom). This food-web dia- gram represents our prior expectation of food-web structure, where solid and dashed arrows represent positive and negative effects, respectively. (B) To manipulate genetic diversity, we used 3 transgenic lines of Arabidopsis (gsm1, AOP2, AOP2/gsoh) that alter the biosynthesis of aliphatic glucosinolates in a common genetic background (Col-0 accession). The chemical phe- notype (3MSO, 4MSO, But-3-enyl, or OH-But-3-enyl) of each Arabidopsis genotype depends on which genes (MAM1, AOP2, and GS-OH) have functional alleles (details given in note 9). The glucosinolate pathway is adapted from Fig. 1 in ref. 1. A B A MAM1 gsm1 AOP2 GS-OH AOP2 AOP2/gsoh AOP2 Fig. 1. Study system. (A) In our experimental food web, the parasitoid Diaeretiella rapae (top) parasitizes the aphids Brevicoryne brassicae (left) and Lipaphis erysimi (right), and these aphids compete for their shared resource Arabidopsis thaliana (bottom). This food-web dia- gram represents our prior expectation of food-web structure, where solid and dashed arrows represent positive and negative effects, respectively. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. (B) To manipulate genetic diversity, we used 3 transgenic lines of Arabidopsis (gsm1, AOP2, AOP2/gsoh) that alter the biosynthesis of aliphatic glucosinolates in a common genetic background (Col-0 accession). The chemical phe- notype (3MSO, 4MSO, But-3-enyl, or OH-But-3-enyl) of each Arabidopsis genotype depends on which genes (MAM1, AOP2, and GS-OH) have functional alleles (details given in note 9). The glucosinolate pathway is adapted from Fig. 1 in ref. 1. 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint cales species (13–17), in a common genetic background (Col-0 accession, Fig. 1B). Together, the Col-0 accession and transgenic lines reproduce most of the natural variation in the chemical phenotypes of Arabidopsis accessions that co-occur throughout Europe and determine ﬁtness under ﬁeld conditions (1, 9, 18). We created experimental plant populations from all possi- cales species (13–17), in a common genetic background (Col-0 accession, Fig. 1B). Together, the Col-0 accession and transgenic lines reproduce most of the natural variation in the chemical phenotypes of Arabidopsis accessions that co-occur throughout Europe and determine ﬁtness under ﬁeld conditions (1, 9, 18). We created experimental plant populations from all possi- ble combinations of Arabidopsis genotypes (n = 11 combinations across 60 experimental food 30 webs), including monocultures (n = 4), two-genotype mixtures (n = 6), and the four-genotype mixture (n = 1). This experimental design allowed us to test both a general effect of genetic diversity and isolate the contribution of allelic variation at speciﬁc genes. ble combinations of Arabidopsis genotypes (n = 11 combinations across 60 experimental food 30 webs), including monocultures (n = 4), two-genotype mixtures (n = 6), and the four-genotype mixture (n = 1). This experimental design allowed us to test both a general effect of genetic diversity and isolate the contribution of allelic variation at speciﬁc genes. ble combinations of Arabidopsis genotypes (n = 11 combinations across 60 experimental food 30 webs), including monocultures (n = 4), two-genotype mixtures (n = 6), and the four-genotype mixture (n = 1). This experimental design allowed us to test both a general effect of genetic diversity and isolate the contribution of allelic variation at speciﬁc genes. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. To test the sensitivity of these genetic effects to climate change, we conducted the experi- ment under two different, constant temperature regimes (20◦C and 23◦C). We chose these tem- 35 peratures to reﬂect the warming this food web is expected to experience over the next 25–50 years (19). More generally, warming can simultaneously modify processes at different biolog- ical scales (6). This is because temperature fundamentally determines an organism’s metabolic rate, which can scale up to alter the strength and stability of trophic interactions with other species (20). 40 After adding the experimental food web to each plant population, we tracked the population dynamics of each species for 17 weeks, allowing for multiple generations of aphids (⇠16 gener- ations) and parasitoids (⇠8 generations). This allowed us to track critical transitions in the food web over time (Fig. 2). In the context of this paper, critical transitions refer to local extinctions that simplify the food web (21). 45 Warming had an immediate effect on food-web persistence (Fig. 2, Table S1). Speciﬁcally, a 1◦C increase in warming accelerated the extinction of the aphid Brevicoryne brassicae, resulting in a simpler food chain (yellow to green in Fig. 2, S1–S3). However, warming did not alter the risk of a critical transition in the remaining food chain (green to either blue or purple in Fig. 2, Table S2). Although counterintuitive, warming increased the odds that the food web was 50 4 Warming (+1°C) Genetic diversity (+1 genotype) +59% -11% n.t. -26% +8% n.t. -9% -22% -53% -19% Fig. 2. Critical transitions in the experimental food web. Each color corresponds to a dif- ferent food-web structure, and arrows between two colors indicate a critical transition. The thickness of an arrow is proportional to the percent change in probability of a critical transi- tion for every one unit increase in temperature (left) or genetic diversity (right). Solid arrows indicate positive changes, while dashed arrows indicate negative changes. Black arrows denote statistically clear effects (P < 0.05), while grey arrows are unclear. Rare critical transitions were not tested (labeled n.t.) for differences between treatments. Details on the statistical tests for each critical transition are provided in Tables S1–S2. Genetic diversity (+1 genotype) Warming (+1°C) Fig. 2. Critical transitions in the experimental food web. Each color corresponds to a dif- ferent food-web structure, and arrows between two colors indicate a critical transition. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. The thickness of an arrow is proportional to the percent change in probability of a critical transi- tion for every one unit increase in temperature (left) or genetic diversity (right). Solid arrows indicate positive changes, while dashed arrows indicate negative changes. Black arrows denote statistically clear effects (P < 0.05), while grey arrows are unclear. Rare critical transitions were not tested (labeled n.t.) for differences between treatments. Details on the statistical tests for each critical transition are provided in Tables S1–S2. 5 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint reduced to only the aphid Lipaphis erysimi (blue in Fig. 2), rather than completely collapsing (purple in Fig. 2), by the end of the experiment (multinomial GLM ANOVA: F2,20 = 4.03, P = 0.034, Fig. S4). Although warming accelerated the transition of the intial food web toward a simpler food chain, it was genetic diversity that governed the risk of a critical transition in the remaining 55 food chain (Fig. 2, Table S2). Speciﬁcally, adding another genotype to the plant population reduced the risk of the food chain completely collapsing by 53% (green to purple in Fig. 2). This reduced risk of a critical transition tripled the odds of the food chain persisting to the end of the experiment (relative to complete collapse, multinomial GLM ANOVA: F2,18 = 3.61, P = 0.048, Fig. S4). Therefore, despite the initial effect of warming, the net effect of genetic 60 diversity on food-web persistence was larger than that of warming (Fig. S4). Moreover, there was no clear evidence that temperature modiﬁed the effects of genetic diversity on any of the possible critical transitions (all P > 0.40 for ‘temp:rich’ in Tables S1–S2), suggesting that the positive effect of genetic diversity was robust to experimental warming. To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. We found that adding Col-0 or gsm1 to the plant population reduced the overall risk of a critical transition in this food chain by 45% and 48%, respectively (Fig. 3B). To understand the source of this genetic diversity effect, we isolated the role of each speciﬁc 65 genotype (Fig. 3). To do this, we leveraged the fact that the genetic diversity effect (adding an additional genotype in Fig. 3A) corresponds to the average genotype-speciﬁc effect (i.e., average of green, blue, red, and orange points in Fig. 3B). We focused on the effect of genetic diversity on the overall risk of a critical transition in the food chain (green in Fig. 3A), i.e., To understand the source of this genetic diversity effect, we isolated the role of each speciﬁc 65 genotype (Fig. 3). To do this, we leveraged the fact that the genetic diversity effect (adding an additional genotype in Fig. 3A) corresponds to the average genotype-speciﬁc effect (i.e., average of green, blue, red, and orange points in Fig. 3B). We focused on the effect of genetic diversity on the overall risk of a critical transition in the food chain (green in Fig. 3A), i.e., To understand the source of this genetic diversity effect, we isolated the role of each speciﬁc 65 genotype (Fig. 3). To do this, we leveraged the fact that the genetic diversity effect (adding an additional genotype in Fig. 3A) corresponds to the average genotype-speciﬁc effect (i.e., average of green, blue, red, and orange points in Fig. 3B). We focused on the effect of genetic diversity on the overall risk of a critical transition in the food chain (green in Fig. 3A), i.e., a transition to either an aphid only (blue in Fig. 3A) or completely collapsed state (purple in 70 Fig. 3A). The rationale for this choice is that there was more certainty in this overall effect (P = 0.015) compared to the reduced risk of a complete collapse (P = 0.044 for green to purple transition in Fig. 2). We found that adding Col-0 or gsm1 to the plant population reduced the overall risk of a critical transition in this food chain by 45% and 48%, respectively (Fig. 3B). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. Although warming accelerated the transition of the intial food web toward a simpler food chain, it was genetic diversity that governed the risk of a critical transition in the remaining 55 food chain (Fig. 2, Table S2). Speciﬁcally, adding another genotype to the plant population reduced the risk of the food chain completely collapsing by 53% (green to purple in Fig. 2). This reduced risk of a critical transition tripled the odds of the food chain persisting to the end of the experiment (relative to complete collapse, multinomial GLM ANOVA: F2,18 = 3.61, P = 0.048, Fig. S4). Therefore, despite the initial effect of warming, the net effect of genetic 60 diversity on food-web persistence was larger than that of warming (Fig. S4). Moreover, there was no clear evidence that temperature modiﬁed the effects of genetic diversity on any of the possible critical transitions (all P > 0.40 for ‘temp:rich’ in Tables S1–S2), suggesting that the positive effect of genetic diversity was robust to experimental warming. = 0.048, Fig. S4). Therefore, despite the initial effect of warming, the net effect of genetic 60 diversity on food-web persistence was larger than that of warming (Fig. S4). Moreover, there was no clear evidence that temperature modiﬁed the effects of genetic diversity on any of the possible critical transitions (all P > 0.40 for ‘temp:rich’ in Tables S1–S2), suggesting that the positive effect of genetic diversity was robust to experimental warming. To understand the source of this genetic diversity effect, we isolated the role of each speciﬁc 65 genotype (Fig. 3). To do this, we leveraged the fact that the genetic diversity effect (adding an additional genotype in Fig. 3A) corresponds to the average genotype-speciﬁc effect (i.e., average of green, blue, red, and orange points in Fig. 3B). We focused on the effect of genetic diversity on the overall risk of a critical transition in the food chain (green in Fig. 3A), i.e., a transition to either an aphid only (blue in Fig. 3A) or completely collapsed state (purple in 70 Fig. 3A). The rationale for this choice is that there was more certainty in this overall effect (P = 0.015) compared to the reduced risk of a complete collapse (P = 0.044 for green to purple transition in Fig. 2). To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. These genotypes differ in that Col-0 has a functional allele at the MAM1 gene whereas gsm1 75 = 0.015) compared to the reduced risk of a complete collapse (P = 0.044 for green to purple transition in Fig. 2). We found that adding Col-0 or gsm1 to the plant population reduced the overall risk of a critical transition in this food chain by 45% and 48%, respectively (Fig. 3B). These genotypes differ in that Col-0 has a functional allele at the MAM1 gene, whereas gsm1 75 6 6 A B -35% Genetic diversity (+1 genotype) ● ● ● ● −40 0 40 Col−0 gsm1 AOP2 AOP2/gsoh Risk of critical transition (Δ %) Fig. 3. Identifying a keystone gene. (A) Genetic diversity reduced the overall risk of a crit- ical transition in the food chain (green) to either an aphid only (blue) or completely collapsed (purple) state by 35%. (B) We then isolated the contribution of each genotype to this genetic diversity effect. Points correspond to mean estimates, while thick and thin bars correspond to standard errors and 95% conﬁdence intervals, respectively. Note that the effect of genetic di- versity (-35%) corresponds to the average genotype-speciﬁc effect (i.e., average of green, blue, red, and orange points). Both Col-0 and gsm1 have a non-functional AOP2 gene and clearly de- crease the risk of a critical transition. In contrast, both AOP2 and AOP2/gsoh have a functional AOP2 gene and unclear effects. This suggests that AOP2 functions as a keystone gene in this food web. B -35% enetic diversity (+1 genotype) ● ● ● ● −40 0 40 Col−0 gsm1 AOP2 AOP2/gsoh Risk of critical transition (Δ %) B ● ● ● ● −40 0 40 Col−0 gsm1 AOP2 AOP2/gsoh Risk of critical transition (Δ %) B A Genetic diversity (+1 genotype) Fig. 3. Identifying a keystone gene. (A) Genetic diversity reduced the overall risk of a crit- ical transition in the food chain (green) to either an aphid only (blue) or completely collapsed (purple) state by 35%. (B) We then isolated the contribution of each genotype to this genetic diversity effect. Points correspond to mean estimates, while thick and thin bars correspond to standard errors and 95% conﬁdence intervals, respectively. Note that the effect of genetic di- versity (-35%) corresponds to the average genotype-speciﬁc effect (i.e., average of green, blue, red, and orange points). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. Both Col-0 and gsm1 have a non-functional AOP2 gene and clearly de- crease the risk of a critical transition. In contrast, both AOP2 and AOP2/gsoh have a functional AOP2 gene and unclear effects. This suggests that AOP2 functions as a keystone gene in this food web. 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint has a non-functional allele. The MAM1 gene controls the elongation of the glucosinolate side chain (Fig. 1B, ref. 13, 15), and inﬂuences herbivory from aphids (10) and other insects (15). These independent contributions suggest that these genotypes have complimentary effects on food-chain persistence. Interestingly, Col-0 and gsm1 both lack a functional AOP2 gene, which modiﬁes the glucosinolate side chain (Fig. 1B, ref. 14, 16). The two genotypes that have a 80 functional AOP2 gene had unclear effects on food-chain persistence (Fig. 3B). Analagous to a keystone species that determines species diversity in a food web (22), our results indicate that AOP2 functions as a keystone gene in this food web by shaping species diversity through differences in food-web persistence (23). To understand how genetic change translates into food-web persistence, we applied theory 85 on the structural stability of food webs. Structural stability quantiﬁes the range of ecological conditions that allow species to stably coexist (24,25). At the boundary of this range, the food web undergoes a critical transition to a simpler community (21). The geometry of intra- and in- terspeciﬁc interactions deﬁne the location of critical boundaries, while species’ intrinsic growth To understand how genetic change translates into food-web persistence, we applied theory 85 on the structural stability of food webs. Structural stability quantiﬁes the range of ecological conditions that allow species to stably coexist (24,25). At the boundary of this range, the food web undergoes a critical transition to a simpler community (21). The geometry of intra- and in- terspeciﬁc interactions deﬁne the location of critical boundaries, while species’ intrinsic growth rates determine the proximity of the food web to a critical boundary. The distance, or to be 90 precise the normalized angle, from a critical boundary measures the vulnerability of a food web to a critical transition. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. We used our time series data to quantify the effect of genetic diversity on interactions and species’ intrinsic growth rates, and thus the risk of a critical transition (details in Supplementary Material and Table S3). rates determine the proximity of the food web to a critical boundary. The distance, or to be 90 precise the normalized angle, from a critical boundary measures the vulnerability of a food web to a critical transition. We used our time series data to quantify the effect of genetic diversity on interactions and species’ intrinsic growth rates, and thus the risk of a critical transition (details in Supplementary Material and Table S3). We found that genetic diversity buffered the remaining food chain from a critical transition 95 in a speciﬁc way (Fig. 4). This buffering effect was not because genetic diversity altered the location of the critical boundary (border of grey area in Fig. 4). Rather it was because genetic diversity moved the vector of intrinsic growth rates (solid arrows) upward and into the region where the parasitoid and aphid coexist (grey area in Fig. 4; Bayesian multivariate autoregressive We found that genetic diversity buffered the remaining food chain from a critical transition 95 in a speciﬁc way (Fig. 4). This buffering effect was not because genetic diversity altered the location of the critical boundary (border of grey area in Fig. 4). Rather it was because genetic diversity moved the vector of intrinsic growth rates (solid arrows) upward and into the region where the parasitoid and aphid coexist (grey area in Fig. 4; Bayesian multivariate autoregressive 8 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint −0.6 −0.4 −0.2 0.0 0.00 0.25 0.50 0.75 1.00 Aphid growth rate (normalized) Parasitoid growth rate (normalized) 1 genotype 2 genotypes 4 genotypes Extinction Coexistence Fig. 4. Genetic diversity buffers the remaining food chain from its critical boundary. The grey area represents the range of intrinsic growth rates (normalized to length 1) where the aphid and parasitoid coexist. The white area represents the range of intrinsic growth rates where the parasitoid goes extinct. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. Increasing genetic diversity moves the vector of intrinsic growth rates (solid arrows) upward and into the region of parameter space where the aphid and parasitoid coexist (grey area). Note that normalizing the vector of intrinsic growth rates to length 1 is necessary for accurately visualizing and calculating the proximity of the food chain to its critical boundary. −0.6 −0.4 −0.2 0.0 0.00 0.25 0.50 0.75 1.00 Aphid growth rate (normalized) Parasitoid growth rate (normalized) 1 genotype 2 genotypes 4 genotypes Extinction Coexistence Fig. 4. Genetic diversity buffers the remaining food chain from its critical boundary. The grey area represents the range of intrinsic growth rates (normalized to length 1) where the aphid and parasitoid coexist. The white area represents the range of intrinsic growth rates where the parasitoid goes extinct. Increasing genetic diversity moves the vector of intrinsic growth rates (solid arrows) upward and into the region of parameter space where the aphid and parasitoid coexist (grey area). Note that normalizing the vector of intrinsic growth rates to length 1 is necessary for accurately visualizing and calculating the proximity of the food chain to its critical boundary. 9 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint due to a concordant increase in the aphid’s intrinsic growth rate (Bayesian MAR(1) model: 97% of posterior estimates > 0), and to a lesser extent the parasitoid’s growth rate (Bayesian MAR(1) model: 87% of posterior estimates > 0). Although this analysis assumes that this food chain is at a stable equilibrium, our results hold under non-equilibrium conditions (i.e., persistence from different initial conditions, Fig. S6). 105 The buffering effect of genetic diversity was underlied by Col-0 and gsm1’s positive effect on aphid and parasitoid intrinsic growth rates (Fig. S7). To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. This positive relationship was likely due to the faster relative growth rate of Col-0 and gsm1 genotypes compared to genotypes with a functional AOP2 gene (Fig. S8; note that genetic diversity did not affect plant growth, ANOVA: F1,9 = 0.10, P = 0.759, Table S4). Work in similar environmental conditions has 110 shown that natural accessions of Arabidopsis with a functional AOP2 gene produce more total aliphatic glucosinolates and have slower growth rates (10), which has been shown to reduce the intrinsic growth rate of other aphid species (26). Note that the AOP2 gene also has known pleiotropic effects on phenology, jasmonic acid signaling, and circadian rhythms (27,28), which ANOVA: F1,9 = 0.10, P = 0.759, Table S4). Work in similar environmental conditions has 110 shown that natural accessions of Arabidopsis with a functional AOP2 gene produce more total aliphatic glucosinolates and have slower growth rates (10), which has been shown to reduce the intrinsic growth rate of other aphid species (26). Note that the AOP2 gene also has known pleiotropic effects on phenology, jasmonic acid signaling, and circadian rhythms (27,28), which ANOVA: F1,9 = 0.10, P = 0.759, Table S4). Work in similar environmental conditions has 110 shown that natural accessions of Arabidopsis with a functional AOP2 gene produce more total aliphatic glucosinolates and have slower growth rates (10), which has been shown to reduce the intrinsic growth rate of other aphid species (26). Note that the AOP2 gene also has known pleiotropic effects on phenology, jasmonic acid signaling, and circadian rhythms (27,28), which may ultimately mediate the dynamics of plant growth and aphid herbivory we observed. The 115 direct plant genetic effect on the aphid’s growth rate is also the likely cause of the positive indirect effect on the parasitoid’s growth rate. For example, different plant genotypes of a closely related crucifer species alter the intrinsic growth rates of aphids and parasitoids in a coordinated manner (29). This positive correspondence in intrinsic growth rates is likely quite general among insect herbivores and their parasitoids (30). 120 may ultimately mediate the dynamics of plant growth and aphid herbivory we observed. The 115 direct plant genetic effect on the aphid’s growth rate is also the likely cause of the positive indirect effect on the parasitoid’s growth rate. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. This suggests that the loss of genetic diversity we are witnessing (34) may accelerate the local extinction of species across multiple trophic levels. 130 Yet, these results also present an opportunity for conservation and ecosystem restoration. For 135 example, assisted migration of pre-adapted plant genotypes is becoming a well recognized strat- egy for restoring and preserving forest ecosystems into the future (35). Our results suggest that maximizing genetic diversity within pre-adapted populations may foster the structural integrity of terrestrial food webs in an uncertain and changing world. To whom correspondence should be addressed; E-mail: matthew.barbour@ieu.uzh. For example, different plant genotypes of a closely related crucifer species alter the intrinsic growth rates of aphids and parasitoids in a coordinated manner (29). This positive correspondence in intrinsic growth rates is likely quite general among insect herbivores and their parasitoids (30). 120 These results hint at a general mechanism that may underlie a common pattern —plant populations with higher genetic diversity harbor more species-rich food webs (31, 32). Our work suggests that this pattern may be caused by an increased probability of having plants with chemical phenotypes that promote reproduction in herbivores and their natural enemies. While we have shown that this mechanism prevents critical transitions in a simple food chain, 125 While we have shown that this mechanism prevents critical transitions in a simple food chain, 125 10 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint it is possible that this mechanism operates as well in more diverse food webs. Given that food chains of plants, insect herbivores, and their parasitoids comprise ⇠40% of all described Eukaryotes (33), this mechanism may have far reaching consequences on the persistence and functioning of terrestrial ecosystems. Taken together, our results show that genetic diversity in plant defense metabolism can in- 130 crease the persistence of food webs in the face of climate warming. Although climate warming had a strong initial effect on food-web persistence, genetic diversity served as biological in- surance against subsequent collapse. This suggests that the loss of genetic diversity we are witnessing (34) may accelerate the local extinction of species across multiple trophic levels. Taken together, our results show that genetic diversity in plant defense metabolism can in- 130 crease the persistence of food webs in the face of climate warming. Although climate warming had a strong initial effect on food-web persistence, genetic diversity served as biological in- surance against subsequent collapse. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint References and Notes: 140 1. R. Kerwin, et al., eLife 4 (2015). 2. M. A. Barbour, et al., Proc. Natl. Acad. Sci. U.S.A. 113, 2128–2133 (2016). 3. H. L. Barker, et al., Mol. Ecol. 28, 4404–4421 (2019). 4. R. T. Paine, Am. Nat. 100, 65–75 (1966). 5. K. S. McCann, Food Webs (Princeton University Press, 2011). 145 6. J. P. Harmon, N. A. Moran, A. R. Ives, Science 323, 1347–1350 (2009). 7. B. R. Scheffers, et al., Science 354, aaf7671-1 (2016). 1. R. Kerwin, et al., eLife 4 (2015). 1. R. Kerwin, et al., eLife 4 (2015). 1. R. Kerwin, et al., eLife 4 (2015). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint 1. R. Kerwin, et al., eLife 4 (2015). 2. M. A. Barbour, et al., Proc. Natl. Acad. Sci. U.S.A. 113, 2128–2133 (2016). 3. H. L. Barker, et al., Mol. Ecol. 28, 4404–4421 (2019). 4. R. T. Paine, Am. Nat. 100, 65–75 (1966). 5. K. S. McCann, Food Webs (Princeton University Press, 2011). 6. J. P. Harmon, N. A. Moran, A. R. Ives, Science 323, 1347–1350 (2009). 7. B. R. Scheffers, et al., Science 354, aaf7671-1 (2016). 11 8. J. B. Hughes, G. C. Daily, P. R. Ehrlich, Science 278, 689–692 (1997). 9. As noted in Fig. 1B, the Col-0 genotype (green) has a functional MAM1 gene, a natu- ral non-functional AOP2 gene, and a functional GS-OH gene, resulting in the accumu- 150 lation of primarily 4-Methylsulﬁnylbutyl (4MSO). Note that AOP2 is epistatic to GS- OH, resulting in an effectively non-functional GS-OH gene in Col-0. The gsm1 genotype (blue) creates a non-functional MAM1 gene, resulting in an accumulation of primarily 3- Methylsulﬁnylpropyl (3MSO). The AOP2 genotype (orange) inserts a functional AOP2 gene and also allows GS-OH to function, resulting in an accumulation of primarily OH- 155 But-3-enyl. The AOP2/gsoh genotype (red) has a non-functional GS-OH gene, resulting in an accumulation of primarily But-3-enyl. Note that transgenic lines mimic the chemi- cal phenotype of diverse natural accessions that co-occur throughout Europe (e.g., gsm1 ⇠ Oy-0 and Wa-1, AOP2 ⇠NFA-10 and Sq-1, AOP2/gsoh ⇠T670 and IP-Mot-0, in ref. 18). 150 10. T. Z¨ust, et al., Science 338, 116–119 (2012). 160 11. Y. Sato, R. Shimizu-Inatsugi, M. Yamazaki, K. K. Shimizu, A. J. Nagano, BMC Plant Biol. 19, 163 (2019). 12. D. B. Stouffer, J. Bascompte, Ecol. Lett. 13, 154–161 (2010). 13. G. W. Haughn, L. Davin, M. Giblin, E. W. Underhill, Plant Physiol. 97, 217–226 (1991). 14. D. J. Kliebenstein, V. M. Lambrix, M. Reichelt, J. Gershenzon, T. Mitchell-Olds, Plant 165 Cell 13, 681–693 (2001). 15. J. Kroymann, S. Donnerhacke, D. Schnabelrauch, T. Mitchell-Olds, Proc. Natl. Acad. Sci. U.S.A. 100, 14587–14592 (2003). 16. B. G. Hansen, et al., Plant Physiol. 148, 2096–2108 (2008). 12 17. D. J. Kliebenstein, N. I. Cacho, Advances in Botanical Research (Academic Press, 2016), 170 vol. 80, chap. 3. 18. E. K. F. Chan, H. C. Rowe, J. A. Corwin, B. Joseph, D. J. Kliebenstein, PLoS Biol. 9, e1001125 (2011). 19. E. M. Zubler, et al., Clim. Change 125, 237–252 (2014). 20. B. Gilbert, et al., Ecol. Lett. 17, 902–914 (2014). 175 21. J. J. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint 1. R. Kerwin, et al., eLife 4 (2015). 36, 672–683 (2018). 49. A. R. Ives, B. Dennis, K. L. Cottingham, S. R. Carpenter, Ecol. Monogr. 73, 301–330 (2003). 210 50. P.-C. Brkner, J. Stat. Softw. 80, 1–28 (2017). 50. P.-C. Brkner, J. Stat. Softw. 80, 1–28 (2017). 51. R Core Team, R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria (2019). 52. M. D. Hoffman, A. Gelman, J. Mach. Learn. Res. 15, 1593–1623 (2014). 53. B. Carpenter, et al., J. Stat. Softw. 76, 1–32 (2017). 215 54. F. Jahan, H. Abbasipour, A. Askarianzadeh, G. Hassanshahi, A. Saeedizadeh, J. Insect Sci. 14 (2014). 55. R. Taghizadeh, J. Agric. Sci. Technol. 21, 627–636 (2019). 56. O. Kostenko, et al., Ecol. Entomol. 40, 748–758 (2015). 57. S. Saavedra, et al., Ecol. Monogr. 87, 470–486 (2017). 220 58. C. Song, R. P. Rohr, S. Saavedra, J. Theor. Biol. 450, 30–36 (2018). 1. R. Kerwin, et al., eLife 4 (2015). Lever, et al., Ecol. Lett. 23, 2–15 (2020). 22. R. T. Paine, Am. Nat. 103, 91–93 (1969). 22. R. T. Paine, Am. Nat. 103, 91–93 (1969). 23. L. H. Skovmand, et al., Trends Ecol. Evol. 33, 689–700 (2018). 24. U. Bastolla, et al., Nature 458, 1018–1020 (2009). 25. R. P. Rohr, S. Saavedra, J. Bascompte, Science 345, 1253497 (2014). 180 26. T. Z¨ust, B. Joseph, K. K. Shimizu, D. J. Kliebenstein, L. A. Turnbull, Proc. Royal Soc. B 278, 2598–2603 (2011). 27. R. E. Kerwin, et al., Plant Cell 23, 471–485 (2011). 28. M. Burow, et al., Mol. Plant 8, 1201–1212 (2015). 29. A. Karami, Y. Fathipour, A. A. Talebi, G. V. P. Reddy, Arthropod Plant Interact. 12, 291– 185 301 (2018). 30. C. S. Awmack, S. R. Leather, Annu. Rev. Entomol. 47, 817–844 (2002). 31. G. M. Crutsinger, et al., Science 313, 966–968 (2006). 13 32. J. Koricheva, D. Hayes, Funct. Ecol. 32, 1704–1717 (2018). 32. J. Koricheva, D. Hayes, Funct. Ecol. 32, 1704–1717 (2018). 33. D. A. Grimaldi, M. S. Engel, Evolution of the Insects (Cambridge University Press, 2005). 190 34. D. M. Leigh, A. P. Hendry, E. V´azquez-Dom´ınguez, V. L. Friesen, Evol. Appl. 12, 1505– 1512 (2019). 35. S. N. Aitken, J. B. Bemmels, Evol. Appl. 9, 271–290 (2016). 36. G. Li, C. F. Quiros, Theor. Appl. Genet. 106, 1116–1121 (2003). 37. 1001 Genomes Consortium, Cell 166, 481–491 (2016). 195 37. 1001 Genomes Consortium, Cell 166, 481–491 (2016). 195 38. M. Begert, C. Frei, Int. J. Climatol. 38, 2792–2807 (2018). 38. M. Begert, C. Frei, Int. J. Climatol. 38, 2792–2807 (2018). 39. T. Liu, B. Yue, Southwest. Entomol. 25, 91–99 (2000). 40. S. Satar, U. Kersting, M. R. Ulusoy, Turk. J. Agric. For. 29, 341–346 (2005). 41. H. R. S. Moayeri, H. Madadi, H. Pouraskari, A. Enkegaard, Eur. J. Entomol. 110, 109–113 (2013). 0 42. L. Rivero, et al., Methods Mol. Biol. 1062, 3–25 (2014). 43. W. N. Venables, B. D. Ripley, Modern Applied Statistics With S (Springer, 2002). 44. J. D. Kalbﬂeisch, R. L. Prentice, The Statistical Analysis of Failure Time Data (Wiley, 2011). 45. S. H. Hurlbert, Ecol. Monogr. 54, 187–211 (1984). 205 46. B. Schmid, M. Baruffol, Z. Wang, P. A. Niklaus, J. Plant Ecol. 10, 91–110 (2017). 47. A. Colin Cameron, D. L. Miller, J. Hum. Resour. 50, 317–372 (2015). 14 48. J. E. Pustejovsky, E. Tipton, J. Bus. Econ. Stat. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint Acknowledgements We thank Daniel Trujillo-Villegas and Xenia Muenger for their help conducting the experi- ment, and Matthias Furler for greenhouse support. Bernhard Schmid gave valuable feedback on the experimental design. Antonio Ferrera gave insight into quantifying the proximity of a 225 critical boundary. This study was supported by the University Research Priority Program on Global Change and Biodiversity of the University of Zurich. Funding: Swiss National Science 15 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint Foundation grant 31003A 160671 to J. Bascompte. Author Contributions: M.A. Barbour contributed to conceptualization, data curation, formal analysis, investigation, methodology, Foundation grant 31003A 160671 to J. Bascompte. Author Contributions: M.A. Barbour contributed to conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources (insects), visualization, writing - original draft, writing - re- 230 view and editing; D.J. Kliebenstein contributed to resources (plant genotypes), writing - review and editing; J. Bascompte contributed to conceptualization, funding acquisition, project admin- istration, writing - review and editing. Competing interests: Authors declare no competing interests. Data and materials availability: All data and code to reproduce the reported results is publically available on GitHub (https://mabarbour.github.io/genes-to-foodweb-stability/) and 235 have been archived on Zenodo (http://doi.org/10.5281/zenodo.3904601). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.23.167387 doi: bioRxiv preprint Supplementary materials Materials and Methods Figs. S1 to S8 Tables S1 to S4 240 References (36-58) Materials and Methods 16
https://openalex.org/W2905476261	https://repositorium.sdum.uminho.pt/bitstream/1822/60439/1/energies-11-03453.pdf	English	null	A Novel Multilevel Bidirectional Topology for On-Board EV Battery Chargers in Smart Grids	Energies	2,018	cc-by	19,832	Received: 31 October 2018; Accepted: 6 December 2018; Published: 10 December 2018 Received: 31 October 2018; Accepted: 6 December 2018; Published: 10 December 2018 Abstract: This paper proposes a novel on-board electric vehicle (EV) battery charger (EVBC) based on a bidirectional multilevel topology. The proposed topology is formed by an AC-DC converter for the grid-side interface and by a DC-DC converter for the battery-side interface. Both converters are interfaced by a split DC-link used to achieve distinct voltage levels in both converters. Characteristically, the proposed EVBC operates with sinusoidal grid-side current, unitary power factor, controlled battery-side current or voltage, and controlled DC-link voltages. The grid-side converter operates with ﬁve voltage levels, while the battery-side operates with three voltage levels. An assessment, for comparison with classical multilevel converters for EVBCs is considered along the paper, illustrating the key beneﬁts of the proposed topology. As the proposed EVBC is controlled in bidirectional mode, targeting the EV incorporation into smart grids, the grid-to-vehicle (G2V) and vehicle-to-grid (V2G) operation modes are discussed and evaluated. Both converters of the proposed EVBC use discrete-time predictive control algorithms, which are described in the paper. An experimental validation was performed under real operating conditions, employing a developed laboratory prototype. Keywords: multilevel converter; electric vehicle; on-board battery charger; power factor correction; power quality; smart grid A Novel Multilevel Bidirectional Topology for On-Board EV Battery Chargers in Smart Grids Rafael S. Leite, João L. Afonso and Vítor Monteiro * Industrial Electronics Department, School of Engineering, University of Minho, 4800-058 Guimarães, P a73417@alunos.uminho.pt (R.S.L.); jla@dei.uminho.pt (J.L.A.) * Correspondence: vmonteiro@dei.uminho.pt; Tel.: +351-253-510-392 energies energies energies www.mdpi.com/journal/energies 1. Introduction For instance, considering the grid-side converter, a ﬁve-level topology with reduced switching devices and based on the active neutral point clamped in presented in [16] for grid-tied solar photovoltaic applications; a ﬁve-level Vienna-type structure is proposed in [17] for active rectiﬁers; a unidirectional ﬁve-level based on the T-rectiﬁer topology is analyzed in [18] for high-speed gen-set applications; an improved ﬁve-level topology is presented in [19] for active rectiﬁers or grid-tied applications; a unidirectional ﬁve-level topology is proposed in [20] for power factor correction converters, including applications of EVBC; a symmetric cascade ﬁve-level topology is proposed in [21] for grid-tied inverters; based on the previous structure, a bidirectional ﬁve-level topology is proposed in [19], allowing the operation as active converter or as grid-tied converter; a modular unidirectional ﬁve-level topology is offered in [22] for applications of renewable energy sources; a ﬁve-level topology for renewables applications is proposed in [23]; a ﬁve-level topology based on the neutral point clamped arrangement is presented in [24] for motor drivers; a novel ﬁve-level topology is proposed in [25] for unidirectional EVBC; and a ﬁve-level topology with reduced switching devices is proposed in [20] for active rectiﬁer applications. In terms of the battery-side converter, two-level topologies are usually employed for the battery-side coupling converter. However, it should be noted that DC-DC multi-level topologies can be applied for other applications, for instance, in [26] is presented a novel multilevel boost converter for applications of photovoltaics or fuel cell generation systems, in [27] a review of DC-DC four- and three-level topologies is presented, and in [28] a two-level interleaved and intercoupled boost converter for high power applications is analyzed. The three-level topology is presented in [29], however, the application is for the voltage balancing of series connected batteries. On the other hand, in this paper, the split DC-link is used as interface for the grid-side converter, where the voltage of the capacitors is always controlled by the grid-side power converter. Moreover, the control algorithm is completely different, in order to obtain a reduced current ripple, where the proposed approach consists in using the converter controlled by current controlling only two switching devices during each operation mode. 1. Introduction The electric vehicle (EV) is considered as the central element to support electric mobility in smart grids, serving to help to address major energy concerns. From a global perspective, different options of EVs can be considered distinguished by the energy storage system, as battery EVs (BEVs) or fuel cell EVs (FCEVs), and by the external interface for the charging process, as plug-in EVs (PHEV) [1–3]. Within the scope of this paper, the ﬁnal application is for EVs using batteries as the energy storage system, where the main advantage is the capacity of the energy storage system and the main drawback is the required charging time. The relevance of the EV for this purpose is carefully addressed and evaluated in [4–6] in terms of power electronics and control methodologies for the grid-side. As demonstrated in [7,8], since the EV batteries are charged from the power grid (independently of the on-board or off-board technology), power quality is an imperative feature for assuring the grid stability. In this perspective, advanced contributions for the EV controlled action in smart grids, and bearing in mind power quality issues, are presented in [9]. Additionally, the opportunity to operate in bidirectional mode, as well as to operate in the four quadrants in terms of power quality will also be decisive for contributing to establish energy management strategies in a smart grid perspective. These new contributions for the EV operation in four-quadrants and framed in smart grids, are examined in [10,11]. The ﬂexible incorporation of an EV into the energy management of Energies 2018, 11, 3453; doi:10.3390/en11123453 www.mdpi.com/journal/energies www.mdpi.com/journal/energies 2 of 21 Energies 2018, 11, 3453 a smart home is presented in [12], perspective an advanced communication toward to control the charging and discharging processes. Classically, on-board EV battery chargers (EVBC) are projected with two- or three-level topologies for the grid-side coupling converter [13], however, by increasing the levels, the size of the passive ﬁlters can be reduced, as demonstrated in [14,15] for other types of applications, different from EVBC. Nonetheless, the levels cannot be augmented indeterminately. By establishing a tradeoff between power density and required hardware and software, ﬁve-level topologies are identiﬁed as interesting solutions for different purposes. 1. Introduction It should be noted that in [29] are controlled four switching devices, therefore, controlling only two it is possible to reduce the switching losses and the control complexity (e.g., it is not necessary to deal with any type of dead-time for the switching devices). The above-mentioned ﬁve-level topologies for the grid-side converter were validated in the scope of diverse applications, but not all were validated neither compared in the scope of an EVBC. The same occurs with the battery-side converter. Moreover, no EVBC was validated employing multilevel topologies in the grid-side and the battery-side converters. In this sense, this paper proposes an on-board EVBC multilevel topology. The internal constitution of a conventional on-board EVBC is presented in Figure 1, where is highlighted the bidirectional power ﬂow, between the batteries and the grid, in order to accomplish with the grid-to-vehicle and vehicle-to-grid operations in smart grids. The key contributions of this paper are: (a) a novel EVBC based on a multilevel topology; (b) an analysis of the proposed EVBC in terms of operation targeting smart grids; and (c) an experimental validation using a dedicated developed on-board EVBC. The paper is outlined as follows: A description of the hardware topology of the proposed EVBC is presented in Section 2. The discrete-time predictive control algorithms used for the grid-side converter and for the battery-side converter are presented in Section 3. The foremost experimental results considering diverse operating states for smart grids are presented in Section 4. The main conclusions are discussed in Section 5. 3 of 21 3 of 21 3 of 21 Energies 2018, 11, 3453 Energies 2018, 11, x FOR Energies 2018, 11, x FOR Figure 1. Internal constitution of a conventional on-board electric vehicle battery charger (EVBC). Power Converters Composed by two bidirectional multilevel converters: an ac-dc grid- side and a dc-dc battery-side. Communication Interface of communication in order to provide the state of the bidirectional converters Control Algorithms Digital control system of the converters for the grid interface and for the battery interface. on-board EV battery charger (internal constitution) Communication Power Power grid Battery Battery Management System (BMS) Figure 1. Internal constitution of a conventional on-board electric vehicle battery charger (EVBC). Figure 1. Internal constitution of a conventional on-board electric vehicle battery charger (EVBC). Power Converters Composed by two bidirectional multilevel converters: an ac-dc grid- side and a dc-dc battery-side. 1. Introduction Communication Interface of communication in order to provide the state of the bidirectional converters Control Algorithms Digital control system of the converters for the grid interface and for the battery interface. on-board EV battery charger (internal constitution) Communication Power Power grid Battery Battery Management System (BMS) on-board EV battery charger (internal constitution) on-board EV battery charger (internal constitution) Control Algorithms Digital control system of the converters for the grid interface and for the battery interface. Control Algorithms Digital control system of the converters for the grid interface and for the battery interface. Power Converters Composed by two bidirectional multilevel converters: an ac-dc grid- side and a dc-dc battery-side. Power Converters Composed by two bidirectional multilevel converters: an ac-dc grid- side and a dc-dc battery-side. Communication Interface of communication in order to provide the state of the bidirectional converters Communication Interface of communication in order to provide the state of the bidirectional converters Figure 1. Internal constitution of a conventional on-board electric vehicle battery charger (EVBC). Figure 1. Internal constitution of a conventional on-board electric vehicle battery charger (EVBC). Figure 1. Internal constitution of a conventional on-board electric vehicle battery charger (EVBC). 2 EV Battery Charger: Topology Description 2. EV Battery Charger: Topology Description 2. EV Battery Charger: Topology Description y g p gy p Figure 2 shows the global electrical schematic of the proposed EVBC. This topology consists of a grid-side converter and a battery-side converter, both with a multilevel characteristic supported by a split DC-link formed by two sets of capacitors (C1 and C2). In terms of other components, the EVBC consists of twelve insulated-gate bipolar transistors (IGBTs) (used as controlled switching devices, eight for the grid-side converter and four the battery-side converter), an inductive coupling filter (L1, L2), and a LC passive filter interfacing the batteries (L3, L4 and C3). Figure 2 shows the global electrical schematic of the proposed EVBC. This topology consists of a grid-side converter and a battery-side converter, both with a multilevel characteristic supported by a split DC-link formed by two sets of capacitors (C1 and C2). In terms of other components, the EVBC consists of twelve insulated-gate bipolar transistors (IGBTs) (used as controlled switching devices, eight for the grid-side converter and four the battery-side converter), an inductive coupling ﬁlter (L1, L2), and a LC passive ﬁlter interfacing the batteries (L3, L4 and C3). Figure 2 shows the global electrical schematic of the proposed EVBC. This topology consists of a grid-side converter and a battery-side converter, both with a multilevel characteristic supported by a split DC-link formed by two sets of capacitors (C1 and C2). In terms of other components, the EVBC consists of twelve insulated-gate bipolar transistors (IGBTs) (used as controlled switching devices, eight for the grid-side converter and four the battery-side converter), an inductive coupling filter (L1, L2), and a LC passive filter interfacing the batteries (L3, L4 and C3). Fi 2 T l f th d b d l t i hi l b tt h (EVBC) dc-dc battery-side converter ac-dc grid-side converter iev S3 S4 S5 S6 S1 S2 S7 S8 vL1 L1 vg vcv_AC C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 vL2 L2 Figure 2. Topology of the proposed on-board electric vehicle battery charger (EVBC). dc-dc battery-side converter ac-dc grid-side converter iev S3 S4 S5 S6 S1 S2 S7 S8 vL1 L1 vg vcv_AC C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 vL2 L2 Figure 2. Topology of the proposed on-board electric vehicle battery charger (EVBC). 2.1. Topology Description: Grid-Side Converter Topology Description: Grid-Side Converter In the development of power electronics systems, electrical grid power quality issues are, more than ever, a major concern. Since the voltage levels produced by grid-side multilevel converters are directly proportional to the quality of the obtained grid current, multilevel converters have emerged as contributors for this concern. The circuit topology of the grid-side multilevel converter proposed for the on-board EVBC is presented in Figure 2. The proposed topology emerged as a derivation of the traditional full-bride rectiﬁer with four devices connected to the split DC-link as the power factor correction (PFC) three-level DC-DC converter. This topology can produce ﬁve distinct voltage levels (+vdc, +vdc/2, 0, −vdc/2, −vdc) at the terminals of the converter (vcv_AC). As can be seen, each IGBT is applied to a maximum voltage of +vdc. The conﬁguration of the topology allows to switch necessarily only six of the eight IGBTs, during the full operation as active rectiﬁer or grid-tied inverter, hence, decreasing the switching losses. When the grid side converter operates as active rectiﬁer, Figure 3, during the positive half cycle of the power grid voltage, the IGBTs (S1, S4) are always switched on and the IGBTs (S2, S3) are always switched off. When the IGBT S6 is switched on, the IGBT S5 is switched to state the voltage levels 0 and +vdc/2. On the other hand, when the IGBT S5 is switched off, the IGBT S6 is switched to state the voltage levels +vdc/2 and +vdc. During the negative half cycle of the power grid voltage, the IGBTs (S2, S3) are always switched on and the IGBTs (S1, S4) are always switched off. When the IGBT S5 is switched on, the IGBT S6 is switched to state the voltage levels 0 and −vdc/2. Finally, when the IGBT S6 is switched off, the IGBT S5 is switched to state the voltage levels −vdc/2 and −vdc. When the grid side converter operates as an inverter, Figure 4, during the positive half cycle of the power grid voltage, the IGBTs (S1, S4) are always switched on and the IGBTs (S2, S3) are always switched off. When the IGBT S8 is switched off, the IGBT S7 is switched to state the voltage levels 0 and +vdc/2. On the other hand, when the IGBT S7 is switched on, the IGBT S8 is switched to state the voltage levels +vdc/2 and +vdc. 2 EV Battery Charger: Topology Description 2. EV Battery Charger: Topology Description 2. EV Battery Charger: Topology Description Fi T l f h d b d l h l b h (EVBC) dc-dc battery-side converter ac-dc grid-side converter Figure 2. Topology of the proposed on-board electric vehicle battery charger (EVBC). Figure 2. Topology of the proposed on-board electric vehicle battery charger (EVBC). Figure 2. Topology of the proposed on-board electric vehicle battery charger (EVBC). The values of the parameters constituting the on-board EVBC, as well as the specifications of th t th t t k i t t h h i t i i T bl 1 The values of the parameters constituting the on-board EVBC, as well as the specifications of the system that was taken into account when choosing components, are given in Table 1. The values of the parameters constituting the on-board EVBC, as well as the speciﬁcations of the system that was taken into account when choosing components, are given in Table 1. Figure 2. Topology of the proposed on-board electric vehicle battery charger (EVBC). The values of the parameters constituting the on-board EVBC, as well as the specifications of h h k i h h i i i T bl 1 The values of the parameters constituting the on-board EVBC, as well as the specifications of the system that was taken into account when choosing components, are given in Table 1. The values of the parameters constituting the on-board EVBC, as well as the speciﬁcations of the system that was taken into account when choosing components, are given in Table 1. 4 of 21 Energies 2018, 11, 3453 Table 1. Parameters and speciﬁcations of the on-board EVBC. Table 1. Parameters and speciﬁcations of the on-board EVBC. Table 1. Parameters and speciﬁcations of the on-board EVBC. Parameter Value Unit Inductive Passive Filter L1, L2 5 mH Inductive Passive Filter L3, L4 2.5 mH Capacitive Passive Filter C1, C2 2.24 mF Capacitive Passive Filter C3 20 µF Nominal Input Voltage (grid-side) vg 230 ± 10% V Nominal Grid Frequency f 50 ± 1% Hz Nominal Input Current (grid-side) iev 16 A Total Harmonic Distortion (grid current) - <5% - Total Power Factor - 1 - Nominal DC-link Voltage vdc 400 V Nominal Output Voltage (battery-side) vbat 200–400 V Nominal Output Current (battery-side) ibat 10 A Switching Frequency fsw 20 kHz Sampling Frequency fs 40 kHz 5 of 21 5 of 21 Operation stages for the grid-side converter during the operation as active rectiﬁer: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 3. Operation stages for the grid-side converter during the operation as active rectifier: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: (a) v AC 0; (b) v AC +vd /2; (c) v AC +vd ; (d) v AC 0; (e) v AC vd /2; (f) v AC vd Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: ( ) 0 (b) /2 ( ) (d) 0 ( ) /2 (f) Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. ( ) _ ; ( ) _ / ; ( ) _ ; ( ) _ ; ( ) _ / ; ( ) _ A predictive current control technique, with a fixed switching frequency of 20 kHz, was applied to obtain a sinusoidal EVBC current and in phase with the grid voltage (or phase opposition in vehicle-to-grid mode). Since this current control is identified as a linear current control, the modulation technique is applied individually. In this sense, Figure 5 shows the pulse-width modulation (PWM) modulation technique arrangement used for the grid-side converter. 5 of 21 5 of 21 The proposed PWM modulation technique requires only one carrier signal and two reference signals to control eight IGBTs. This figure shows the adapted voltage reference (ref1, ref2), the triangular carrier signal (vcarrier), the PWM signals of the IGBTs, the voltage levels produced by the converter (vcv_AC) and the power grid voltage (vg). The pulses signal of the IGBTs (S1, S2, S3, S4) are exclusively dependent of the instantaneous value of the power grid voltage. During the operation as grid-tied inverter, the IGBTs (S7, S8) have an opposite a command signal as the IGBTs (S5, S6), respectively. In the modulation strategy, the voltages references (ref1, ref2) are adapted, based on a digital codification, from the modulating signal sM established by the Equation (1), where vcv_AC are the voltage levels produced by the converter and ma the amplitude modulation index. The voltage levels produced by A predictive current control technique, with a fixed switching frequency of 20 kHz, was applied to obtain a sinusoidal EVBC current and in phase with the grid voltage (or phase opposition in vehicle-to-grid mode). Since this current control is identified as a linear current control, the modulation technique is applied individually. In this sense, Figure 5 shows the pulse-width modulation (PWM) modulation technique arrangement used for the grid-side converter. The proposed PWM modulation technique requires only one carrier signal and two reference signals to control eight IGBTs. This figure shows the adapted voltage reference (ref1, ref2), the triangular carrier signal (vcarrier), the PWM signals of the IGBTs, the voltage levels produced by the converter (vcv_AC) and the power grid voltage (vg). The pulses signal of the IGBTs (S1, S2, S3, S4) are exclusively dependent of the instantaneous value of the power grid voltage. During the operation as grid-tied inverter, the IGBTs (S7, S8) have an opposite a command signal as the IGBTs (S5, S6), respectively. In the modulation strategy, the voltages references (ref1, ref2) are adapted, based on a digital codification, from the modulating signal sM established by the Equation (1), where vcv_AC are the voltage levels produced by the converter and ma the amplitude modulation index. 5 of 21 5 of 21 Energies 2018, 11, x FOR PEER REVIEW 5 of 21 Figure 3. Operation stages for the grid-side converter during the operation as active rectifier: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: (a) vcv AC = 0; (b) vcv AC = +vdc/2; (c) vcv AC = +vdc; (d) vcv AC = 0; (e) vcv AC = −vdc/2; (f) vcv AC = −vdc. Figure 3. Operation stages for the grid-side converter during the operation as active rectiﬁer: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 3. Operation stages for the grid-side converter during the operation as active rectifier: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 4. Operation stages for the grid-side converter during the operation as grid-tied inverter: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 3. Operation stages for the grid-side converter during the operation as active rectifier: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 3. Operation stages for the grid-side converter during the operation as active rectiﬁer: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 3. Operation stages for the grid-side converter during the operation as active rectifier: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 3. Operation stages for the grid-side converter during the operation as active rectifier: (a) vcv_AC = 0; (b) vcv_AC = +vdc/2; (c) vcv_AC = +vdc; (d) vcv_AC = 0; (e) vcv_AC = −vdc/2; (f) vcv_AC = −vdc. Figure 3. 2.1. Topology Description: Grid-Side Converter During the negative half cycle of the power grid voltage, the IGBTs (S2, S3) are always switched on and the IGBTs (S1, S4) are always switched off. When the IGBT S7 is switched off, the IGBT S8 is switched to state the voltage levels 0 and −vdc/2. Finally, when the IGBT S8 is switched on, the IGBT S7 is switched to state the voltage levels −vdc/2 and −vdc. Energies 2018, 11, 3453 Energies 2018, 11, x FOR 5 of 21 5 of 21 5 of 21 5 of 21 The voltage levels produced by the converter are obtained in the control algorithm explained in Section 3 1 A predictive current control technique, with a ﬁxed switching frequency of 20 kHz, was applied to obtain a sinusoidal EVBC current and in phase with the grid voltage (or phase opposition in vehicle-to-grid mode). Since this current control is identiﬁed as a linear current control, the modulation technique is applied individually. In this sense, Figure 5 shows the pulse-width modulation (PWM) modulation technique arrangement used for the grid-side converter. The proposed PWM modulation technique requires only one carrier signal and two reference signals to control eight IGBTs. This ﬁgure shows the adapted voltage reference (ref 1, ref 2), the triangular carrier signal (vcarrier), the PWM signals of the IGBTs, the voltage levels produced by the converter (vcv_AC) and the power grid voltage (vg). The pulses signal of the IGBTs (S1, S2, S3, S4) are exclusively dependent of the instantaneous value of the power grid voltage. During the operation as grid-tied inverter, the IGBTs (S7, S8) have an opposite a command signal as the IGBTs (S5, S6), respectively. In the modulation strategy, the voltages references (ref 1, ref 2) are adapted, based on a digital codiﬁcation, from the modulating signal sM established by the Equation (1), where vcv_AC are the voltage levels produced by the converter and ma the amplitude modulation index. The voltage levels produced by the converter are obtained in the control algorithm, explained in Section 3.1. (1) (1) algorithm, explaine algorithm, explaine = 2 sM = 2vcv_ACma. 6 of 21 6 of 21 6 of 21 6 of 21 Energies 2018, 11, 3453 Energies 2018 11 x FOR Figure 5. Modified pulse-width modulation (PWM) strategy for the grid-side converter during the operation as active rectifier or as grid-tied inverter 0.00 0.01 0.02 0.03 0.04 0.05 Time (s) +vdc +vdc/2 -vdc/2 -vdc 0 vg vcarrier S6 S5 ref2 ref1 vcv_AC S1,4 S2,3 S8 S7 Figure 5. Modiﬁed pulse-width modulation (PWM) strategy for the grid-side converter during the operation as active rectiﬁer or as grid-tied inverter. Figure 5. Modified pulse-width modulation (PWM) strategy for the grid-side converter during the operation as active rectifier or as grid-tied inverter. 0.00 0.01 0.02 0.03 0.04 0.05 Time (s) +vdc +vdc/2 -vdc/2 -vdc 0 vg vcarrier S6 S5 ref2 ref1 vcv_AC S1,4 S2,3 S8 S7 Figure 5. 2 2 Topology Description: Battery-Side Converter 2.2. Topology Description: Battery-Side Converter 2.2. Topology Description: Battery Side Converter The circuit topology of the battery-side converter implemented in the on-board EVBC is presented in Figure 2. The topology consists in four IGBTs connected to the split DC-link and a passive LC filter interfacing the batteries. Furthermore, this topology can produce three distinct voltage levels (+vdc, +vdc/2, 0) at the terminals of the converter (vcv_DC). When the converter operates as a buck-type converter, the energy power flows of the DC-link to the batteries during the charging process. In this mode, the IGBTs (S9, S10) and the anti-parallel diodes of the IGBTs (S11, S12) are used. When only one of the IGBTs (S9, S10) is switched on, the inductors (L3, L4) and the batteries stores energy from the split DC-link (C1 or C2). When both IGBTs (S9, S10) are switched on, the inductors (L3, L4) and the batteries store energy from the DC-link (C1, C2). When both IGBTs (S9, S10) are switched off, the stored energy in the inductors (L3, L4) is released to the batteries. On the other hand, when the converter operates as boost-type converter, the energy power flows of the batteries to the DC-link, during the discharging process. During this mode, the IGBTs (S11, S12) and the anti-parallel diodes of the IGBTs (S9, S10) are used. When both IGBTs (S11, S12) are switched on, the inductors (L3, L4) stores energy from the batteries. When one of the IGBTs (S11, S12) is switched on, the split DC-link (C1 or C2) stores energy from the batteries and the inductors (L3, L4). Finally, when both IGBTs (S11, S12) are switched off, the DC-link (C1, C2) stores energy from the batteries and the inductors (L3, L4). Figures 6 and 7 show the operation stages for the battery-side converter during the operation as buck-type and boost type respectively The circuit topology of the battery-side converter implemented in the on-board EVBC is presented in Figure 2. The topology consists in four IGBTs connected to the split DC-link and a passive LC ﬁlter interfacing the batteries. Furthermore, this topology can produce three distinct voltage levels (+vdc, +vdc/2, 0) at the terminals of the converter (vcv_DC). When the converter operates as a buck-type converter, the energy power ﬂows of the DC-link to the batteries during the charging process. In this mode, the IGBTs (S9, S10) and the anti-parallel diodes of the IGBTs (S11, S12) are used. 5 of 21 5 of 21 Modiﬁed pulse-width modulation (PWM) strategy for the grid-side converter during the operation as active rectiﬁer or as grid-tied inverter. ref1 Figure 5. Modified pulse-width modulation (PWM) strategy for the grid-side converter during the operation as active rectifier or as grid-tied inverter. Figure 5. Modiﬁed pulse-width modulation (PWM) strategy for the grid-side converter during the operation as active rectiﬁer or as grid-tied inverter. 2 2 Topology Description: Battery Side Converter 2.2. Topology Description: Battery-Side Converter 2 2 Topology Description: Battery-Side Converter 2.2. Topology Description: Battery-Side Converter 2 2 Topology Description: Battery-Side Converter 2.2. Topology Description: Battery-Side Converter When only one of the IGBTs (S9, S10) is switched on, the inductors (L3, L4) and the batteries stores energy from the split DC-link (C1 or C2). When both IGBTs (S9, S10) are switched on, the inductors (L3, L4) and the batteries store energy from the DC-link (C1, C2). When both IGBTs (S9, S10) are switched off, the stored energy in the inductors (L3, L4) is released to the batteries. On the other hand, when the converter operates as boost-type converter, the energy power ﬂows of the batteries to the DC-link, during the discharging process. During this mode, the IGBTs (S11, S12) and the anti-parallel diodes of the IGBTs (S9, S10) are used. When both IGBTs (S11, S12) are switched on, the inductors (L3, L4) stores energy from the batteries. When one of the IGBTs (S11, S12) is switched on, the split DC-link (C1 or C2) stores energy from the batteries and the inductors (L3, L4). Finally, when both IGBTs (S11, S12) are switched off, the DC-link (C1, C2) stores energy from the batteries and the inductors (L3, L4). Figures 6 and 7 show the operation stages for the battery-side converter during the operation as buck-type and boost-type, respectively. 7 of 21 7 of 21 Energies 2018, 11, 3453 Energies 2018, 11, x FOR Figure 6. Operation stages for the battery-side converter during the operation as a buck-type converter: (a) The stored energy in L3, L4 is released to the batteries; (b) L3, L4 and the batteries stores energy from C1; (c) L3, L4 and the batteries stores energy from C2; (d) L3, L4 and the batteries store (b) (a) (d) (c) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 vL3 L3 vL4 L4 vL3 L3 vL4 L4 vL3 L3 vL4 L4 Figure 6. 2 2 Topology Description: Battery-Side Converter 2.2. Topology Description: Battery-Side Converter Operation stages for the battery-side converter during the operation as a buck-type converter: (a) The stored energy in L3, L4 is released to the batteries; (b) L3, L4 and the batteries stores energy from C1; (c) L3, L4 and the batteries stores energy from C2; (d) L3, L4 and the batteries store Figure 6. Operation stages for the battery-side converter during the operation as a buck-type converter: (a) The stored energy in L3, L4 is released to the batteries; (b) L3, L4 and the batteries stores energy from C1; (c) L3, L4 and the batteries stores energy from C2; (d) L3, L4 and the batteries store energy from C1, C2. converter: (a) The stored energy in L3, L4 is released to the batteries; (b) L3, L4 and the batteries stores energy from C1; (c) L3, L4 and the batteries stores energy from C2; (d) L3, L4 and the batteries store energy from C1, C2. C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 L4 (b) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 v energy from C1, C2. C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 L4 (a) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 energy from C1, C2. Figure 7. Operation stages for the battery-side converter during the operation as a boost-type converter: (a) L3, L4 stores energy from the batteries; (b) C2 stores energy from the batteries and L3, L4; (b) (a) (d) (c) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 vL3 L3 vL4 L4 vL3 L3 vL4 L4 vL3 L3 vL4 L4 Figure 7. 2 2 Topology Description: Battery-Side Converter 2.2. Topology Description: Battery-Side Converter Operation stages for the battery-side converter during the operation as a buck-type converter: (a) The stored energy in L3, L4 is released to the batteries; (b) L3, L4 and the batteries stores energy from C1; (c) L3, L4 and the batteries stores energy from C2; (d) L3, L4 and the batteries store energy from C1, C2. Figure 6. Operation stages for the battery-side converter during the operation as a buck-type converter: (a) The stored energy in L3, L4 is released to the batteries; (b) L3, L4 and the batteries stores energy from C1; (c) L3, L4 and the batteries stores energy from C2; (d) L3, L4 and the batteries store energy from C1, C2. (b) (a) (d) (c) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat L3 vL4 L4 L3 vL4 L4 vL3 L3 vL4 L4 vL3 L3 vL4 L4 (b) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 (b) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat L3 vL4 L4 vL3 L3 (a) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 v (a) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL4 L4 vL3 L3 (c) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat L3 vL4 L4 Figure 6. Operation stages for the battery-side (c) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL4 L4 (d) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat L3 vL4 L4 converter during the operation as a buck-type (d) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL4 L4 (d) perat (c) stag Figure 6. 2 2 Topology Description: Battery-Side Converter 2.2. Topology Description: Battery-Side Converter Operation stages for the battery-side converter during the operation as a boost-type converter: (a) L3, L4 stores energy from the batteries; (b) C2 stores energy from the batteries and L3, L4; (c) C1 stores energy from the batteries and L3, L4; (d) C1, C2 stores energy from the batteries and L3, L4. (b) (a) (d) (c) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 vL3 L3 vL4 L4 vL3 L3 vL4 L4 vL3 L3 vL4 L4 Figure 7. Operation stages for the battery-side converter during the operation as a boost-type converter: (a) L3, L4 stores energy from the batteries; (b) C2 stores energy from the batteries and L3, L4; (c) C1 stores energy from the batteries and L3, L4; (d) C1, C2 stores energy from the batteries and L3, L4. (b) (a) (a) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL4 vL3 L3 L4 (c) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL3 L3 vL4 L4 (b) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat vL4 vL3 L3 L4 (d) C2 vdc 2 C1 vdc 2 S9 S10 S11 S12 C3 vcv_DC1 vcv_DC2 vbat ibat L3 vL4 L4 (d) (c) Figure 7. Operation stages for the battery-side converter during the operation as a boost-type converter: (a) L3 L4 stores energy from the batteries; (b) C2 stores energy from the batteries and L3 L4; (d) (c) vL4 vL4 Figure 7. Operation stages for the battery-side converter during the operation as a boost-type converter: (a) L3, L4 stores energy from the batteries; (b) C2 stores energy from the batteries and L3, L4; (c) C1 stores energy from the batteries and L3, L4; (d) C1, C2 stores energy from the batteries and L3, L4. Figure 7. 3.1. Control Algorithm: Grid-Side Converter Based on the voltages shown in Figure 2, Equation (2) can be established, where vg represents the instantaneous value of the grid voltage, vL1 and vL2 the instantaneous value of the inductance voltage, and vcv_AC is the instantaneous value (i.e., the voltage produced during each sampling period of the DSP) of the voltage produced by the converter: (2) vg = vcv_AC + vL1 + vL2. (2) It should be noted that, as represented in Figure 2, it was used a mutual coupling inductance. Therefore, replacing the inductance voltage by its intrinsic equation, and rewriting the equation as a function of the voltage produced by the converter, it is obtained: vcv_AC = vg −L1 diev dt −L2 diev dt . (3) (3) Applying the progressive Euler method, illustrated in Equation (4), the derivative component of the current can be approximated by considering a very low ∆t in order to obtain a good prediction of the system behavior: diev(t) dt = iev(t + ∆t) −iev(t) ∆t . (4) (4) Applying the Equation (4) in the Equation (3), and assuming a sampling frequency of fs = 1/Ts, results in the digital control Equation (5), where the term k represents the current sample and [k + 1] represents the next sample: vcv_AC[k] = vg[k] −(L1 + L2)iev[k + 1] −iev[k] Ts . (5) (5) Since the law of predictive control consists of a closed-loop control and, if the reference current at time [k + 1] is to be equal to the current produced by the converter at time [k], the equation that translates the current control implemented can be deﬁned by: Since the law of predictive control consists of a closed-loop control and, if the reference current at time [k + 1] is to be equal to the current produced by the converter at time [k], the equation that translates the current control implemented can be deﬁned by: vcv_AC[k] = vg[k] −(L1 + L2)iev∗[k] −iev[k] Ts . (6) (6) Since, the EVBC is proposed to operate with a sinusoidal current and unitary power factor in the grid-side converter, the instantaneous value of the power grid voltage is directly proportional to the EVBC current. However, aiming to prevent the inclusion of the harmonic distortion of the grid voltage into the current, it is used a phase-locked loop (PLL). 3. EV Battery Charger: Control Algorithms This section presents the speciﬁcations and the methodology used for the control algorithms implementation, both for the grid-side and the battery-side converters. The control algorithm was designed for a digital platform, based on a Texas Instruments digital signal processor (DSP) F28335 (Texas Instruments, Inc., Dallas, TX, USA) and considering a sampling frequency of 40 kHz, obtained with a timer interruption. 2 2 Topology Description: Battery-Side Converter 2.2. Topology Description: Battery-Side Converter Operation stages for the battery-side converter during the operation as a boost-type converter: (a) L3, L4 stores energy from the batteries; (b) C2 stores energy from the batteries and L3, L4; (c) C1 stores energy from the batteries and L3, L4; (d) C1, C2 stores energy from the batteries and L3, L4. (c) C1 stores energy from the batteries and L3, L4; (d) C1, C2 stores energy from the batteries and L3, L4. Similar to the algorithm applied in the grid-side converter, in the battery-side converter was also applied a predictive current control technique and a modulation technique with a fixed switching frequency of 20 kHz, to control the current and voltage in the batteries during the charging or discharging processes. In the modulation technique, two 180° phase-shifted carriers were used in order to reduce the ripple of the EV battery current and hence the frequency EV Similar to the algorithm applied in the grid-side converter, in the battery-side converter was also applied a predictive current control technique and a modulation technique with a fixed switching frequency of 20 kHz, to control the current and voltage in the batteries during the charging or discharging processes. In the modulation technique, two 180° phase-shifted carriers were used, in order to reduce the ripple of the EV battery current and, hence, the frequency EV battery current is held twice of the switching frequency. Similar to the algorithm applied in the grid-side converter, in the battery-side converter was also applied a predictive current control technique and a modulation technique with a ﬁxed switching frequency of 20 kHz, to control the current and voltage in the batteries during the charging or discharging processes. In the modulation technique, two 180◦phase-shifted carriers were used, in order to reduce the ripple of the EV battery current and, hence, the frequency EV battery current is held twice of the switching frequency. 8 of 21 Energies 2018, 11, 3453 3.1. Control Algorithm: Grid-Side Converter Thus, instead of the grid voltage, it is used the output signal from the PLL, resulting in: iev = Gevvpll, (7) (7) where vpll is in phase with the power grid voltage and Gev represents the equivalent conductance of the EVBC from the grid-side point of view, which can be deﬁned according to the mean value of active power (Pev) and the rms value of the power grid voltage (Vg): where vpll is in phase with the power grid voltage and Gev represents the equivalent conductance of the EVBC from the grid-side point of view, which can be deﬁned according to the mean value of active power (Pev) and the rms value of the power grid voltage (Vg): Energies 2018, 11, 3453 Energies 2018, 11, 3453 9 of 21 Gev = PevVG−2. (8) (8) Applying the Equation (8) in the Equation (7), the reference of the EVBC current is obtained according to: 2 Applying the Equation (8) in the Equation (7), the reference of the EVBC current is obtained according to: iev∗= PevVG−2vpll. (9) (9) The active power of the EVBC can be divided in two parts, namely the power to regulate the DC-link voltage and the power to regulate the batteries. Furthermore, because of the split DC-link, two proportional-integral (PI) are used to regulate the DC-link voltage independently in both capacitors (pdc1, pdc2). Therefore, during the G2V operation mode, the reference of the EVBC current can be deﬁned as: (10) iev∗= (pdc1 + pdc1 + pbat)vpllVG−2. (10) On the other hand, during the V2G operation mode, the reference of the EVBC current is established according to Equation (11), where ibat* represent the reference of current to discharge the batteries. On the other hand, during the V2G operation mode, the reference of the EVBC current is established according to Equation (11), where ibat* represent the reference of current to discharge the batteries iev∗= (pdc1 + pdc1 + ibat∗vbat)vpllVG−2. (11) (11) iev∗= (pdc1 + pdc1 + ibat∗vbat)vpllVG−2. 3.2. Control Algorithm: Battery-Side Converter During the process of charging the batteries, the battery-side converter operates as buck converter, controlling the charging current or the charging voltage for the batteries. In this way, based on the representations of the currents and voltages between the DC-link and the batteries (cf. Figure 2), it is possible to establish the Equation (12), where vcv_DC represents the voltage produced by the converter (i.e., the sum between vcv_DC1 and vcv_DC2), vL3 and vL4 represent the voltages in the inductances (L3 and L4), respectively, and vbat represents the voltage in the batteries: (12) vcv_DC = vbat + vL3 + vL4. (12) Since the current in the inductance L3 is the same as that in the inductance L4, the Equation (12) can be rewritten, replacing the voltage in the inductance by its intrinsic equation: Since the current in the inductance L3 is the same as that in the inductance L4, the Equation (12) can be rewritten, replacing the voltage in the inductance by its intrinsic equation: vcv_DC = vbat + (L3 + L4)diL3,L4 dt . (13) (13) Applying the progressive Euler method, the Equation (13) can be established in discrete time as vcv_DC[k] = vbat[k] + (L3 + L4)(iL3,L4[k + 1] −iL3,L4[k])Ts−1. (14) (14) Since it is desired that the reference current at time [k + 1] should be equal to the current produced by the converter at time [k], it is obtained: Since it is desired that the reference current at time [k + 1] should be equal to the current produced by the converter at time [k], it is obtained: vcv_DC[k] = vbat[k] + (L3 + L4)(iL3,L4∗[k] −iL3,L4[k])Ts−1. (15) (15) When the same converter operates as buck converter, but controlling the charging voltage of the batteries, the reference voltage is established as: When the same converter operates as buck converter, but controlling the charging voltage of the batteries, the reference voltage is established as: vcv_DC[k] = vbat∗[k] −(L3 + L4)(iL3,L4[k] −iL3,L4[k −1])Ts−1. (16) (16) The aforementioned equations were deﬁned for the battery-side converter operating as buck converter, i.e., charging the batteries from the grid (G2V mode). On the other hand, a set of equations should also be deﬁned for the battery-side converter operating as boost converter, i.e., discharging the batteries to the grid (V2G mode). 4. EV Battery Charger: Experimental Validation This section introduces the experimental validation, where is presented the hardware description, as well as the most relevant experimental results. 3.2. Control Algorithm: Battery-Side Converter Based on the representations of the currents and voltages in Figure 2, The aforementioned equations were deﬁned for the battery-side converter operating as buck converter, i.e., charging the batteries from the grid (G2V mode). On the other hand, a set of equations should also be deﬁned for the battery-side converter operating as boost converter, i.e., discharging the batteries to the grid (V2G mode). Based on the representations of the currents and voltages in Figure 2, Energies 2018, 11, 3453 10 of 21 when the converter intends to discharge the batteries with a constant current, the following relation can be established: converter intends to discharge the batteries with a constant current, the following relation ablished: (17) vcv_DC = vbat −vL3 −vL4. (17) Applying the same aforementioned modeling reasoning, the discrete implementation of Equation (17) results in: vcv_DC[k] = vbat[k] −(L3 + L4)(−iL3,L4∗[k] −iL3,L4[k])T−1. (18) (18) From Equation (18), it is important to note that it is necessary to identify the positive direction of the current in the batteries (Figure 2), which is why the negative signal −iL3,L4[k + 1] is applied to the digital implementation of this equation. The obtained signal (vcv_DC[k]) is compared with two carriers in order to obtain the duty-cycle of the gate signals for S9 and S10, which is the same, but two carriers, delayed by 180 degrees, are employed. Applying this strategy, the frequency of the resultant ripple is the double of the switching frequency. The duty-cycle is determined according to the current control algorithm, e.g., in the previous equations is deﬁned the a voltage that is compared with the carriers in order to obtain a current in the EV battery i(L3,L4)[k] equal to the reference current i(L3,L4)[k]. Applying this strategy, the current ripple in the EV battery can be reduced when compared with a traditional buck or boost converter. In circumstances where the battery-side converter is responsible for controlling the voltage on the DC-link, the Equation (19) is implemented, where δcv_DC represents the duty-cycle that the converter must produce, vdc* represents the reference voltage for the DC-link, and vbat represents the battery voltage: δcv_DC[k] = vdc∗[k] −vbat[k] vdc∗[k] . (19) (19) 4.1. Description of the Developed Prototype Laboratorial prototype of the developed EVBC. Figure 9. Laboratorial prototype of the developed EVBC. Figure 9. Laboratorial prototype of the developed EVBC. Figure 9. Laboratorial prototype of the developed EVBC. Figure 9. Laboratorial prototype of the developed EVBC. The converters of the developed prototype were sized to meet the key requirements of the proposed on-board EVBC in terms of power quality, and the specifications given in Table 1. In this sense, the choice of components was mainly based on their operational characteristics, such as the maximum operating frequency and the rated current and voltage. In addition to the electrical aspects of the concerned application, mechanical and thermal aspects, such as dimensions, encapsulation and thermal dissipation of components, have also been taken into account, since the entire system is integrated in a metal box (Bernstein CA 380 (Porta Westfalica, Germany)), with dimensions of 330 × 230 × 110 mm. As it can be seen, two heatsinks were used, one for each converter, which were fixed on each side of the metal box. In order to prevent electrical noise, it was decided to place the gate driver boards as close as possible to the power semiconductors, each one responsible for a power electronics converter. In the control system, electrical noise was also taken into account in order to optimize the signal-to-noise ratio, i.e., to minimize undesired signals superimposed on a measured signal. Based on the developed three-dimensional modeling and the implemented prototype, an estimation of the internal volume distribution associated to the different main parts of the on-board EVBC was established, which is presented in Figure 10. The converters of the developed prototype were sized to meet the key requirements of the proposed on-board EVBC in terms of power quality, and the speciﬁcations given in Table 1. In this sense, the choice of components was mainly based on their operational characteristics, such as the maximum operating frequency and the rated current and voltage. In addition to the electrical aspects of the concerned application, mechanical and thermal aspects, such as dimensions, encapsulation and thermal dissipation of components, have also been taken into account, since the entire system is integrated in a metal box (Bernstein CA 380 (Porta Westfalica, Germany)), with dimensions of 330 × 230 × 110 mm. As it can be seen, two heatsinks were used, one for each converter, which were ﬁxed on each side of the metal box. 4.1. Description of the Developed Prototype After the computer validation, a laboratory prototype of the on-board EVBC was implemented, which is mainly divided in two parts: the digital control platform and the power hardware. The digital control platform includes the DSP board (F28335), the current and voltage sensors (models LTSR15 NP and CYHVS025A from LEM (Geneva, Switzerland) and from ChenYang (Finsing, Germany), respectively), as well as the printed circuit boards of the signal conditioning, error detection, command, and gate drivers (developed with HCPL 3120 optocouplers from Avago). On the other hand, the power hardware includes both converters, constituted by discrete IGBTs (FGA25N120ANTD from Fairchild (Sunnyvale, CA, USA)) and by the DC-link capacitors (EETUQ2W561DA from Panasonic (Kadoma, Japan)). Since the main aim of the implementation was the development of a laboratory prototype as close as possible of the reality, namely in compactness and robustness, a three-dimensional modeling was carried out to determine the best method and solution of the component layout for its implementation. Figure 8 shows the internal and external view of the three-dimensional modeling of the EVBC. With this modeling, it was possible to implement the ﬁnal laboratorial prototype, which is presented in Figure 9. However, it should be noted that the laboratory prototype was developed just aiming to validate the structure of the topology and to perform a laboratorial experimental validation. In fact, after the proof-of-concept in terms of topology and operation modes, some improvements are required focusing in the optimization of the switching devices (e.g., employing SiC devices) and in the optimization of the passive ﬁlters (e.g., employing a inductor-capacitor-inductor, LCL, ﬁlter) toward a pre-industrialized prototype. 11 of 21 ward a Energies 2018, 11, 3453 passive filters ( i d i li d ndustrialized prototype. Figure 8. Three-dimensional modeling of the proposed on-board EVBC. Figure 8. Three-dimensional modeling of the proposed on-board EVBC. ies 2018, 11, x FOR PEER REVIEW 11 o Figure 9. Laboratorial prototype of the developed EVBC. Figure 9. Laboratorial prototype of the developed EVBC. gies 2018, 11, x FOR PEER REVIEW 11 o Figure 9. Laboratorial prototype of the developed EVBC. Figure 8. Three-dimensional modeling of the proposed on-board EVBC. Figure 8. Three-dimensional modeling of the proposed on-board EVBC. FOR PEER REVIEW x FOR PEER REVIEW Figure 8. Three-dimensional modeling of the proposed on-board EVBC. Figure 8. Three-dimensional modeling of the proposed on-board EVBC. OR PEER REVIEW Figure 9. Laboratorial prototype of the developed EVBC. Figure 9. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation Figure 12 shows the experimental results during the initial stage of the EV charging process. First, during the time interval (1) the EVBC is not connected to the power grid. At time instant (2), the EVBC is connected to the power grid. In this stage, the DC-link start the pre-charge process through the anti-parallel diodes of the IGBTs (S1, S2, S3, S4), operating the grid-side converter as a traditional full-bridge rectifier. The pre-charge circuit contains a resistor to limit the typical peak currents of the capacitors. After the DC-link pre-charge process, at time instant (3), this resistor is bypassed so that the DC-link voltage remains close to the peak value of the grid voltage. At time instant (4), the split DC-link voltage, in each capacitor, is regulated to the operation voltage and only after stabilizing the DC-link voltage, at time instant (5), the current battery increases progressively (i.e., during the constant current algorithm), which is controlled by the battery-side converter Figure 12 shows the experimental results during the initial stage of the EV charging process. First, during the time interval (1) the EVBC is not connected to the power grid. At time instant (2), the EVBC is connected to the power grid. In this stage, the DC-link start the pre-charge process through the anti-parallel diodes of the IGBTs (S1, S2, S3, S4), operating the grid-side converter as a traditional full-bridge rectiﬁer. The pre-charge circuit contains a resistor to limit the typical peak currents of the capacitors. After the DC-link pre-charge process, at time instant (3), this resistor is bypassed so that the DC-link voltage remains close to the peak value of the grid voltage. At time instant (4), the split DC-link voltage, in each capacitor, is regulated to the operation voltage and only after stabilizing the DC-link voltage, at time instant (5), the current battery increases progressively (i.e., during the constant current algorithm), which is controlled by the battery-side converter operating as buck converter. (i.e., during the constant current algorithm), which is controlled by the battery side converter operating as buck converter. As clearly shown in Figure 13, the grid voltage is distorted due to the nonlinear electrical appliances linked in the electrical installation. 4.1. Description of the Developed Prototype In order to prevent electrical noise, it was decided to place the gate driver boards as close as possible to the power semiconductors, each one responsible for a power electronics converter. In the control system, electrical noise was also taken into account in order to optimize the signal-to-noise ratio, i.e., to minimize undesired signals superimposed on a measured signal. Based on the developed three-dimensional modeling and the implemented prototype, an estimation of the internal volume distribution associated to the different main parts of the on-board EVBC was established, which is presented in Figure 10. The converters of the developed prototype were sized to meet the key requirements of the proposed on-board EVBC in terms of power quality, and the specifications given in Table 1. In this sense, the choice of components was mainly based on their operational characteristics, such as the maximum operating frequency and the rated current and voltage. In addition to the electrical aspects of the concerned application, mechanical and thermal aspects, such as dimensions, encapsulation and thermal dissipation of components, have also been taken into account, since the entire system is integrated in a metal box (Bernstein CA 380 (Porta Westfalica, Germany)), with dimensions of 330 × 230 × 110 mm. As it can be seen, two heatsinks were used, one for each converter, which were fixed on each side of the metal box. In order to prevent electrical noise, it was decided to place the gate driver boards as close as possible to the power semiconductors, each one responsible for a power electronics converter. In the control system, electrical noise was also taken into account in order to optimize the signal-to-noise ratio, i.e., to minimize undesired signals superimposed on a measured signal. Based on the developed three-dimensional modeling and the implemented prototype, an estimation of the internal volume distribution associated to the different main parts of the on-board EVBC was established, which is presented in Figure 10. Figure 10 Internal volume distribution of the developed on-board EVBC Figure 10. Internal volume distribution of the developed on-board EVBC. Figure 10. Internal volume distribution of the developed on-board EVBC. Fi I l l d b f h d l d b d EVBC Figure 10. Internal volume distribution of the developed on-board EVBC. Figure 10. Internal volume distribution of the developed on-board EVBC. Energies 2018, 11, 3453 12 of 21 12 of 21 4.2. Experimental Results In the on-board EVBC laboratorial prototype described above, the G2V operation mode was initially validated, followed by the V2G. Besides the validation of both operations mode, the experimental results also demonstrate the validation of the PLL algorithm, modulation and current control strategy and DC-link voltage control. Figure 11 presents the general view of the laboratorial setup that was used during the experimental results, which were registered with a digital oscilloscope Yokogawa model DL708E (Yokogawa Electric, Tokyo, Japan), with a Fluke 435 power quality analyzer (Fluke Corporation, Everett, WA, USA), and with a FLIR i7 infrared thermal imaging camera (FLIR Systems, Wilsonville, OR, USA). Energies 2018, 11, x FOR PEER REVIEW 12 of 21 Figure 11. Laboratory setup used to obtain the experimental results. Figure 11. Laboratory setup used to obtain the experimental results. Figure 11. Laboratory setup used to obtain the experimental results. Figure 11. Laboratory setup used to obtain the experimental results. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation imum amplitude of the power grid voltage, and the ripple in the voltages of the capacitors ha uency that is double the frequency of the power grid. In this operation mode the measu iency was 92%. Figure 12. Experimental results during the initial stage of the electric vehicle (EV) battery charging process: EV battery current (ibat: 1 A/div); DC-link voltages of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div); EVBC current (iev: 2 A/div) Figure 12. Experimental results during the initial stage of the electric vehicle (EV) battery charging process: EV battery current (ibat: 1 A/div); DC-link voltages of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div); EVBC current (iev: 2 A/div). ximum amplitude of the power grid voltage, and the ripple in the voltages of the capacitors h uency that is double the frequency of the power grid. In this operation mode the measu ciency was 92%. V/div); EVBC current (iev: 2 A/div). As clearly shown in Figure 13, the grid voltage is distorted due to the nonlinear electri pliances linked in the electrical installation. However, during a steady-state operation, with t opted current control strategy, the EVBC current is kept with a sinusoidal waveform and in pha th the grid voltage (i.e., operating with a unitary power factor, as shown in the zoom det Figure 13. Experimental results in grid-to-vehicle (G2V) mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 100 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 5 V/div, ∆vdc2: 5 V/div). Figure 13. Experimental results in grid-to-vehicle (G2V) mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 100 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 5 V/div, ∆vdc2: 5 V/div). Figure 13. Experimental results in grid-to-vehicle (G2V) mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 100 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 5 V/div, ∆vdc2: 5 V/div). Figure 13. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation However, during a steady-state operation, with the adopted current control strategy, the EVBC current is kept with a sinusoidal waveform and in phase with the grid voltage (i.e., operating with a unitary power factor, as shown in the zoom detail presented in this ﬁgure). Furthermore, the DC-link voltage in both capacitors presents an acceptable ripple for this type of application (i.e., about 10%). The average voltage value in each DC-link capacitor is controlled according to each reference, assuming a value which is greater than the maximum amplitude of the power grid voltage, and the ripple in the voltages of the capacitors has a frequency that is double the frequency of the power grid. In this operation mode the measured efﬁciency was 92%. The experimental results shown in Figure 14 were attained to verify the switching states of the grid-side converter according the Figure 3. The IGBTs S1, S2, S3, and S4, as well as the IGBTs S5 and S6, have a ﬁxed switching frequency of 50 Hz and 20 kHz, respectively. This ﬁgure also shows the digital signal obtained from the PLL algorithm, which is sinusoidal and in phase with grid voltage. This signal was visualized in the oscilloscope using an external digital-to-analog converter (DAC), model TLV5610 from Texas Instruments. 13 of 21 onverter Energies 2018, 11, 3453 (i.e., during the c operating as buck erating as buck converter. Figure 12. Experimental results during the initial stage of the electric vehicle (EV) battery charging process: EV battery current (ibat: 1 A/div); DC-link voltages of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div); EVBC current (iev: 2 A/div). As clearly shown in Figure 13, the grid voltage is distorted due to the nonlinear electri pliances linked in the electrical installation. However, during a steady-state operation, with t opted current control strategy, the EVBC current is kept with a sinusoidal waveform and in pha th the grid voltage (i.e., operating with a unitary power factor, as shown in the zoom det Figure 12. Experimental results during the initial stage of the electric vehicle (EV) battery charging process: EV battery current (ibat: 1 A/div); DC-link voltages of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div); EVBC current (iev: 2 A/div). rgies 2018, 11, x FOR PEER REVIEW 13 of sented in this figure). Furthermore, the DC-link voltage in both capacitors presents an acceptab ple for this type of application (i.e., about 10%). 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation The average voltage value in each DC-lin acitor is controlled according to each reference, assuming a value which is greater than t ximum amplitude of the power grid voltage, and the ripple in the voltages of the capacitors has quency that is double the frequency of the power grid. In this operation mode the measur ciency was 92%. Figure 13. Experimental results in grid-to-vehicle (G2V) mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 100 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 5 V/div, ∆vdc2: 5 V/div). Figure 13. Experimental results in grid-to-vehicle (G2V) mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 100 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 5 V/div, ∆vdc2: 5 V/div). Figure 12. Experimental results during the initial stage of the electric vehicle (EV) battery charging process: EV battery current (ibat: 1 A/div); DC-link voltages of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div); EVBC current (iev: 2 A/div) Figure 12. Experimental results during the initial stage of the electric vehicle (EV) battery charging process: EV battery current (ibat: 1 A/div); DC-link voltages of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div); EVBC current (iev: 2 A/div). ergies 2018, 11, x FOR PEER REVIEW 13 of esented in this figure). Furthermore, the DC-link voltage in both capacitors presents an acceptab pple for this type of application (i.e., about 10%). The average voltage value in each DC-lin pacitor is controlled according to each reference, assuming a value which is greater than th aximum amplitude of the power grid voltage, and the ripple in the voltages of the capacitors has equency that is double the frequency of the power grid. In this operation mode the measure ficiency was 92%. Figure 12. Experimental results during the initial stage of the electric vehicle (EV) battery charging process: EV battery current (ibat: 1 A/div); DC-link voltages of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div); EVBC current (iev: 2 A/div) Figure 12. Experimental results during the initial stage of the electric vehicle (EV) battery charging process: EV battery current (ibat: 1 A/div); DC-link voltages of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div); EVBC current (iev: 2 A/div). 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation Experimental results in grid-to-vehicle (G2V) mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 100 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 5 V/div, ∆vdc2: 5 V/div). The experimental results shown in Figure 14 were attained to verify the switching states of the grid-side converter according the Figure 3. The IGBTs S1, S2, S3, and S4, as well as the IGBTs S5 and S6, have a fixed switching frequency of 50 Hz and 20 kHz, respectively. This figure also shows the digital signal obtained from the PLL algorithm, which is sinusoidal and in phase with grid voltage. This signal was visualized in the oscilloscope using an external digital-to-analog converter (DAC), model TLV5610 from Texas Instruments. The PWM technique developed for the battery-side converter consists in two carriers with a 180◦ phase-shifting. With this phase-shifting strategy, one of the gate signals is applied to IGBT S9 while the other is applied to the IGBT S10, to reduce the ripple of the EV battery current. Thus, as it can be seen in Figure 15, the frequency of the current ripple in the inductor L3 (iL3) is 40 kHz, which is twice of the switching frequency. This experimental result shows the relation of the current in the inductor and the gate-emitter voltage, vge_S9 and vge_S10, of the IGTBs S9 and S10. During this result, the registered value of the ripple current in the inductor was 0.16 A. 14 of 21 r (DAC), Energies 2018, 11, 3453 This signal was vi model TLV5610 fr Figure 14. Experimental results during the G2V operation mode: Gate-emitter voltage of the grid-side insulated-gate bipolar transistors (IGBTs) (S1, S2, S3, S4, S5, S6: 5 V/div); Output digital signal of the phase-locked loop (PLL) (vPLL: 150 V/div). Figure 14. Experimental results during the G2V operation mode: Gate-emitter voltage of the grid-side insulated-gate bipolar transistors (IGBTs) (S1, S2, S3, S4, S5, S6: 5 V/div); Output digital signal of the phase-locked loop (PLL) (vPLL: 150 V/div). ergies 2018, 11, x FOR PEER REVIEW 14 of The PWM technique developed for the battery-side converter consists in two carriers with 0° phase-shifting. With this phase-shifting strategy, one of the gate signals is applied to IGBT hile the other is applied to the IGBT S10, to reduce the ripple of the EV battery current. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S9 and S10 (vge_s9: 5 V/div and vge_10: 5 V/div) during a time interval of 100 µs. Figure 15. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S9 and S10 (vge_s9: 5 V/div and vge_10: 5 V/div) during a time interval of 100 µs. Figure 15. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S9 and S10 (vge_s9: 5 V/div and vge_10: 5 V/div) during a time interval of 100 µs. For further details on the relationship between the grid voltage and the EVBC grid-side current, it was used the x-y mode of the oscilloscope, as shown in Figure 16. Channel 4 is used in the x-axis and the channel 2 is used in the y-axis, representing the grid voltage and EVBC grid-side current, respectively. Thus, for the grid-side, this result shows the EVBC current in function of the grid voltage. It is relevant to highlight that this variation is not linear due to the harmonic distortion present in the grid voltage. For further details on the relationship between the grid voltage and the EVBC grid-side current, it was used the x-y mode of the oscilloscope, as shown in Figure 16. Channel 4 is used in the x-axis and the channel 2 is used in the y-axis, representing the grid voltage and EVBC grid-side current, respectively. Thus, for the grid-side, this result shows the EVBC current in function of the grid voltage. It is relevant to highlight that this variation is not linear due to the harmonic distortion present in the grid voltage. present in the grid voltage. The experimental result shown in Figure 17 was obtained in x-y mode in order to identify the DC-link voltage regulation and to clearly identify the ﬁve distinct voltage levels (+vdc, +vdc/2, 0, −vdc/2, −vdc) produced by the grid-side converter. Thus, the DC-link voltage ripple (∆vdc1, ∆vdc2) and the voltage levels (vcv_AC), used in the y-axis, are a function of the grid voltage (vg), used in the x-axis. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation Experimental results during the G2V operation mode: Gate-emitter voltage of the grid-side insulated-gate bipolar transistors (IGBTs) (S1, S2, S3, S4, S5, S6: 5 V/div); Output digital signal of the phase-locked loop (PLL) (vPLL: 150 V/div). ergies 2018, 11, x FOR PEER REVIEW 14 of The PWM technique developed for the battery-side converter consists in two carriers with 0° phase-shifting. With this phase-shifting strategy, one of the gate signals is applied to IGBT hile the other is applied to the IGBT S10, to reduce the ripple of the EV battery current. Thus, as n be seen in Figure 15, the frequency of the current ripple in the inductor L3 (iL3) is 40 kHz, which wice of the switching frequency. This experimental result shows the relation of the current in t ductor and the gate-emitter voltage, vge_S9 and vge_S10, of the IGTBs S9 and S10. During this result, t gistered value of the ripple current in the inductor was 0.16 A. Figure 14. Experimental results during the G2V operation mode: Gate-emitter voltage of the grid-side insulated-gate bipolar transistors (IGBTs) (S1, S2, S3, S4, S5, S6: 5 V/div); Output digital signal of the phase-locked loop (PLL) (vPLL: 150 V/div). Figure 14. Experimental results during the G2V operation mode: Gate-emitter voltage of the grid-side insulated-gate bipolar transistors (IGBTs) (S1, S2, S3, S4, S5, S6: 5 V/div); Output digital signal of the phase-locked loop (PLL) (vPLL: 150 V/div). ce of the switching frequency. This experimental result shows the relation of the current in uctor and the gate-emitter voltage, vge_S9 and vge_S10, of the IGTBs S9 and S10. During this result, stered value of the ripple current in the inductor was 0.16 A. Figure 15. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S9 and S10 (vge_s9: 5 V/div and vge_10: 5 V/div) during a time interval of 100 µs. Figure 15. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S9 and S10 (vge_s9: 5 V/div and vge_10: 5 V/div) during a time interval of 100 µs. Figure 15. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S9 and S10 (vge_s9: 5 V/div and vge_10: 5 V/div) during a time interval of 100 µs. Figure 15. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation Thus, as n be seen in Figure 15, the frequency of the current ripple in the inductor L3 (iL3) is 40 kHz, which ice of the switching frequency. This experimental result shows the relation of the current in t ductor and the gate-emitter voltage, vge_S9 and vge_S10, of the IGTBs S9 and S10. During this result, t gistered value of the ripple current in the inductor was 0.16 A. Figure 15. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S9 and S10 (vge_s9: 5 V/div and vge_10: 5 V/div) during a time interval of 100 µs. Figure 15. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S9 and S10 (vge_s9: 5 V/div and vge_10: 5 V/div) during a time interval of 100 µs. Figure 14. Experimental results during the G2V operation mode: Gate-emitter voltage of the grid-side insulated-gate bipolar transistors (IGBTs) (S1, S2, S3, S4, S5, S6: 5 V/div); Output digital signal of the phase-locked loop (PLL) (vPLL: 150 V/div). Figure 14. Experimental results during the G2V operation mode: Gate-emitter voltage of the grid-side insulated-gate bipolar transistors (IGBTs) (S1, S2, S3, S4, S5, S6: 5 V/div); Output digital signal of the phase-locked loop (PLL) (vPLL: 150 V/div). ergies 2018, 11, x FOR PEER REVIEW 14 of The PWM technique developed for the battery-side converter consists in two carriers with 0° phase-shifting. With this phase-shifting strategy, one of the gate signals is applied to IGBT hile the other is applied to the IGBT S10, to reduce the ripple of the EV battery current. Thus, as n be seen in Figure 15, the frequency of the current ripple in the inductor L3 (iL3) is 40 kHz, which wice of the switching frequency. This experimental result shows the relation of the current in th ductor and the gate-emitter voltage, vge_S9 and vge_S10, of the IGTBs S9 and S10. During this result, th gistered value of the ripple current in the inductor was 0.16 A. Figure 14. Experimental results during the G2V operation mode: Gate-emitter voltage of the grid-side insulated-gate bipolar transistors (IGBTs) (S1, S2, S3, S4, S5, S6: 5 V/div); Output digital signal of the phase-locked loop (PLL) (vPLL: 150 V/div). Figure 14. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation Experimental results in x-y mode showing the DC-link voltage ripple (∆vdc1: 5 V/div, ∆vdc2: 5 V/div) (y-axis) and the voltage levels produced by the grid-side converter (vcv_AC: 50 V/div) (y-axis), both in function of the power grid voltage (vg: 100 V/div) (x-axis). Using the Fluke power quality analyzer, in Figure 18a,b shows the harmonic spectrum of th wer grid voltage and the EVBC current, measured total harmonic distortion (THD%) of 3.5% an %, respectively. In power electronics systems, the thermal characteristic is a factor that directl Figure 17. Experimental results in x-y mode showing the DC-link voltage ripple (∆vdc1: 5 V/div, ∆vdc2: 5 V/div) (y-axis) and the voltage levels produced by the grid-side converter (vcv_AC: 50 V/div) (y-axis), both in function of the power grid voltage (vg: 100 V/div) (x-axis). Using the Fluke power quality analyzer, in Figure 18a,b shows the harmonic spectrum of th wer grid voltage and the EVBC current, measured total harmonic distortion (THD%) of 3.5% an %, respectively. In power electronics systems, the thermal characteristic is a factor that directl t th f f it S t l th th l diti f th EVBC th i t Figure 16. Experimental results in x-y mode showing the EVBC current (iev: 5 A/div) in function of the power grid voltage (vg: 100 V/div). Figure 16. Experimental results in x-y mode showing the EVBC current (iev: 5 A/div) in function of the power grid voltage (vg: 100 V/div). voltage of the capacitor C1 is regulated, and during the negative half-cycle of the grid voltage, tage of the capacitor C2 is regulated. Figure 17. Experimental results in x-y mode showing the DC-link voltage ripple (∆vdc1: 5 V/div, ∆vdc2: 5 V/div) (y-axis) and the voltage levels produced by the grid-side converter (vcv_AC: 50 V/div) (y-axis), both in function of the power grid voltage (vg: 100 V/div) (x-axis). Figure 17. Experimental results in x-y mode showing the DC-link voltage ripple (∆vdc1: 5 V/div, ∆vdc2: 5 V/div) (y-axis) and the voltage levels produced by the grid-side converter (vcv_AC: 50 V/div) (y-axis), both in function of the power grid voltage (vg: 100 V/div) (x-axis). Figure 17. Experimental results in x-y mode showing the DC-link voltage ripple (∆vdc1: 5 V/div, ∆vdc2: 5 V/div) (y-axis) and the voltage levels produced by the grid-side converter (vcv_AC: 50 V/div) (y-axis), both in function of the power grid voltage (vg: 100 V/div) (x-axis). Figure 17. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation In order to keep the DC-link regulated and balanced, during the positive half-cycle of the grid voltage, the voltage of the capacitor C1 is regulated, and during the negative half-cycle of the grid voltage, the voltage of the capacitor C2 is regulated. 15 of 21 stortion Energies 2018, 11, 3453 voltage. It is relev present in the grid Figure 16. Experimental results in x-y mode showing the EVBC current (iev: 5 A/div) in function of the power grid voltage (vg: 100 V/div). Figure 16. Experimental results in x-y mode showing the EVBC current (iev: 5 A/div) in function of the power grid voltage (vg: 100 V/div). ergies 2018, 11, x FOR PEER REVIEW 15 of The experimental result shown in Figure 17 was obtained in x-y mode in order to identify t C-link voltage regulation and to clearly identify the five distinct voltage levels (+vdc, +vdc/2, 0, −vd dc) produced by the grid-side converter. Thus, the DC-link voltage ripple (∆vdc1, ∆vdc2) and t ltage levels (vcv_AC), used in the y-axis, are a function of the grid voltage (vg), used in the x-axis. der to keep the DC-link regulated and balanced, during the positive half-cycle of the grid voltag e voltage of the capacitor C1 is regulated, and during the negative half-cycle of the grid voltage, t ltage of the capacitor C2 is regulated. Figure 16. Experimental results in x-y mode showing the EVBC current (iev: 5 A/div) in function of the power grid voltage (vg: 100 V/div). Figure 16. Experimental results in x-y mode showing the EVBC current (iev: 5 A/div) in function of the power grid voltage (vg: 100 V/div). rgies 2018, 11, x FOR PEER REVIEW 15 of 2 The experimental result shown in Figure 17 was obtained in x-y mode in order to identify th C-link voltage regulation and to clearly identify the five distinct voltage levels (+vdc, +vdc/2, 0, −vdc dc) produced by the grid-side converter. Thus, the DC-link voltage ripple (∆vdc1, ∆vdc2) and th tage levels (vcv_AC), used in the y-axis, are a function of the grid voltage (vg), used in the x-axis. I der to keep the DC-link regulated and balanced, during the positive half-cycle of the grid voltag voltage of the capacitor C1 is regulated, and during the negative half-cycle of the grid voltage, th tage of the capacitor C2 is regulated. Figure 17. 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation Experimental results in x-y mode showing the DC-link voltage ripple (∆vdc1: 5 V/div, ∆vdc2: 5 V/div) (y-axis) and the voltage levels produced by the grid-side converter (vcv_AC: 50 V/div) (y-axis), both in function of the power grid voltage (vg: 100 V/div) (x-axis). Using the Fluke power quality analyzer, in Figure 18a,b shows the harmonic spectrum of the power grid voltage and the EVBC current, measured total harmonic distortion (THD%) of 3.5% and 2.8%, respectively. In power electronics systems, the thermal characteristic is a factor that directly affects the performance of it. So, to analyse the thermal conditions of the EVBC, the experimental results of temperature measurements during the G2V operation mode are presented in Figure 19. Figure 19a shows the overall thermal distribution of the implemented EVBC, Figure 19b shows the measured temperature of IGBT S9 (switched at a fixed frequency of 20 kHz), where was registered a temperature value of 47.8 °C, and Figure 19c shows the measured temperature of the IGBT S11 (only the antiparallel diode is used in this context), where was registered a temperature value of 36.8 °C. Using the Fluke power quality analyzer, in Figure 18a,b shows the harmonic spectrum of the power grid voltage and the EVBC current, measured total harmonic distortion (THD%) of 3.5% and 2.8%, respectively. In power electronics systems, the thermal characteristic is a factor that directly affects the performance of it. So, to analyse the thermal conditions of the EVBC, the experimental results of temperature measurements during the G2V operation mode are presented in Figure 19. Figure 19a shows the overall thermal distribution of the implemented EVBC, Figure 19b shows the measured temperature of IGBT S9 (switched at a fixed frequency of 20 kHz), where was registered a temperature value of 47.8 ◦C, and Figure 19c shows the measured temperature of the IGBT S11 (only the antiparallel diode is used in this context), where was registered a temperature value of 36.8 ◦C. 16 of 21 S11 (only 6 8 °C 16 of 21 S11 (only 6 °C Energies 2018, 11, 3453 temperature value h ll l d Figure 18. Experimental results in G2V mode of the total harmonic distortion and spectral analysis: (a) power grid voltage (vg); and (b) EVBC current (iev). Figure 18. Experimental results in G2V mode of the total harmonic distortion and spectral analysis: (a) power grid voltage (vg); and (b) EVBC current (iev). 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation 4.2.1. Experimental Results: Grid-To-Vehicle (G2V) Operation ergies 2018, 11, x FOR PEER REVIEW 16 of Figure 18. Experimental results in G2V mode of the total harmonic distortion and spectral analysis: (a) power grid voltage (vg); and (b) EVBC current (iev). Figure 18. Experimental results in G2V mode of the total harmonic distortion and spectral analysis: (a) power grid voltage (vg); and (b) EVBC current (iev). ergies 2018, 11, x FOR PEER REVIEW 16 of Figure 19. Experimental results of the temperature measurements during G2V operation mode: (a) overall thermal distribution of the developed EVBC prototype; (b) temperature at the IGBT S9; and (c) temperature at the IGBT S11. Figure 19. Experimental results of the temperature measurements during G2V operation mode: (a) overall thermal distribution of the developed EVBC prototype; (b) temperature at the IGBT S9; and (c) temperature at the IGBT S11. Figure 18. Experimental results in G2V mode of the total harmonic distortion and spectral analysis: (a) power grid voltage (vg); and (b) EVBC current (iev). Figure 18. Experimental results in G2V mode of the total harmonic distortion and spectral analysis: (a) power grid voltage (vg); and (b) EVBC current (iev). gies 2018, 11, x FOR PEER REVIEW 16 o Figure 19. Experimental results of the temperature measurements during G2V operation mode: (a) overall thermal distribution of the developed EVBC prototype; (b) temperature at the IGBT S9; and (c) temperature at the IGBT S11. Figure 19. Experimental results of the temperature measurements during G2V operation mode: (a) overall thermal distribution of the developed EVBC prototype; (b) temperature at the IGBT S9; and (c) temperature at the IGBT S11. Figure 19. Experimental results of the temperature measurements during G2V operation mode: (a) overall thermal distribution of the developed EVBC prototype; (b) temperature at the IGBT S9; and (c) temperature at the IGBT S11. Figure 19. Experimental results of the temperature measurements during G2V operation mode: (a) overall thermal distribution of the developed EVBC prototype; (b) temperature at the IGBT S9; and (c) temperature at the IGBT S11. 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation nergies 2018, 11, x FOR PEER REVIEW 17 of rid. Furthermore, the five distinct voltage levels (+vdc, +vdc/2, 0, −vdc/2, −vdc) produced by the grid-si onverter can be seen in this figure. Figure 20. Experimental results showing the modulation technique applied to the IGBTs S7 and S8, namely, the reference signals adopted for the modulation (refs7: 1 V/div, refs8: 1 V/div) and the voltage gate-emitter of the respective IGBT (vge_s7: 5 V/div and vge_s8: 5 V/div). Figure 20. Experimental results showing the modulation technique applied to the IGBTs S7 and S8, namely, the reference signals adopted for the modulation (refs7: 1 V/div, refs8: 1 V/div) and the voltage gate-emitter of the respective IGBT (vge_s7: 5 V/div and vge_s8: 5 V/div). d. Furthermore, the five distinct voltage levels (+vdc, +vdc/2, 0, −vdc/2, −vdc) produced by the grid nverter can be seen in this figure. Figure 20. Experimental results showing the modulation technique applied to the IGBTs S7 and S8, namely, the reference signals adopted for the modulation (refs7: 1 V/div, refs8: 1 V/div) and the voltage gate-emitter of the respective IGBT (vge_s7: 5 V/div and vge_s8: 5 V/div). Figure 20. Experimental results showing the modulation technique applied to the IGBTs S7 and S8, namely, the reference signals adopted for the modulation (refs7: 1 V/div, refs8: 1 V/div) and the voltage gate-emitter of the respective IGBT (vge_s7: 5 V/div and vge_s8: 5 V/div). d. Furthermore, the five distinct voltage levels (+vdc, +vdc/2, 0, −vdc/2, −vdc) produced by the grid-s nverter can be seen in this figure. Similar to the G2V operation mode, Figure 21 shows the switching states of the grid-sid nverter according to Table 2 during the V2G operation mode. In this result, the IGBTs S1, S2, S nd S4, as well as the IGBTs S7 and S8, have a fixed switching frequency of 50 Hz and 20 kH spectively. In this operation mode, the IGBTs (S5, S6) are always switched off, reason why they a ot represented in this figure. Figure 22 shows the V2G operation mode during a steady operation of the EVBC grid-sid urrent (iev), the grid voltage (vg), the voltage levels assumed by the grid-side converter (vcv_AC), an e DC-link voltage of both capacitors (vdc1, vdc2). The EVBC grid-side current is sinusoidal, but hase opposition with the grid voltage, meaning that the power follows from the batteries to th Figure 21. 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation Similar to the G2V operation mode, Figure 21 shows the switching states of the grid-sid nverter according to Table 2 during the V2G operation mode. In this result, the IGBTs S1, S2, S d S4, as well as the IGBTs S7 and S8, have a fixed switching frequency of 50 Hz and 20 kH pectively. In this operation mode, the IGBTs (S5, S6) are always switched off, reason why they a t represented in this figure. Figure 22 shows the V2G operation mode during a steady operation of the EVBC grid-sid rrent (iev), the grid voltage (vg), the voltage levels assumed by the grid-side converter (vcv_AC), an e DC-link voltage of both capacitors (vdc1, vdc2). The EVBC grid-side current is sinusoidal, but ase opposition with the grid voltage, meaning that the power follows from the batteries to th Figure 20. Experimental results showing the modulation technique applied to the IGBTs S7 and S8, namely, the reference signals adopted for the modulation (refs7: 1 V/div, refs8: 1 V/div) and the voltage gate-emitter of the respective IGBT (vge_s7: 5 V/div and vge_s8: 5 V/div). ergies 2018, 11, x FOR PEER REVIEW 17 of id. Furthermore, the five distinct voltage levels (+vdc, +vdc/2, 0, −vdc/2, −vdc) produced by the grid-si nverter can be seen in this figure. Figure 21. Experimental results during vehicle-to-grid (V2G) operation mode: Gate-emitter voltage of the grid-side IGBTs (S1, S2, S3, S4, S7, S8: 5 V/div), and output digital signal of the PLL (vPLL: 150 V/div). Figure 21. Experimental results during vehicle-to-grid (V2G) operation mode: Gate-emitter voltage of the grid-side IGBTs (S1, S2, S3, S4, S7, S8: 5 V/div), and output digital signal of the PLL (vPLL: 150 V/div). For further details, Figure 23 presents an experimental result, during a time interval of 50 m the EVBC grid-side current (iev), the grid voltage (vg), and the DC-link voltage ripple in bot Figure 20. Experimental results showing the modulation technique applied to the IGBTs S7 and S8, namely, the reference signals adopted for the modulation (refs7: 1 V/div, refs8: 1 V/div) and the voltage gate-emitter of the respective IGBT (vge_s7: 5 V/div and vge_s8: 5 V/div). Figure 20. Experimental results showing the modulation technique applied to the IGBTs S7 and S8, namely, the reference signals adopted for the modulation (refs7: 1 V/div, refs8: 1 V/div) and the voltage gate-emitter of the respective IGBT (vge_s7: 5 V/div and vge_s8: 5 V/div). 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation The developed EVBC was also validated during the V2G operation mode. Furthermore, once the IGBTs S7 and S8 of the grid-side converter has a fixed switching frequency of 20 kHz, to validate the modulation technique applied in these IGBTs, Figure 20 presents the reference signal adapted and the gate-emitter voltage of the respective IGBT. This voltage is a resulting signal of the comparison between the carrier signal and the reference signal. In this result, the reference signal was acquired using an external DAC The developed EVBC was also validated during the V2G operation mode. Furthermore, once the IGBTs S7 and S8 of the grid-side converter has a ﬁxed switching frequency of 20 kHz, to validate the modulation technique applied in these IGBTs, Figure 20 presents the reference signal adapted and the gate-emitter voltage of the respective IGBT. This voltage is a resulting signal of the comparison between the carrier signal and the reference signal. In this result, the reference signal was acquired using an external DAC. was acquired using an external DAC. Similar to the G2V operation mode, Figure 21 shows the switching states of the grid-side converter according to Figure 3 during the V2G operation mode. In this result, the IGBTs S1, S2, S3, and S4, as well as the IGBTs S7 and S8, have a ﬁxed switching frequency of 50 Hz and 20 kHz, respectively. In this operation mode, the IGBTs (S5, S6) are always switched off, reason why they are not represented in this ﬁgure. Figure 22 shows the V2G operation mode during a steady operation of the EVBC grid-side current (iev), the grid voltage (vg), the voltage levels assumed by the grid-side converter (vcv_AC), and the DC-link voltage of both capacitors (vdc1, vdc2). The EVBC grid-side current is sinusoidal, but in phase opposition with the grid voltage, meaning that the power follows from the batteries to the grid. Furthermore, the ﬁve distinct voltage levels (+vdc, +vdc/2, 0, −vdc/2, −vdc) produced by the grid-side converter can be seen in this ﬁgure. 17 of 21 ce signal Energies 2018, 11, 3453 comparison betwe was acquired using as acquired using an external DAC. Figure 20. Experimental results showing the modulation technique applied to the IGBTs S7 and S8, namely, the reference signals adopted for the modulation (refs7: 1 V/div, refs8: 1 V/div) and the voltage gate-emitter of the respective IGBT (vge_s7: 5 V/div and vge_s8: 5 V/div). 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation Experimental results during vehicle-to-grid (V2G) operation mode: Gate-emitter voltage of the grid-side IGBTs (S1, S2, S3, S4, S7, S8: 5 V/div), and output digital signal of the PLL (vPLL: 150 V/div). Figure 21. Experimental results during vehicle-to-grid (V2G) operation mode: Gate-emitter voltage of the grid-side IGBTs (S1, S2, S3, S4, S7, S8: 5 V/div), and output digital signal of the PLL (vPLL: 150 V/div). Figure 21. Experimental results during vehicle-to-grid (V2G) operation mode: Gate-emitter voltage of the grid-side IGBTs (S1, S2, S3, S4, S7, S8: 5 V/div), and output digital signal of the PLL (vPLL: 150 V/div). Figure 21. Experimental results during vehicle-to-grid (V2G) operation mode: Gate-emitter voltage of the grid-side IGBTs (S1, S2, S3, S4, S7, S8: 5 V/div), and output digital signal of the PLL (vPLL: 150 V/div). For further details, Figure 23 presents an experimental result, during a time interval of 50 ms, of the EVBC grid-side current (iev), the grid voltage (vg), and the DC-link voltage ripple in both capacitors (∆vdc1, ∆vdc2). With the detail of the current zero-crossing, it is possible to state that during these results the EVBC operates with unitary power factor. As aforementioned, during the positive half-cycle of the power grid voltage, the voltage of the capacitor C1 is regulated and during the negative half-cycle of the power gird voltage, the voltage of the capacitor C2 is regulated, which are controlled by the grid-side converter. Moreover, as it can be seen in this ﬁgure, the DC-link voltage has a voltage ripple of 3%. 18 of 21 150 Energies 2018, 11, 3453 of the grid-side V/div) Figure 22. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). For further details, Figure 23 presents an experimental result, during a time interval of 50 ms, EVBC grid-side current (iev), the grid voltage (vg), and the DC-link voltage ripple in bo pacitors (∆vdc1, ∆vdc2). With the detail of the current zero-crossing, it is possible to state that durin se results the EVBC operates with unitary power factor. 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 50 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 2 V/div, ∆vdc2: 2 V/div). Regarding the battery-side converter, the same modulation technique implemented in the G2V operation mode was used, namely the application of two 180° phase-shifted carrier signal. This strategy was adopted to reduce the ripple amplitude of the batteries current. In this sense, Figure 24 shows the current ripple in the inductor L3 according to the gate-emitter voltages, vge_S11 and vge_S12, of the IGBTs S11, S12. It is important to note that during this operation mode, the IGBTs S9 and S10 are always off, reason why they are not shown in the figure. As it can be seen, the measured current ripple in the inductor L3 was 0.13 A for a frequency of 40 kHz, which is twice of the switching frequency. According to this result, when the IGBT S9 or the IGBT S10 is on, the inductor stores energy and during the state transition of one the IGBTs, the inductor releases this energy. Using the power quality analyzer, in Figure 25a,b, the harmonic spectrum of the power grid voltage and the EVBC current is shown, measured THD% of 4.2% and 3.5%, respectively. These figures were obtained employing a Fluke 435 power quality analyzer, and the high value of THD in the power grid voltage is caused by distorted voltage drop in the line impedance, which is produced by di t t d t d b l li l d t d t th l t i l i t ll ti Regarding the battery-side converter, the same modulation technique implemented in the G2V operation mode was used, namely the application of two 180◦phase-shifted carrier signal. This strategy was adopted to reduce the ripple amplitude of the batteries current. In this sense, Figure 24 shows the current ripple in the inductor L3 according to the gate-emitter voltages, vge_S11 and vge_S12, of the IGBTs S11, S12. It is important to note that during this operation mode, the IGBTs S9 and S10 are always off, reason why they are not shown in the ﬁgure. 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). Figure 22. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). gies 2018, 11, x FOR PEER REVIEW 18 of For further details, Figure 23 presents an experimental result, during a time interval of 50 ms, e EVBC grid-side current (iev), the grid voltage (vg), and the DC-link voltage ripple in bo pacitors (∆vdc1, ∆vdc2). With the detail of the current zero-crossing, it is possible to state that duri ese results the EVBC operates with unitary power factor. As aforementioned, during the positi lf-cycle of the power grid voltage, the voltage of the capacitor C1 is regulated and during t gative half-cycle of the power gird voltage, the voltage of the capacitor C2 is regulated, which a ntrolled by the grid-side converter. Moreover, as it can be seen in this figure, the DC-link volta s a voltage ripple of 3%. Figure 23. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 50 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 2 V/div ∆vdc2: 2 V/div) Figure 23. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 50 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 2 V/div, ∆vdc2: 2 V/div). Figure 23. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 50 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC link voltages ripple in both capacitors (∆vd 1: 2 V/div ∆vd 2: 2 V/div) Figure 23. 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). ergies 2018, 11, x FOR PEER REVIEW 18 of 2 Figure 22. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). For further details, Figure 23 presents an experimental result, during a time interval of 50 ms, e EVBC grid-side current (iev), the grid voltage (vg), and the DC-link voltage ripple in bo pacitors (∆vdc1, ∆vdc2). With the detail of the current zero-crossing, it is possible to state that durin ese results the EVBC operates with unitary power factor. As aforementioned, during the positi lf-cycle of the power grid voltage, the voltage of the capacitor C1 is regulated and during th gative half-cycle of the power gird voltage, the voltage of the capacitor C2 is regulated, which a ntrolled by the grid-side converter. Moreover, as it can be seen in this figure, the DC-link volta s a voltage ripple of 3%. Figure 22. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). rgies 2018, 11, x FOR PEER REVIEW 18 of Figure 23. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 50 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and Figure 23. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 50 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC link voltages ripple in both capacitors (∆vd 1: 2 V/div ∆vd 2: 2 V/div) Figure 22. 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation As aforementioned, during the positi f-cycle of the power grid voltage, the voltage of the capacitor C1 is regulated and during th gative half-cycle of the power gird voltage, the voltage of the capacitor C2 is regulated, which a ntrolled by the grid-side converter. Moreover, as it can be seen in this figure, the DC-link volta s a voltage ripple of 3%. Figure 22. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). rgies 2018, 11, x FOR PEER REVIEW 18 of Figure 23. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 50 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 2 V/div ∆vdc2: 2 V/div) Figure 23. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the grid voltage (vg: 50 V/div), a detail of zero crossing between the current and the voltage (iev, vg), and the DC-link voltages ripple in both capacitors (∆vdc1: 2 V/div, ∆vdc2: 2 V/div). Figure 22. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). Figure 22. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). ergies 2018, 11, x FOR PEER REVIEW 18 of Figure 22. Experimental results in V2G operation mode showing the EVBC current (iev: 5 A/div), the power grid voltage (vg: 50 V/div), the voltage levels produced by the grid-side converter (vcv_AC: 100 V/div) and the DC-link voltage of both capacitors (vdc1: 20 V/div, vdc2: 20 V/div). Figure 22. 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation As it can be seen, the measured current ripple in the inductor L3 was 0.13 A for a frequency of 40 kHz, which is twice of the switching frequency. According to this result, when the IGBT S9 or the IGBT S10 is on, the inductor stores energy and during the state transition of one the IGBTs, the inductor releases this energy. Using the power quality analyzer, in Figure 25a,b, the harmonic spectrum of the power grid voltage and the EVBC current is shown, measured THD% of 4.2% and 3.5%, respectively. These ﬁgures were obtained employing a Fluke 435 power quality analyzer, and the high value of THD in the power grid voltage is caused by distorted voltage drop in the line impedance, which is produced by distorted current consumed by several nonlinear loads connected to the electrical installation. 19 of 21 uced by n Energies 2018, 11, 3453 grid voltage is ca distorted current c Figure 24. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S11 and S12 (vge_s11: 5 V/div and vge_12: 5 V/div) during a time interval of 100 µs Figure 24. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S11 and S12 (vge_s11: 5 V/div and vge_12: 5 V/div) during a time interval of 100 µs. ergies 2018, 11, x FOR PEER REVIEW 19 of Figure 24. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S11 and S12 (vge_s11: 5 V/div and vge_12: 5 V/div) during a time interval of 100 µs. Figure 24. Experimental results showing the current in the inductor L3 (iL3: 0.1 A/div) and the gate-emitter voltage of the IGBTs S11 and S12 (vge_s11: 5 V/div and vge_12: 5 V/div) during a time interval of 100 µs. rgies 2018, 11, x FOR PEER REVIEW 19 o µ Figure 25. Experimental results in V2G operation mode of the total harmonic distortion and spectral analysis: (a) Power grid voltage (vg); and (b) EVBC current (iev). Figure 25. Experimental results in V2G operation mode of the total harmonic distortion and spectral analysis: (a) Power grid voltage (vg); and (b) EVBC current (iev). Figure 25. 5. Conclusions 5. Conclusions A novel on-board bidirectional EV battery charger (EVBC) was presented. It is constituted by a grid-side converter capable to operate with five voltage levels, and by a battery-side converter capable to operate with three voltage levels. The distinct voltage levels for both converters are obtained using a split DC-link. In order to ensure power quality features, the proposed EVBC operates with grid-side current controlled to improve power factor, and to preserve the battery lifetime the EVBC operates with battery-side controlled current or voltage. Throughout the paper is described the proposed hardware topology, the discrete-time predictive control algorithms used for the grid-side converter and for the battery-side converter, the developed full-scale laboratorial prototype of the EVBC, and the foremost experimental results considering operating modes for smart grids. The obtained results allow validating the key contributions of the paper, mainly, in terms of the bidirectional operation of the novel EVBC based on a multilevel topology. As the EVBC is controlled targeting the EV incorporation into smart grids, the grid-to-vehicle (G2V) and vehicle-to-grid (V2G) operation modes are discussed and evaluated A novel on-board bidirectional EV battery charger (EVBC) was presented. It is constituted by a grid-side converter capable to operate with ﬁve voltage levels, and by a battery-side converter capable to operate with three voltage levels. The distinct voltage levels for both converters are obtained using a split DC-link. In order to ensure power quality features, the proposed EVBC operates with grid-side current controlled to improve power factor, and to preserve the battery lifetime the EVBC operates with battery-side controlled current or voltage. Throughout the paper is described the proposed hardware topology, the discrete-time predictive control algorithms used for the grid-side converter and for the battery-side converter, the developed full-scale laboratorial prototype of the EVBC, and the foremost experimental results considering operating modes for smart grids. The obtained results allow validating the key contributions of the paper, mainly, in terms of the bidirectional operation of the novel EVBC based on a multilevel topology. As the EVBC is controlled targeting the EV incorporation into smart grids, the grid-to-vehicle (G2V) and vehicle-to-grid (V2G) operation modes are discussed and evaluated. 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation 4.2.2. Experimental Results: Vehicle-To-Grid (V2G) Operation Experimental results in V2G operation mode of the total harmonic distortion and spectral analysis: (a) Power grid voltage (vg); and (b) EVBC current (iev). Figure 25. Experimental results in V2G operation mode of the total harmonic distortion and spectral analysis: (a) Power grid voltage (vg); and (b) EVBC current (iev). Funding: This work has been supported by COMPETE: POCI-01-0145-FEDER-007043 and FCT—Fundação para a Ciência e Tecnologia within the Project Scope: UID/CEC/00319/2013. This work is financed by the ERDF—European Regional Development Fund through the Operational Programme for Competitiveness and Internationalisation—COMPETE 2020 Programme, and by National Funds through the Portuguese funding agency, FCT—Fundação para a Ciência e a Tecnologia, within project Funding: This work has been supported by COMPETE: POCI-01-0145-FEDER-007043 and FCT—Fundação para a Ciência e Tecnologia within the Project Scope: UID/CEC/00319/2013. This work is financed by the ERDF—European Regional Development Fund through the Operational Programme for Competitiveness and Internationalisation—COMPETE 2020 Programme, and by National Funds through the Portuguese funding agency, FCT—Fundação para a Ciência e a Tecnologia, within project SAICTPAC/0004/2015—POCI—01-0145-FEDER-016434. This work is part of the FCT project 0302836 NORTE-01-0145-FEDER-030283. References 1. Idaho National Laboratory. Charging and Driving Behavior of Nissan Leaf Drivers in the EV Project with Access to Workplace Charging; EV Project; Idaho National Laboratory: Idaho Falls, ID, USA, 2014; pp. 1–4. 1. Idaho National Laboratory. Charging and Driving Behavior of Nissan Leaf Drivers in the EV Project with Access to Workplace Charging; EV Project; Idaho National Laboratory: Idaho Falls, ID, USA, 2014; pp. 1–4. 2. Matthé, R.; Eberle, U. The Voltec System—Energy Storage and Electric Propulsion. In Lithium-Ion Batteries—Advances and Applications; Elsevier: Amsterdam, The Netherlands, 2014; pp. 151–176. 2. Matthé, R.; Eberle, U. The Voltec System—Energy Storage and Electric Propulsion. In Lithium-Ion Batteries—Advances and Applications; Elsevier: Amsterdam, The Netherlands, 2014; pp. 151–176. 3. Robledo, C.B.; Oldenbroek, V.; Abbruzzese, F.; van Wijk, A.J.M. Integrating a hydrogen fuel cell electric vehicle with vehicle-to-grid technology, photovoltaic power and a residential building. Appl. Energy 2018, 215, 615–629. [CrossRef] 3. Robledo, C.B.; Oldenbroek, V.; Abbruzzese, F.; van Wijk, A.J.M. Integrating a hydrogen fuel cell electric vehicle with vehicle-to-grid technology, photovoltaic power and a residential building. Appl. Energy 2018, 215, 615–629. [CrossRef] 4. Boulanger, A.G.; Chu, A.C.; Maxx, S.; Waltz, D.L. Vehicle Electriﬁcation: Status and Issues. Proc. IEEE 2011, 99, 1116–1138. [CrossRef] 4. Boulanger, A.G.; Chu, A.C.; Maxx, S.; Waltz, D.L. Vehicle Electriﬁcation: Status and Issues. Proc. IEEE 2011, 99, 1116–1138. [CrossRef] 5. Rajashekara, K. Present Status and Future Trends in Electric Vehicle Propulsion Technologies. IEEE J. Emerg. Sel. Top. Power Electron. 2013, 1, 3–10. [CrossRef] 5. Rajashekara, K. Present Status and Future Trends in Electric Vehicle Propulsion Technologies. IEEE J. Emerg. Sel. Top. Power Electron. 2013, 1, 3–10. [CrossRef] p 6. Milberg, J.; Schlenker, A. Plug into the Future. IEEE Power Energy Mag. 2011, 9, 56–65. [CrossRef] 7. Monteiro, V.; Ferreira, J.C.; Melendez, A.A.N.; Couto, C.; Afonso, J.L. Experimental Validation of a Novel Architecture Based on a Dual-Stage Converter for Off-Board Fast Battery Chargers of Electric Vehicles. IEEE Trans. Veh. Technol. 2018, 67, 1000–1011. [CrossRef] 8. Basu, M.; Gaughan, K.; Coyle, E. Harmonic distortion caused by EV battery chargers in the distribution systems network and its remedy. In Proceedings of the International Conference UPEC—Universities Power Engineering Conference, Bristol, UK, 6–8 September 2004; pp. 869–873. g g p pp 9. Monteiro, V.; Pinto, J.G.; Afonso, J.L. Operation Modes for the Electric Vehicle in Smart Grids and Smart Homes: Present and Proposed Modes. IEEE Trans. Veh. Technol. 2016, 65, 1007–1020. [CrossRef] 10. References Kisacikoglu, M.C.; Ozpineci, B.; Tolbert, L.M. Examination of a PHEV Bidirectional Charger System for V2G Reactive Power Compensation. In Proceedings of the IEEE APEC Applied Power Electronics Conference and Exposition, Palm Springs, CA, USA, 21–25 February 2010; pp. 458–465. g p g y Reactive Power Compensation. In Proceedings of the IEEE APEC Applied Power Electronics Conference and Exposition, Palm Springs, CA, USA, 21–25 February 2010; pp. 458–465. 11. Monteiro, V.; Ferreira, J.C.; Meléndez, A.A.N.; Afonso, J.L. Electric Vehicles On-Board Battery Charger for the Future Smart Grids. In Technological Innovation for the Internet of Things, 1st ed.; Camarinha-Matos, L.M., Tomic, S., Graça, P., Eds.; Springer: Berlin, Germany, 2013; Chapter 38; pp. 351–358. 12. Multin, M.; Allerding, F.; Schmeck, H. Integration of Electric Vehicles in Smart Homes—An ICT-based Solution for V2G Scenarios. In Proceedings of the IEEE ISGT PES Innovative Smart Grid Technologies, Washington, DC, USA, 16–20 January 2012; pp. 1–8. 13. Wong, N.; Kazerani, M. A Review of Bidirectional On-Board Charger Topologies for Plug-In Vehicles. In Proceedings of the IEEE CCECE Canadian Conference on Electrical and Computer Engineering, Montreal, QC, Canada, 29 April–2 May 2012; pp. 1–6. 14. Mei, J.; Xiao, B.; Shen, K.; Tolbert, L.M.; Zheng, J.Y. Modular Multilevel Inverter with new Modulation Method and its Application to Photovoltaic Grid-Connected Generator. IEEE Trans. Power Electron. 2013, 28, 5063–5073. [CrossRef] 15. Tolbert, L.M.; Peng, F.Z.; Habetler, T.G. Multilevel Converters for Large Electric Drives. IEEE Trans. Ind. Appl. 1999, 35, 36–44. [CrossRef] 16. Wang, H.; Kou, L.; Liu, Y.-F.; Sen, P.C. A New Six-Switch Five-Level Active Neutral Point Clamped Inverter for PV Applications. IEEE Trans. Power Electron. 2017, 32, 6700–6715. [CrossRef] 17. Monteiro, V.; Meléndez, A.A.N.; Afonso, J.L. Novel Single-Phase Five-Level VIENNA-Type Rectiﬁer with Model Predictive Current Control. In Proceedings of the IEEE IECON Industrial Electronics Conference, Beijing, China, 29 October–1 November 2017; pp. 6413–6418. j g, , ; pp 18. Grbovic, P.; Lidozzi, A.; Solero, L.; Crescimbini, F. Five-Level Unidirectional T-Rectiﬁer for High Speed Gen-Set Applications. IEEE Trans. Ind. Appl. 2016, 52, 1642–1651. [CrossRef] 19. Monteiro, V.; Ferreira, J.C.; Meléndez, A.A.N.; Afonso, J.L. Model Predictive Control Applied to an Improved Five-Level Bidirectional Converter. IEEE Trans. Ind. Electron. 2016, 63, 5879–5890. [CrossRef] 20. Monteiro, V.; Meléndez, A.A.N.; Ferreira, J.C.; Couto, C.; Afonso, J.L. Experimental Validation of a Proposed Single-Phase Five-Level Active Rectiﬁer Operating with Model Predictive Current Control. 5. Conclusions 5. Conclusions Author Contributions: All authors contributed equally to the conceptualization and writing of the pape Author Contributions: All authors contributed equally to the conceptualization and writing of the pape Funding: This work has been supported by COMPETE: POCI-01-0145-FEDER-007043 and FCT—Fundação para a Ciência e Tecnologia within the Project Scope: UID/CEC/00319/2013. This work is financed by the ERDF—European Regional Development Fund through the Operational Programme for Competitiveness and Internationalisation—COMPETE 2020 Programme, and by National Funds through the Portuguese funding agency, FCT—Fundação para a Ciência e a Tecnologia, within project Funding: This work has been supported by COMPETE: POCI-01-0145-FEDER-007043 and FCT—Fundação para a Ciência e Tecnologia within the Project Scope: UID/CEC/00319/2013. This work is financed by the ERDF—European Regional Development Fund through the Operational Programme for Competitiveness and Internationalisation—COMPETE 2020 Programme, and by National Funds through the Portuguese funding agency, FCT—Fundação para a Ciência e a Tecnologia, within project SAICTPAC/0004/2015—POCI—01-0145-FEDER-016434. This work is part of the FCT project 0302836 NORTE-01-0145-FEDER-030283. 20 of 21 20 of 21 Energies 2018, 11, 3453 Conﬂicts of Interest: The authors declare no conﬂict of interest. © 2018 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/). References In Proceedings of the IEEE IECON Industrial Electronics Conference, Yokohama, Japan, 9–12 November 2015; pp. 3939–3944. 21 of 21 Energies 2018, 11, 3453 21. Mokhberdoran, A.; Ajami, A. Symmetric and Asymmetric Design and Implementation of New Cascaded Multilevel Inverter Topology. IEEE Trans. Power Electron. 2014, 29, 6712–6724. [CrossRef] 22. Elsheikh, M.G.; Ahmed, M.E.; Abdelkarem, E.; Orabi, M. Single-Phase Five-Level Inverter with Less Number of Power Elements. In Proceedings of the IEEE INTELEC International Telecommunications Energy Conference, Orlando, FL, USA, 5–9 February 2012; pp. 1–8. y pp 23. Loukriz, A.H.; Dudley, S.; Quinlan, T.; Walker, S.D. Experimental Realization of a Single-Phase Five Level Inverter for PV Applications. In Proceedings of the IEEE COMPEL Control and Modeling for Power Electronics, Trondheim, Norway, 27–30 June 2016; pp. 1–6. 24. Wang, K.; Xu, L.; Zheng, Z.; Li, Y. Capacitor Voltage Balancing of a Five-Level ANPC Converter Using Phase-Shifted PWM. IEEE Trans. Power Electron. 2015, 30, 1147–1156. [CrossRef] 25. Monteiro, V.; Pinto, J.G.; Sousa, T.J.C.; Meléndez, A.A.N.; Afonso, J.L. A Novel Single-Phase Five-Level Active Rectiﬁer for On-Board EV Battery Chargers. In Proceedings of the IEEE ISIE International Symposium on Industrial Electronics, Edinburgh, UK, 19–21 June 2017; Volume 4, pp. 582–587. 26. Rosas-Caro, J.C.; Ramírez, J.M.; García-Vite, P.M. Novel DC-DC Multilevel Boost Converter. In Proceedings of the IEEE Power Electronics Specialists Conference, Rhodes, Greece, 15–19 June 2008; pp. 2146–2151. 27. Zhang, F.; Peng, F.Z.; Qian, Z. Study of the Multilevel Converters in DC-DC Applications. In Proceedings of the IEEE Annual Power Electronics Specialists Conference, Aachen, Germany, 20–25 June 2004. 28. Lee, P.; Lee, Y.; Cheng, D.K.W. Steady-state analysis of an interleaved boost converter with coupled inductors. IEEE Trans. Ind. Electron. 2000, 47, 787–795. [CrossRef] 29. Jiang, W.; Fahimi, B. Phase-Shift Controlled Multilevel Bidirectional DC/DC Converter: A Novel Solution to Battery Charge Equalization in Fuel Cell Vehicle. In Proceedings of the IEEE Vehicle Power and Propulsion Conference, Arlington, TX, USA, 9–12 September 2007; pp. 587–590. © 2018 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/). © 2018 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/).
https://openalex.org/W3110806514	https://corpus.ulaval.ca/bitstreams/60d38951-4668-4164-9670-6e1b9966884b/download	English	null	Effects of Daily Raspberry Consumption on Immune-Metabolic Health in Subjects at Risk of Metabolic Syndrome: A Randomized Controlled Trial	Nutrients	2,020	cc-by	14,065	Eﬀects of Daily Raspberry Consumption on Immune-Metabolic Health in Subjects at Risk of Metabolic Syndrome: A Randomized Controlled Trial Maximilien Franck 1,2, Juan de Toro-Martín 1, Véronique Garneau 1, Valérie Guay 1, Michèle Kearney 1, Geneviève Pilon 1,3, Denis Roy 1, Patrick Couture 1,4, Charles Couillard 1,2, André Marette 1,3 and Marie-Claude Vohl 1,2,* 1 Centre Nutrition, Santé et Société (NUTRISS) and Institut sur la Nutrition et les Aliments Fonctionnels (INAF), Université Laval, Québec, QC G1V 0A6, Canada; maximilien.franck.1@ulaval.ca (M.F.); juan.de-toro-martin.1@ulaval.ca (J.d.T.-M.); veronique.garneau@fsaa.ulaval.ca (V.G.); valerie.guay@fsaa.ulaval.ca (V.G.); michele.kearney@fsaa.ulaval.ca (M.K.); genevieve.pilon@criucpq.ulaval.ca (G.P.); denis.roy@fsaa.ulaval.ca (D.R.); patrick.couture@fmed.ulaval.ca (P.C.); charles.couillard@fsaa.ulaval.ca (C.C.); andre.marette@criucpq.ulaval.ca (A.M.) 2 School of Nutrition, Université Laval, Québec, QC G1V 0A6, Canada 3 Quebec Heart and Lung Institute (IUCPQ) Research Center, 2725 Chemin Sainte-Foy, Québec, QC G1V 4G5, Canada 4 Endocrinology and Nephrology Unit, CHU de Québec-Université Laval, Québec, QC G1V 4G2, Canad * Correspondence: marie-claude.vohl@fsaa.ulaval.ca; Tel.: +1-418-656-2131 (ext. 404676) Received: 17 November 2020; Accepted: 15 December 2020; Published: 17 December 2020 Abstract: Consumption of red raspberries has been reported to exert acute beneﬁcial eﬀects on postprandial glycemia, insulinemia, triglyceridemia, and cytokine levels in metabolically disturbed subjects. In a two-arm parallel-group, randomized, controlled trial, 59 subjects with overweight or abdominal obesity and with slight hyperinsulinemia or hypertriglyceridemia were randomized to consume 280 g/day of frozen raspberries or to maintain their usual diet for 8 weeks. Primary analyses measured metabolic diﬀerences between the groups. Secondary analyses performed with omics tools in the intervention group assessed blood gene expression and plasma metabolomic changes following the raspberry supplementation. The intervention did not signiﬁcantly aﬀect plasma insulin, glucose, inﬂammatory marker concentrations, nor blood pressure. Following the supplementation, 43 genes were diﬀerentially expressed, and several functional pathways were enriched, a major portion of which were involved in the regulation of cytotoxicity, immune cell traﬃcking, protein signal transduction, and interleukin production. In addition, 10 serum metabolites were found signiﬁcantly altered, among which β-alanine, trimethylamine N-oxide, and bioactive lipids. Although the supplementation had no meaningful metabolic eﬀects, these results highlight the impact of a diet rich in raspberry on the immune function and phospholipid metabolism, thus providing novel insights into potential immune-metabolic pathways inﬂuenced by regular raspberry consumption. Keywords: berry fruits; metabolic syndrome; multi-omics; immunity; gene expression; sphingolipids; phenolic compounds nutrients nutrients 1. Introduction Both clinical and epidemiological studies highlight the contribution of a plant-food-predominant diet in the maintenance, or even the improvement, of metabolic homeostasis owing to its ﬁber and phytochemical contents [1]. Long-term consumption of a diet poor in these components Nutrients 2020, 12, 3858; doi:10.3390/nu12123858 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 20 Nutrients 2020, 12, 3858 constitutes the main predisposing factor to metabolic homeostasis dysregulation [2,3]. Metabolic dysregulations can be seen as a broad range of intermediate phenotypes, all converging with time toward metabolic syndrome, a cluster of interrelated immune-metabolic abnormalities increasing the risk of developing type 2 diabetes or atherosclerotic diseases [4]. Although public health authorities promote plant-food-predominant diets [5], eﬀorts to increase vegetable and fruit consumption have been hardly eﬃcient. Accordingly, it has been demonstrated that dietary habits are diﬃcult to change over the long term [6], highlighting the need to identify simple nutritional interventions that increase the ﬁber and phytochemical contents of the diet. In this regard, growing evidence has supported the role of berry fruits in the prevention and management of metabolic disorders [7]. Among the most commonly consumed berries, red raspberry (Rb; Rubus idaeus L.) is low in sugar and particularly rich in both ﬁber and phenolic compounds, mostly anthocyanins, and ellagitannins [8]. p p y y g Collectively, in vitro and ex vivo research conducted with Rb extracts or puriﬁed components have revealed various antioxidative, anti-inﬂammatory, and metabolic properties through which Rb components may help treat or improve immune-metabolic abnormalities [9,10]. Several animal studies have conﬁrmed these beneﬁcial eﬀects with both Rb components and the entire fruit [11–13]. Moreover, some of these studies brought to light the immunomodulatory eﬀects of Rb phenolic compounds [14,15]. Altogether, these results are consistent with those observed with black raspberry (Rubus occidentalis), although of reduced amplitude for an equal amount, presumably attributable to its lower phenolic content [16,17]. Unlike the black raspberry, for which the health-promoting eﬀects have been conﬁrmed in numerous human studies [18,19], very few human randomized controlled intervention studies have been undertaken to assess the health impact of whole Rb or its components, and most of them looked only at the short-term metabolic impact [20–25]. In that respect, the aims of this randomized controlled clinical trial were to investigate the health eﬀects of Rb consumption on immune-metabolic features in subjects at risk of developing metabolic syndrome and to delineate the mechanisms underlying these eﬀects through transcriptomics and metabolomics. 1. Introduction In the context of personalized nutrition, this holistic approach is part of the eﬀorts undertaken to further understand the impact of foods or nutrients on metabolic syndrome. 2.1. Study Participants and Eligibility Criteria A two-arm parallel-group, randomized, controlled trial of the eﬀects of Rb consumption on immune-metabolic parameters was conducted at the Institute of Nutrition and Functional Foods (INAF) at Université Laval between January 2018 and August 2019. Study participants from the greater Quebec City metropolitan area were recruited through emails, newspapers, and media advertisements. A total of 468 individuals contacted our research team for information related to the study, among which 119 were scheduled for a screening visit at the clinical investigation unit, and 59 were found to be eligible to participate in the study (Figure 1). The age and medical history of participants were assessed at week 0, before the beginning of the intervention. To be included in the study, subjects had to be men or pre-menopausal women, Caucasian, aged between 18 to 60 years, have a body-mass index (BMI) between 25 and 40 kg/m2 or a waist circumference ≥94 cm for men and ≥80 cm for women, as well as meet at least one of the following criteria: Plasma triglycerides (TG) > 1.35 mmol/L, or insulin concentration > 42 pmol/L, using our new analytic method and corresponding to a threshold value of 60 pmol/L with the former method that was predictive of a higher risk of cardiovascular disease in the Quebec population [26]. Subjects were excluded if they had a diagnosis for diabetes, hypercholesterolemia, or hypertension, had a taste aversion for or were allergic or intolerant to Rb, were taking medication known to aﬀect study parameters, had taken antibiotics, supplements, or natural health products on a regular basis over the past 3 months, had undergone surgery in the last 3 months, or had planned surgery during the duration of the study. Nicotine usage, non-conventional dietary patterns (e.g., vegan, gluten-free 3 of 20 Nutrients 2020, 12, 3858 020 12 FOR PEER R or ketogenic diets), bodyweight loss or gain greater than 5% in the last 3 months, or daily alcohol consumption greater than 2 drinks were also considered exclusion criteria. The study was approved by the Université Laval Ethics Committee in January 2018 (CER-Université 2017–218). Upon recruitment, written informed consent was obtained from all subjects prior to the beginning of the study procedures. This trial was registered at clinicaltrials.gov as NCT03620617. 2.1. Study Participants and Eligibility Criteria Changes in anthropometric and metabolic variables in response to the Rb supplementation were deﬁned as the primary outcomes, whereas pre- and post-supplementation changes in metabolomics and transcriptomics were considered as the secondary outcomes. Université Laval Ethics Committee in January 2018 (CER-Université 2017–218 ent, written informed consent was obtained from all subjects prior to the beginnin ocedures. This trial was registered at clinicaltrials.gov as NCT03620617. Ch metric and metabolic variables in response to the Rb supplementation were defin outcomes, whereas pre- and post-supplementation changes in metabolom tomics were considered as the secondary outcomes. Figure 1. Patient flow diagram for recruitment, randomization, and data collection. Figure 1. Patient ﬂow diagram for recruitment, randomization, and data collection. 1. Patient flow diagram for recruitment, randomization, and data col Figure 1. Patient ﬂow diagram for recruitment, randomization, and data collection. ti l I t ti 2.2. Nutritional Intervention tional Intervention randomization occurred after eligibility was confirmed. Participants were rando ms using an in-house electronic management platform. A sex-stratified block rando a 2:2 treatment allocation for each sex to either the intervention group consumin e control group was used (Figure 2). The randomization process was under the respo d study coordinator. Three clinical coordinators and one graduate student were res cruitment and follow-up of participants in this study. None of the participants dro ey found out which group they had been assigned to. After a 2-week run-in nts assigned to the intervention group were invited to consume 280 g of frozen 2 cups) for 8 weeks. Control group participants were invited to maintain their he The randomization occurred after eligibility was conﬁrmed. Participants were randomized to study arms using an in-house electronic management platform. A sex-stratiﬁed block randomization ensuring a 2:2 treatment allocation for each sex to either the intervention group consuming frozen Rb, or the control group was used (Figure 2). The randomization process was under the responsibility of the lead study coordinator. Three clinical coordinators and one graduate student were responsible for the recruitment and follow-up of participants in this study. None of the participants dropped out when they found out which group they had been assigned to. After a 2-week run-in period, participants assigned to the intervention group were invited to consume 280 g of frozen Rb daily (roughly 2 cups) for 8 weeks. Control group participants were invited to maintain their health and food habits stable for an 8-week period. Participants of both groups were scheduled for clinical visits after the run-in period (week 0), during the intervention (week 4), and at the end of the protocol (week 8). During the protocol, including the run-in period, participants were asked to avoid the use of supplements, natural 4 of 20 Nutrients 2020, 12, 3858 health products, wine, or products with a polyphenolic proﬁle similar to that of Rb and to limit the consumption of berries other than those provided, and any other products containing berries, to 2 portions per week. They were also instructed to limit coﬀee and tea consumption to 1 serving/day, and alcohol to 2 drinks/week. Participants had to complete a journal reporting any deviation from the dietary instructions listed above, as well as the Rb quantity consumed for the intervention group. ti l I t ti 2.2. Nutritional Intervention The count of empty Rb packages returned by the participants was used to assess their compliance with the study protocol. Participants received regular emails to follow-up on any issues that may arise and to enhance adherence to the protocol. 020, 12, x FOR PEER REVIEW Figure 2. Graphical representation of the study protocol. Figure 2. Graphical representation of the study protocol. ure 2 Graphical representation of the study pro Figure 2. Graphical representation of the study protocol. g 2.3. Anthropometric Measurements ropometric Measurements each clinical visit, waist and hip circumferences were measured to the nearest m g to procedures recommended at the Airlie conference [27]. Height was also measu millimeter. Bodyweight was measured using a BWB-800 electronic scale (Tanita, A IL, USA) to an accuracy of 0.1 kg. For weight measurements, participants were aske oor clothes and to remove their shoes. BMI was calculated as weight in kilogram t in meters squared (kg/m2) Systolic (SBP) and diastolic (DBP) blood pressure meas At each clinical visit, waist and hip circumferences were measured to the nearest millimeter according to procedures recommended at the Airlie conference [27]. Height was also measured to the nearest millimeter. Bodyweight was measured using a BWB-800 electronic scale (Tanita, Arlington Heights, IL, USA) to an accuracy of 0.1 kg. For weight measurements, participants were asked to wear light indoor clothes and to remove their shoes. BMI was calculated as weight in kilograms divided by height in meters squared (kg/m2). Systolic (SBP) and diastolic (DBP) blood pressure measurements were assessed while sitting on a chair after a 10 min rest. The mean of 3 measurements performed at 3 min intervals was used for the analyses. t in meters squared (kg/m ). Systolic (SBP essed while sitting on a chair after a 10 m 2.4. Nutritional and Physical Activity Assessments g p ervals was used for the analyses. itional and Physical Activity Assessments ritional habits were assessed at each clinical visit to the clinical investigation unit of a web-based and self-administered Food Frequency Questionnaire (FFQ), vali peaking Canadian adults [28]. The FFQ was based on 136 items grouped into the 8 f s: Dairy products, fruits, vegetables, meat and alternatives, cereals and grain Nutritional habits were assessed at each clinical visit to the clinical investigation unit through the use of a web-based and self-administered Food Frequency Questionnaire (FFQ), validated for French-speaking Canadian adults [28]. The FFQ was based on 136 items grouped into the 8 following categories: Dairy products, fruits, vegetables, meat and alternatives, cereals and grain products, beverages, other foods, and supplements. Participants reported how often they consumed each item per day, per week, per month, or none at all in the last month. Finally, pictures with examples of portion sizes were used to reﬂect a better estimation of the portion consumed by the participant. Physical activity habits were evaluated using the Leisure Time Activity questionnaire, which was completed by participants at the beginning and at the end of the intervention [29]. 2.6. Transcriptomic Analyses Gene expression proﬁling before and after the supplementation was conducted in whole blood, commonly used to assess the eﬀects of dietary interventions in human trials [36]. The eﬀects of Rb supplementation on the gene expression proﬁle was examined in blood samples collected into PAXgene preparation tubes (Qiagen, Valencia, CA, USA) at week 0 and 8, and stored at −80 ◦C until the analyses. Total RNA was extracted, and samples were sent to the Génome Québec Innovation Centre (McGill University, Montreal, Canada) for sequencing. The purity and integrity of RNA samples were assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA samples were converted to cDNA with the Illumina NEB stranded mRNA library preparation kit (Illumina, San Diego, CA, USA) based on the manufacturer’s protocol. The prepared libraries were then sequenced on an Illumina NovaSeq6000 S4 sequencer using paired-end, 100 bp reads. Quality trimming of the raw reads was carried out to remove bases with a Phred33 score < 30 and reads with less than 50 bases using Trim Galore (v0.6.5), a wrapper tool around Cutadapt (v1.15) and FastQC (v0.11.9). Kallisto (0.46.2) with 100 bootstraps and default parameters was run for alignment and normalization. Reads were aligned to the GRCh38 human reference transcriptome [37]. Transcriptomic data reported as counts per million (cpm), were ﬁltered based on a worthwhile number of counts in a minimum number of samples in the edgeR v3.28.1 package [38]. The diﬀerential expression analysis was conducted using a generalization of a paired t-test implemented in the quasi-likelihood functionality of edgeR. Diﬀerentially expressed transcripts between pre- vs. post-intervention at a nominal p-value < 0.05 were considered for further analysis. The functional signiﬁcance of genes showing at least a 25% diﬀerence (1.25-fold change) between pre- and post-supplementation states was explored by pathway enrichment analysis using the clusterProﬁler v3.16.0 R package [39]. The clusterProﬁler package implements statistical methods to analyze and visualize functional proﬁles of genes and gene clusters and produces adjusted p-values using the Benjamini–Hochberg procedure (BH-p) to indicate pathways that were signiﬁcantly enriched. The following pathway databases were used for functional enrichment analysis: Gene Ontology Biological Processes (GO-BP) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Transcriptomic data analysis was implemented in R v3.6.3. es, other foods, and su k th 2.5. Biochemical Analyses per week, per month, or none at all in the last month. Finally, pictures with exa sizes were used to reflect a better estimation of the portion consumed by the pa activity habits were evaluated using the Leisure Time Activity questionnaire, w ed by participants at the beginning and at the end of the intervention [29]. hemical Analyses Fasting plasma samples were collected on weeks 0, 4, and 8, and used to measure total-cholesterol (Total-C), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), TG, glucose, and insulin levels. Total-C and TG concentrations were measured using enzymatic assays [30]. Apolipoprotein B (ApoB) was measured by immunonephelometry assay using a Siemens Dimension Vista™1500 Analyzer. The HDL-C fraction was obtained after precipitation of VLDL and LDL particles in the infranatant with heparin manganese chloride. LDL-C was calculated with the Friedewald formula [31]. High-sensitivity C-reactive protein (CRP) levels were measured by immunoassay [32]. Fasting glycated hemoglobin 5 of 20 Nutrients 2020, 12, 3858 (HbA1c) was measured in plasma by immunoturbidimetry (Biorad HPLC D-100). Oral glucose tolerance tests (OGTTs) using a 75 g glucose solution were conducted twice during the intervention period (weeks 0 and 8). Blood samples were drawn at −15, 0, 15, 30, 60, 90, and 120 min during the OGTT to assess plasma insulin and glucose levels. Glucose concentrations were enzymatically measured [33], while insulin concentrations were measured by chemiluminescence (Siemens Advia Centaur XPT). The homeostatic model assessment of insulin resistance (HOMA-IR) index and the Matsuda index were calculated from OGTT values [34,35]. 2.7. Metabolomic Analyses The quantitative analysis of 630 metabolites from 26 biochemical classes was performed in paired plasma samples from 24 participants before and after the Rb supplementation with the MxP® Quant 500 kit (Biocrates Life Sciences AG, Innsbruck, Austria). The targeted metabolomic proﬁling was performed by the Analytical Facility for Bioactive Molecules at the Hospital for Sick Children (Toronto, Canada). Metabolite data from 24 matched participants were processed using the MetaboAnalystR package (v3.0) [39]. First, 117 features with a constant or single value across samples were found and deleted. Non-informative signals were further ﬁltered out based on the interquartile range estimate, and samples were normalized by quantile normalization. Metabolite data were log-transformed and scaled by Pareto scaling. After quantile normalization, one more feature with a constant value was found and deleted. From the original 630 metabolites analyzed, a total of 382 were ﬁnally retained for statistical analysis. Paired t-tests were used to analyze within-subject changes in plasma metabolite levels between pre- and post-supplementation states. A volcano plot associated with paired t-tests was further used to identify the most diﬀerentially abundant metabolites between pre- and post-supplementation groups. 6 of 20 Nutrients 2020, 12, 3858 A p-value < 0.05, along with a count of signiﬁcant pairs higher than 50% showing at least 25% diﬀerence (1.25-fold change), were the criteria used to consider metabolite plasma levels diﬀering signiﬁcantly between pre- and post-supplementation states. The dimensional reduction was conducted by using the partial least squares discriminant analysis (PLS-DA), a supervised algorithm able to reduce the number of metabolites in high-dimensional metabolomics data to produce robust and easy-to-interpret models. This method was able to diﬀerentiate the class membership through multivariate regression of a given set of metabolites. In the present study, we used a variation of PLS-DA, the multilevel PLS-DA (mPLS-DA), given its ability to exploit the paired structure of the multivariate data obtained before and after the Rb supplementation in the same group of participants [39,40]. Concretely, a sparse mPLS-DA (smPLS-DA) was used to identify the most important metabolites helping to discriminate matched study groups [41]. The smPLS-DA algorithm was implemented using the mixOmics R package (v6.12.1) [42]. Variable importance in projection (VIP) coeﬃcients were computed as a weighted sum of squares of the smPLS-DA loadings to depict the relative importance of each metabolite in the classiﬁcation model. Predictors with large VIP were the most relevant for discriminating class membership [43]. 2.8. Statistical Analyses The normality of distribution of all variables was assessed using skewness and kurtosis, and none of the variables needed transformation before analysis. Descriptive characteristics between the Rb and the control group were presented as means (±SD) or frequencies. Chi-square tests for categorical variables and analyses of variance (general linear model, type III sum of squares) for continuous variables were used to seek for inter-group diﬀerences in baseline characteristics and in changes between follow-up and baseline. The mixed procedure in SAS with special provisions for repeated measures that incorporated data from all visits was used to examine the eﬀects of time, treatment, and their interaction on clinical variables. Data were adjusted for age, sex, BMI, and baseline values. For all analyses, a p-value < 0.05 was considered for statistical signiﬁcance. Statistical analyses were performed using SAS version 3.8 (SAS Institute, Cary, NC, USA). 3.1. Trial Flow, Baseline Characteristics, and Compliance Of the 119 subjects screened for eligibility, 59 were enrolled in the study, 29 were randomized to the Rb group, and 30 to the control group (Figure 2). Eleven participants (6 and 5 from the Rb and control group, respectively) withdrew during follow-up by the inability to comply with study procedures or schedule conﬂict. Therefore, 48 subjects were included in the per-protocol analysis. According to BMI values, 22 subjects had obesity (BMI > 30 kg/m2), 19 were overweight (BMI between 25 and 30 kg/m2), and 7 were in the normal weight range (BMI < 25 kg/m2). The mean BMI of participants was 29.9 kg/m2 (ranging from 22.3–43.7), and the mean age was 32.2 years (ranging from 22 to 57). Upon randomization, baseline characteristics were well-distributed at baseline and, therefore, no signiﬁcant diﬀerences between the Rb group and the control group were observed for age, sex, anthropometric, physiologic, or metabolic variables (Table 1). However, although not statistically signiﬁcant, the HOMA-IR diﬀerence between groups may be considered as clinically meaningful. No adverse events related to Rb supplementation were reported during the study. Adherence to the study protocol was good, with an overall compliance of 92.8%. 7 of 20 Nutrients 2020, 12, 3858 Table 1. Clinical and laboratory baseline characteristics of study participants. Table 1. Clinical and laboratory baseline characteristics of study participants. Variable N Control N Rb p-value Sex (men/women) 24 9/15 24 7/17 0.54 Age (years) 24 31.92 ± 8.05 24 32.46 ± 10.12 0.83 BMI (kg/m2) 24 29.38 ± 3.94 24 30.42 ± 5.00 0.43 Waist circumference (cm) 24 98.10 ± 11.81 24 98.53 ± 13.32 0.90 Fasting glucose (mmol/L) 24 4.84 ± 0.47 23 4.98 ± 0.59 0.38 Fasting insulin (pmol/L) 22 73.91 ± 33.57 19 83.11 ± 43.54 0.21 HbA1C (%) 24 5.05 ± 0.29 24 5.03 ± 0.31 0.85 Plasma TG (mmol/L) 24 1.56 ± 0.78 24 1.46 ± 0.80 0.66 ApoB-100 (g/L) 24 0.88 ± 0.17 24 0.92 ± 0.24 0.48 HDL-C (mmol/L) 24 1.33 ± 0.23 24 1.32 ± 0.40 0.94 LDL-C (mmol/L) 24 2.55 ± 0.74 24 2.65 ± 0.86 0.69 CRP (mg/L) 24 2.72 ± 2.82 23 2.68 ± 2.34 0.95 HOMA-IR 13 1.78 ± 0.76 11 2.84 ± 1.73 0.06 MATSUDA 13 5.79 ± 2.43 11 4.54 ± 2.24 0.21 Data are means ± standard deviations. Sex is presented as counts. 3.2. Food Intake and Physical Activity There was no signiﬁcant diﬀerence in energy intake between groups at any time point during the protocol. Accordingly, no signiﬁcant treatment by time interactions was found in macronutrients or other nutritional compounds, with the exception of those deriving directly from Rb composition, namely glucose, fructose, soluble and insoluble ﬁber (Table 2). According to the FFQ data, adding 2 cups/day of Rb accounted, respectively, for 17.6%, 17.1%, 13.3%, and 25.5% of total dietary glucose, fructose, soluble, and insoluble ﬁbers intakes. Moreover, when analyzing dietary intakes based on food groups (bread and cereals, fruits, vegetables, dairy products, animal proteins), there were no signiﬁcant diﬀerences between groups except for fruit consumption (p < 0.0001, a mean of 2, 3 portions for the control group vs. 6 portions for the Rb group during the intervention). The intervention resulted in a nutritionally signiﬁcant shift (around 3 servings) in fruit consumption in the Rb group. Physical activity remained stable throughout the protocol (Table 2). 3.1. Trial Flow, Baseline Characteristics, and Compliance p-values were obtained by a general linear model, type III sum of squares, and chi-square tests (sex). BMI, body mass index. HbA1C, glycated hemoglobin. TG, triglycerides. ApoB-100, Apolipoprotein B. HDL-C, high-density lipoprotein cholesterol. LDL-C, low-density lipoprotein cholesterol. CRP, C-reactive protein. HOMA-IR, homeostatic model assessment of insulin resistance. Data are means ± standard deviations. Sex is presented as counts. p-values were obtained by a general linear model, type III sum of squares, and chi-square tests (sex). BMI, body mass index. HbA1C, glycated hemoglobin. TG, triglycerides. ApoB-100, Apolipoprotein B. HDL-C, high-density lipoprotein cholesterol. LDL-C, low-density lipoprotein cholesterol. CRP, C-reactive protein. HOMA-IR, homeostatic model assessment of insulin resistance. 3.3. Primary Outcomes We found no signiﬁcant diﬀerences between follow-up and baseline values between control and intervention groups for any of the parameters analyzed (Table 3). The statistical adjustment for age, sex, and BMI had no eﬀect on the lack of diﬀerences between the Rb and control groups (data not shown). Repeated measures analysis revealed a signiﬁcant treatment by time interaction eﬀect on SBP (p = 0.03) and plasma ApoB levels (p = 0.03) (Table 4). Indeed, in the control group, we found a signiﬁcant increase in plasma ApoB concentrations from the beginning of the intervention (0.86–0.16 g/L) to the mid-point visit (0.95–0.17 g/L), before regaining its initial level at the end of the study (0.87–0.19 g/L). Again, a signiﬁcant eﬀect of time was observed for both HDL-C (p = 0.03) and LDL-C (p = 0.01) in the control group. There was no other singular or interaction eﬀect of time and treatment for any other metabolic or anthropometric variable. In accordance to the lack of change in fasting and postprandial plasma insulin and glucose concentrations in response to the intervention, both HOMA-IR and MATSUDA indexes derived from OGGT were not found to diﬀer signiﬁcantly. 8 of 20 Nutrients 2020, 12, 3858 Table 2. Daily dietary intakes by nutrients and by food group over time within and between groups. 3.3. Primary Outcomes Time Week 0 Week 4 Week 8 p-Values Treatment Control Rb Control Rb Control Rb Time tx txTime Energy (kcal) 2132.8 ± 823.2 2059.7 ± 662.1 1992.7 ± 692.4 1983.3 ± 682.1 2060.7 ± 638.7 2075.8 ± 623.2 0.32 0.70 0.83 Lipids (g) 93.5 ± 44.9 82.7 ± 28.6 87.7 ± 40.9 76.4 ± 31.8 91.4 ± 37.7 80.9 ± 28.7 0.25 0.60 0.99 Proteins (g) 90.1 ± 37.3 87.2 ± 30.9 87.4 ± 32.1 84.0 ± 30.1 91.9 ± 28.6 88.3 ± 30.8 0.53 0.68 1.00 Soluble Fiber (g) 8.3 ± 4.1 7.7 a ± 3.3 7.3 x ± 3.4 8.5 y ± 3.1 7.5 x ± 3.1 8.9 by ± 3.1 0.69 0.01 0.02 Insoluble Fiber (g) 15.6 ± 7.8 15.0 x ± 6.8 13.8 a ± 6.9 23.1 by ± 6.6 14.4 x ± 6.2 23.8 by ± 6.3 <0.0001 <0.0001 <0.0001 Fructose (g) 24.1 a ± 14.4 24.9 a ± 9.9 19.3 bx ± 8.9 29.7 by ± 9.4 22.2 x ± 9.0 29.9 by ± 8.9 0.41 <0.0001 0.002 Glucose (g) 23.7 a ± 13.3 25.1 a ± 8.9 19.8 bx ± 8.8 30.2 by ± 7.9 22.2 x ± 8.8 30.9 by ± 7.8 0.18 <0.0001 0.0005 Alcohol (g) 3.2 ± 3.6 2.8 ± 2.8 2.5 ± 2.1 1.9 ± 1.7 2.8 ± 2.5 2.3 ± 1.4 0.07 0.37 0.95 Caﬀeine (mg) 147.7 ± 148.9 103.6 ± 114.9 147.9 ± 142.7 95.2 ± 126.9 150.0 ± 149.7 106.6 ± 142.7 0.59 0.65 0.73 Bread and cereals (serving) 3.9 ± 2.2 4.6 ± 2.3 4.0 ± 2.0 4.1 ± 2.6 4.1 ± 2.3 4.1 ± 1.9 0.45 0.36 0.26 Fruits (serving) 2.9 a ± 2.6 3.1 a ± 1.6 2.2 bx ± 1.7 6.0 by ± 1.3 2.9 x ± 1.9 5.9 by ± 1.3 <0.0001 <0.0001 <0.0001 Vegetables (serving) 3.6 ± 1.9 3.6 ± 2.3 3.3 ± 1.6 2.6 ± 1.6 3.4 ± 1.6 3.2 ± 2.1 0.02 0.10 0.41 Dairy products (serving) 2.0 ± 1.7 2.2 ± 1.1 2.0 ± 1.3 2.3 ± 1.3 1.8 ± 0.9 2.2 ± 1.4 0.69 0.51 0.74 Animal proteins (serving) 2.3 ± 1.2 1.8 ± 0.9 2.2 ± 1.2 1.8 ± 0.8 2.5 ± 1.2 2.0 ± 1.1 0.13 0.39 0.93 Physical activity (AMI) 298.1 ± 147.0 259.0 ± 131.9 280.8 ± 190.5 218.3 ± 159.9 0.13 0.34 0.54 Data are means ± standard deviations. 3.3. Primary Outcomes A mixed model adjusted for age, sex, BMI, and baseline values was used to obtain p-values. Time eﬀect, treatment eﬀect, and the interaction of treatment by time were signiﬁcant at α = 0,05. Signiﬁcant p-values are in bold. Letters stand for signiﬁcant diﬀerences (a,b for time eﬀect and x,y for treatment eﬀect). N = 24 for both Rb and control. tx, treatment. AMI, Activity Metabolic Index. txTime stands for the interaction between treatment and time eﬀects. Table 2. Daily dietary intakes by nutrients and by food group over time within and between groups. Data are means ± standard deviations. A mixed model adjusted for age, sex, BMI, and baseline values was used to ob treatment by time were signiﬁcant at α = 0,05. Signiﬁcant p-values are in bold. Letters stand for signiﬁcant diﬀerences and control. tx, treatment. AMI, Activity Metabolic Index. txTime stands for the interaction between treatment and ti Data are means ± standard deviations. A mixed model adjusted for age, sex, BMI, and baseline values was used to obtain p-values. Time eﬀect, treatment eﬀect, and the interaction of reatment by time were signiﬁcant at α = 0,05. Signiﬁcant p-values are in bold. Letters stand for signiﬁcant diﬀerences (a,b for time eﬀect and x,y for treatment eﬀect). N = 24 for both Rb and control. tx, treatment. AMI, Activity Metabolic Index. txTime stands for the interaction between treatment and time eﬀects. Table 3. Diﬀerences between follow-up and baseline. Variable N Control N Rb p-Value BMI (kg/m2) 24 −0.01 ± 0.60 24 +0.10 ± 0.57 0.43 Waist circumference (cm) 24 −0.18 ± 2.18 24 +0.39 ± 3.12 0.46 Fasting glucose (mmol/L) 23 +0.01 ± 0.35 22 −0.09 ± 0.29 0.27 Fasting insulin (pmol/L) 19 +3.10 ± 22.98 16 +3.12 ± 26.49 0.99 HbA1C (%) 23 −0.03 ± 0.13 23 +0.03 ± 0.14 0.13 Plasma TG (mmol/L) 22 −0.04 ± 0.53 23 −0.17 ± 0.68 0.48 ApoB-100 (g/L) 23 +0.01 ± 0.09 23 −0.03 ± 0.12 0.30 HDL-C (mmol/L) 23 −0.04 ± 0.10 23 −0.01 ± 0.18 0.43 LDL-C (mmol/L) 22 +0.01 ± 0.42 23 +0.03 ± 0.42 0.89 CRP (mg/L) 20 +0.90 ± 2.33 22 −0.04 ± 1.59 0.14 HOMA-IR 13 +0.11 ± 0.74 11 −0.16 ± 0.53 0.33 Matsuda index 13 −0.60 ± 2.08 11 +0.14 ± 1.43 0.33 Data are means ± standard deviations. p-values were obtained by a general linear model, type III sum of squares. 3.3. Primary Outcomes BMI, body mass index. HbA1C, glycated hemoglobin. TG, triglycerides. ApoB-100, Apolipoprotein B. HDL-C, high-density lipoprotein cholesterol. LDL-C, low-density lipoprotein cholesterol. CRP, C-reactive protein. HOMA-IR, homeostatic model assessment of insulin resistance. Table 3. Diﬀerences between follow-up and baseline. Data are means ± standard deviations. p-values were obtained by a general linear model, type III sum of squares. BMI, body mass index. HbA1C, glycated hemoglobin. TG, triglycerides. ApoB-100, Apolipoprotein B. HDL-C, high-density lipoprotein cholesterol. LDL-C, low-density lipoprotein cholesterol. CRP, C-reactive protein. HOMA-IR, homeostatic model assessment of insulin resistance. 9 of 20 Nutrients 2020, 12, 3858 Table 4. Variations of anthropometrics, body composition, and blood pressure parameters over time within and between groups. 3.3. Primary Outcomes Time Week 0 Week 4 Week 8 p-Values Treatment N Control N Rb Control Rb Control Rb Time tx txTime SBP (mmHg) 24 110.8 ± 11.2 24 112.8 ± 11.0 110.7 ± 12.9 113.1 ± 10.4 112.8 ± 11.9 110.9 ± 11.0 0.99 0.37 0.03 DBP (mmHg) 24 68.9 ± 8.7 24 71.9 ± 8.9 68.8 ± 10.4 71.8 ± 9.0 69.6 ± 11.1 70.3 ± 9.3 0.86 0.67 0.34 BMI (kg/m2) 24 29.4 ± 3.9 24 30.4 ± 4.9 29.5 ± 3.9 30.5 ± 5.0 29.4 ± 3.9 30.5 ± 5.1 0.44 0.69 0.69 Waist circumference (cm) 24 98.1 ± 11.8 24 98.5 ± 13.3 98.5 ± 12.1 98.4 ± 13.3 97.9 ± 12.9 98.9 ± 14.1 0.93 0.73 0.23 Hips circumference (cm) 24 108.9 ± 7.5 24 112.1 ± 10.2 109.5 ± 8.4 112.3 ± 10.5 109.3 ± 8.4 112.1 ± 10.5 0.63 0.59 0.77 CRP 20 2.50 ± 2.50 21 2.1 ± 1.64 2.65 ± 2.60 2.86 ± 2.59 2.46 ± 2.42 3.28 ± 2.67 0.30 0.16 0.26 ApoB-100 23 0.86 a ± 0.16 23 0.92 ± 0.24 0.95 b ± 0.17 0.92 y ± 0.21 0.87 a ± 0.19 0.89 ± 0.25 0.002 0.02 0.03 Total-C (mmol/L) 23 4.53 a ± 0.79 23 4.60 ± 0.91 4.85 b ± 0.81 4.70 ± 0.84 4.51 a ± 0.88 4.54 ± 0.86 0.001 0.14 0.22 HDL-C (mmol/L) 23 1.33 ± 0.23 23 1.30 ± 0.40 1.39 a ± 0.29 1.32 ± 0.35 1.29 b ± 0.27 1.30 ± 0.33 0.03 0.49 0.17 LDL-C (mmol/L) 22 2.44 a ± 0.67 23 2.62 ± 0.87 2.78 b ± 0.68 2.74 ± 0.78 2.45 a ± 0.55 2.65 ± 0.82 0.001 0.42 0.14 TG (mmol/L) 22 1.42 ± 0.61 23 1.47 ± 0.82 1.32 ± 0.60 1.39 ± 0.64 1.38 ± 0.61 1.29 ± 0.61 0.37 0.59 0.72 Fasting glucose (mmol/L) 23 4.82 ± 0.47 22 5.00 ± 0.59 4.85 ± 0.49 5.11 ± 0.57 4.83 ± 0.52 4.91 ± 0.57 0.12 0.99 0.25 Fasting insulin (pmol/L) 17 65.3 ± 29.0 16 80.1 ± 37.4 65.9 ± 29.1 77.5 ± 33.2 64.0 ± 23.2 83.3 ± 44.2 0.77 0.59 0.10 HbA1C (%) 23 5.02 ± 0.27 23 5.04 ± 0.32 4.98 ± 0.29 5.03 ± 0.30 5.00 ± 0.27 5.07 ± 0.29 0.11 0.22 0.23 Data are means ± standard deviations. 3.4. Secondary Outcomes 3.4. Secondary Outcomes 3.4.1. Eﬀects of Rb Supplementation on Gene Expression The study of diﬀerential gene expression in blood cells in the Rb group revealed a total of 1384 genes diﬀerentially expressed following Rb supplementation (p < 0.05), from which 119 showed a fold change greater than 1.25. Following multiple testing correction, none of these genes reached the signiﬁcance threshold of FDR-p < 0.05. We then relaxed the signiﬁcance criteria in order to identify those genes diﬀerentially expressed at p < 0.001 (Table 5). A total of 43 genes were identiﬁed, among which 9 were found to be upregulated and 34 downregulated, with fold changes ranging from −1.5 (ADGRG1) to 1.4 (PNPLA4), with 8 of the 43 genes demonstrating at least a 1.25-fold change (Figure 3A). Individual changes in gene expression levels for the top diﬀerentially up- and down-regulated genes presented in Fi 3B ill h i i di id l i bili f i h i i g g p p y p g g p Figure 3B illustrate the inter-individual variability of gene expression changes among parti The pathway enrichment analysis was conducted with all of the 119 genes showing at least a p-value < 0.05 and a fold change > 1.25 in response to the Rb supplementation (Figure 3C). Most diﬀerentially expressed genes were clustered into intracellular signal transduction pathways, as shown by the top-12 signiﬁcantly enriched GO-BP categories, including those involved in the regulation of Ras and Rho signal transduction and several pathways related to the production of interleukins IL-6 and IL-1β. A relevant signal transduction pathway related to MAPK signaling was also found to be signiﬁcantly enriched in the KEGG database. 3.3. Primary Outcomes A mixed model adjusted for age, sex, BMI, and baseline values was used to obtain p-values (except for BMI and waist circumference, which were adjusted for age, sex, and baseline values only). Time eﬀect, treatment eﬀect, and the interaction of treatment by time were signiﬁcant at α = 0.5. Signiﬁcant p-values are in bold. Letters stand for signiﬁcant diﬀerences (a,b for time eﬀect and x,y for treatment eﬀect). tx, treatment. SPB, systolic blood pressure. DBP, diastolic blood pressure. BMI, body mass index. CRP, C-reactive protein. ApoB-100, Apolipoprotein B. Total-C, total cholesterol. HDL-C, high-density lipoprotein cholesterol. LDL-C, low-density lipoprotein cholesterol. TG, triglycerides. HbA1C, glycated hemoglobin. HOMA-IR, homeostatic model assessment insulin resistance. txTime stands for the interaction between treatment and time eﬀects. Data are means ± standard deviations. A mixed model adjusted for age, sex, BMI, and baseline values was used to obtain p-values (except for BMI and waist circumference, which were adjusted for age, sex, and baseline values only). Time eﬀect, treatment eﬀect, and the interaction of treatment by time were signiﬁcant at α = 0.5. Signiﬁcant p-values are in bold. Letters stand for signiﬁcant diﬀerences (a,b for time eﬀect and x,y for treatment eﬀect). tx, treatment. SPB, systolic blood pressure. DBP, diastolic blood pressure. BMI, body mass index. CRP, C-reactive protein. ApoB-100, Apolipoprotein B. Total-C, total cholesterol. HDL-C, high-density lipoprotein cholesterol. LDL-C, low-density lipoprotein cholesterol. TG, triglycerides. HbA1C, glycated hemoglobin. HOMA-IR, homeostatic model assessment insulin resistance. tx*Time stands for the interaction between treatment and time eﬀects. 10 of 20 10 of 20 Nutrients 2020, 12, 3858 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels Paired t-test analysis brought out that, following Rb supplementation, a total of 14 metabolites had signiﬁcantly diﬀerent plasma levels (paired t-test p-value < 0.05), as compared to pre-supplementation levels. This number was reduced to 10 metabolites after applying more restricted statistical signiﬁcance criteria (fold-change >1.25 and signiﬁcant metabolite counts > 50%) (Figure 4A and Table 6). Plasma metabolites showing a signiﬁcant reduction following Rb supplementation are shown on the top-left corner of the paired volcano plot, and those showing a signiﬁcant increase are shown on the top-right corner (Figure 4A). Top-three under- and over-abundant metabolites are shown in paired boxplots in Figure 4B. More speciﬁcally, we identiﬁed β-alanine, deoxycholic acid glycine conjugate (GDCA), and trimethylamine N-oxide (TMAO) among the metabolites showing the most signiﬁcant increase following Rb supplementation. On the other hand, three TG species, TG (16:1/32:1), TG (18:2/32:2) and TG (18:0/32:0), were among the metabolites undergoing a signiﬁcant decrease (respectively −0.96 ± 1.83, −1.04 ± 1.99 and −0.93 ± 2.17 µmol/L, p = 0.02, 0.02 and 0.04). The score plot derived from smPLS-DA shows the complete separation of pre- and post-supplementation groups, without overlap (Figure 4C). Component 1 was the main one responsible for group discrimination, accounting for 5% of the variance, with component 2 accounting for 2.4%, respectively. The 10 metabolites associated with the ﬁrst component and underlying the discrimination between pre- and post-supplementation groups are shown in the loadings panel of the ﬁrst component (Figure 4D). Most of these metabolites were also identiﬁed as diﬀerentially abundant between pre- and post-supplementation groups (Figure 4B). 11 of 20 Nutrients 2020, 12, 3858 Table 5. List of the most signiﬁcant diﬀerentially expressed genes in Rb group after 8 weeks of Rb supplementation. 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels RefSeq Gene Symbol Gene Name Nominal p-Value FDR FC NM_001114759 ZNF683 Zinc ﬁnger protein 683 4.5 × 10−6 0.07 −1.28 NM_001470 GABBR1 Gamma-aminobutyric acid type B receptor subunit 1 7.4 × 10−6 0.07 1.22 NM_033423 GZMH Granzyme H 2.5 × 10−5 0.12 −1.19 NM_031950 FGFBP2 Fibroblast growth factor binding protein 2 2.6 × 10−5 0.12 −1.20 NM_030760 S1PR5 Sphingosine-1-phosphate receptor 5 4.8 × 10−5 0.18 −1.21 NM_139355 MATK Megakaryocyte-associated tyrosine kinase 8.4 × 10−5 0.26 −1.16 NM_020395 INTS12 Integrator complex subunit 12 9.5 × 10−5 0.26 1.39 NM_001144884 SLC30A7 Solute carrier family 30 member 7 1.7 × 10−4 0.28 1.23 NM_005170 ASCL2 Achaete-scute family bHLH transcription factor 2 1.8 × 10−4 0.28 −1.16 NM_025069 ZNF703 Zinc ﬁnger protein 703 1.9 × 10−4 0.28 −1.14 NM_001083116 PRF1 Perforin 1 2.1 × 10−4 0.28 −1.25 NM_138360 CARMIL3 Capping protein regulator and myosin 1 linker 3 2.1 × 10−4 0.28 −1.29 NM_005601 NKG7 Natural killer cell granule protein 7 (1) 2.2 × 10−4 0.28 −1.20 NM_001024401 SBK1 SH3 domain binding kinase 1 2.2 × 10−4 0.28 −1.12 NM_006653 FRS3 Fibroblast growth factor receptor substrate 3 2.2 × 10−4 0.28 1.17 NR_110601 PGS1 Phosphatidylglycerophosphate synthase 1 2.9 × 10−4 0.33 1.20 NR_110030 LINC01215 Long intergenic non-protein coding RNA 1215 3.0 × 10−4 0.33 1.24 NM_001363693 NKG7 Natural killer cell granule protein 7 (2) 3.2 × 10−4 0.33 −1.24 NM_001145770 ADGRG1 Adhesion G protein-coupled receptor G1 3.5 × 10−4 0.34 −1.52 NR_024618 LINC02035 Long intergenic non-protein coding RNA 2035 3.6 × 10−4 0.34 1.14 NM_000234 LIG1 DNA ligase 1 3.9 × 10−4 0.34 −1.23 NM_001122630 CDKN1C Cyclin dependent kinase inhibitor 1C 3.9 × 10−4 0.34 -1.18 NM_170783 ZNRD1 Zinc ribbon domain containing 1 4.1 × 10−4 0.34 −1.22 NM_013432 TONSL Tonsoku like, DNA repair protein 4.8 × 10−4 0.36 −1.15 NM_001145777 FKBP5 FKBP prolyl isomerase 5 4.9 × 10−4 0.36 1.19 NM_198053 CD247 CD247 molecule 5.1 × 10−4 0.36 −1.10 NM_032737 LMNB2 Lamin B2 5.2 × 10−4 0.36 −1.09 NM_004650 PNPLA4 Patatin like phospholipase domain containing 4 (1) 6.2 × 10−4 0.37 −1.30 NM_001271822 SERPINB6 Serpin family B member 6 6.2 × 10−4 0.37 −1.17 NM_001358511 PRDX5 Peroxiredoxin 5 6.4 × 10−4 0.37 −1.29 NM_005686 SOX13 SRY-box transcription factor 13 6.4 × 10−4 0.37 −1.14 NM_017931 TTC38 Tetratricopeptide repeat domain 38 6.4 × 10−4 0.37 −1.12 NM_001142389 PNPLA4 Patatin like phospholipase domain containing 4 (2) 6.8 × 10−4 0.37 1.43 NM_012483 GNLY Granulysin 6.9 × 10−4 0.37 −1.18 NM_001004310 FCRL6 Fc receptor like 6 6.9 × 10−4 0.37 −1.20 NM_024310 PLEKHF1 Pleckstrin homology and FYVE domain containing 1 7.2 × 10−4 0.37 −1.16 NM_013351 TBX21 T-box transcription factor 21 7.3 × 10−4 0.37 −1.18 NM_007182 RASSF1 Ras association domain family member 1 7.6 × 10−4 0.37 −1.14 NM_006056 NMUR1 Neuromedin U receptor 1 7.7 × 10−4 0.37 −1.13 NM_001110556 FLNA Filamin A 8.9 × 10−4 0.42 −1.12 NM_004669 CLIC3 Chloride intracellular channel 3 9.3 × 10−4 0.42 −1.22 NM_001136044 TMUB1 Transmembrane and ubiquitin-like domain containing 1 9.3 × 10−4 0.42 −1.11 NM_001335 CTSW Cathepsin W 9.5 × 10−4 0.42 −1.14 RefSeq stands for the reference sequence accession number. 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels A negative fold change stands for a decrease in gene expression levels following Rb supplementation. FDR stands for False Discovery Rate-adjusted p-values obtained by paired t-test. FC stands for fold change. Numbers in parentheses in the Gene name column stand for t i t i t utrients 2020, 12, 3858 12 of 2 TG (18:0/32:0) y y (9Z-tetradecenoyl)-glycerol Triacylglycerols 0.04 3 p-values stand for paired t-tests. HMDB, Human Metabolome Database. Figure 3. Gene expression change between pre- and post-supplementation states in the Rb group. Panel (A) shows the log2 average abundance of transcripts in counts per million mapped reads (log CPM) on the x-axis and the log2 fold-change (log FC) on the y-axis. Non-significant genes are represented by grey dots. Over- and under-expressed genes (FC > 1.25) with unadjusted significant differences (paired t-test p-value < 0.05) are colored in green and red, respectively. Significant differentially expressed genes from paired t-tests (p-value < 0.001) and showing at least a 1.25 FC are labeled with gene names. The dashed lines represent 1.25 FC. Panel (B) shows the top differentially expressed genes between pre- and post-supplementation states in the Rb group. Box and whisker plots show median, first, and third quartiles, and maximum and minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementation. The three transcripts, which exhibited the most significant (p-value < 0.001) over- and under-expression derived from paired t-tests (post vs. Figure 3. Gene expression change between pre- and post-supplementation states in the Rb group. Panel (A) shows the log2 average abundance of transcripts in counts per million mapped reads (log CPM) on the x-axis and the log2 fold-change (log FC) on the y-axis. Non-signiﬁcant genes are represented by grey dots. Over- and under-expressed genes (FC > 1.25) with unadjusted signiﬁcant diﬀerences (paired t-test p-value < 0.05) are colored in green and red, respectively. Signiﬁcant diﬀerentially expressed genes from paired t-tests (p-value < 0.001) and showing at least a 1.25 FC are labeled with gene names. The dashed lines represent 1.25 FC. Panel (B) shows the top diﬀerentially expressed genes between pre- and post-supplementation states in the Rb group. Box and whisker plots show median, ﬁrst, and third quartiles, and maximum and minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementation. 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels The three transcripts, which exhibited the most signiﬁcant (p-value < 0.001) over- and under-expression derived from paired t-tests (post vs. pre), are shown on the top and bottom rows, respectively. Green and red lines stand for increasing or decreasing gene expression levels between pre- and post-supplementation states within individual paired samples. Panel (C) shows network plots of enriched terms following the Rb supplementation. Network plots depict the linkages among diﬀerentially regulated gene clusters and functional enriched terms in the Gene Ontology Biological Processes (GO-BP) (left) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (right) pathway databases. The size of the grey dots is proportional to the number of genes in the enriched pathway (from 7 to 13 genes), and the red-to-green color gradient of gene dots represents the direction of the gene expression fold-change following the Rb supplementation. Nutrients 2020, 12, 3858 TG (18:0/32:0) 12 of 20 p values stand for paired t tests. HMDB, Human Metabolome Database. igure 3. Gene expression change between pre- and post-supplementation states in the Rb group anel (A) shows the log2 average abundance of transcripts in counts per million mapped reads (log CPM) on the x-axis and the log2 fold-change (log FC) on the y-axis. Non-significant genes are epresented by grey dots. Over- and under-expressed genes (FC > 1.25) with unadjusted significan ifferences (paired t-test p-value < 0.05) are colored in green and red, respectively. Significan ifferentially expressed genes from paired t-tests (p-value < 0.001) and showing at least a 1.25 FC are abeled with gene names. The dashed lines represent 1.25 FC. Panel (B) shows the top differentially xpressed genes between pre- and post-supplementation states in the Rb group. Box and whisker lots show median, first, and third quartiles, and maximum and minimum values for the 24 sample airs before (Pre) and after (Post) the Rb supplementation. The three transcripts, which exhibited the most significant (p-value < 0.001) over- and under-expression derived from paired t-tests (post vs Figure 3. Gene expression change between pre- and post-supplementation states in the Rb group. Panel (A) shows the log2 average abundance of transcripts in counts per million mapped reads (log CPM) on the x-axis and the log2 fold-change (log FC) on the y-axis. Non-signiﬁcant genes are represented by grey dots. Over- and under-expressed genes (FC > 1.25) with unadjusted signiﬁcant diﬀerences (paired t-test p-value < 0.05) are colored in green and red, respectively. 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels Signiﬁcant diﬀerentially expressed genes from paired t-tests (p-value < 0.001) and showing at least a 1.25 FC are labeled with gene names. The dashed lines represent 1.25 FC. Panel (B) shows the top diﬀerentially expressed genes between pre- and post-supplementation states in the Rb group. Box and whisker plots show median, ﬁrst, and third quartiles, and maximum and minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementation. The three transcripts, which exhibited the most signiﬁcant (p-value < 0.001) over- and under-expression derived from paired t-tests (post vs. pre), are shown on the top and bottom rows, respectively. Green and red lines stand for increasing or decreasing gene expression levels between pre- and post-supplementation states within individual paired samples. Panel (C) shows network plots of enriched terms following the Rb supplementation. Network plots depict the linkages among diﬀerentially regulated gene clusters and functional enriched terms in the Gene Ontology Biological Processes (GO-BP) (left) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (right) pathway databases. The size of the grey dots is proportional to the number of genes in the enriched pathway (from 7 to 13 genes), and the red-to-green color gradient of gene dots represents the direction of the gene expression fold-change following the Rb supplementation. igure 3. Gene expression change between pre- and post-supplementation states in the Rb group Panel (A) shows the log2 average abundance of transcripts in counts per million mapped reads (log CPM) on the x-axis and the log2 fold-change (log FC) on the y-axis. Non-significant genes are epresented by grey dots. Over- and under-expressed genes (FC > 1.25) with unadjusted significan differences (paired t-test p-value < 0.05) are colored in green and red, respectively. Significan differentially expressed genes from paired t-tests (p-value < 0.001) and showing at least a 1.25 FC are abeled with gene names. The dashed lines represent 1.25 FC. Panel (B) shows the top differentially xpressed genes between pre- and post-supplementation states in the Rb group. Box and whisker plots show median, first, and third quartiles, and maximum and minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementation. The three transcripts, which exhibited the most significant (p-value < 0.001) over- and under-expression derived from paired t-tests (post vs Figure 3. Gene expression change between pre- and post-supplementation states in the Rb group. 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels Most important metabolites in group discrimination are ordered according to their loading weights (horizontal bars), from bottom to top. Bar color indicates the group for which the mean value is the highest for each feature (orange and green stand for pre- and post-supplementation groups, respectively). gure 4. Impact of Rb supplementation on plasma metabolite levels. Panel (A) shows a volcano paired comparisons between metabolite plasma levels in pre- and post-supplementation gro n the x-axis, a count of significant sample pairs is shown. On the y-axis, the minus logarithm aired t-test p-values is shown. Metabolites showing statistically significant changes following th upplementation (p-value < 0.05 and fold change > 1.25) are depicted as blue dots on the right (incr nd left (decrease) top corners. Top-three significantly different metabolites of both increasing ecreasing values are labeled. Panel (B) shows top metabolites showing significant changes follow b supplementation. Box and whisker plots show median, first, and third quartiles, and maxim nd minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementa he three metabolites, which exhibited the most significant decrease and increase following upplementation, are shown on the top and bottom rows, respectively. Green and red lines stand creasing or decreasing plasma metabolite levels between pre- and post-supplementation st ithin individual paired samples. Panel (C) shows a bi-dimensional score plot depicting the dis asma metabolomic profile between pre- (red dots) and post-supplementation (green dots) pa articipants. The two principal components of the smPLS-DA model along with their correspond ariance in group discrimination are shown on y- and x-axis, respectively. Panel (D) shows a load ot representing the top 10 metabolites selected on the first component of the smPLS-DA mo ost important metabolites in group discrimination are ordered according to their loading wei Figure 4. Impact of Rb supplementation on plasma metabolite levels. Panel (A) shows a volcano plot of paired comparisons between metabolite plasma levels in pre- and post-supplementation groups. On the x-axis, a count of signiﬁcant sample pairs is shown. On the y-axis, the minus logarithm of paired t-test p-values is shown. Metabolites showing statistically signiﬁcant changes following the Rb supplementation (p-value < 0.05 and fold change > 1.25) are depicted as blue dots on the right (increase) and left (decrease) top corners. Top-three signiﬁcantly diﬀerent metabolites of both increasing and decreasing values are labeled. Panel (B) shows top metabolites showing signiﬁcant changes following Rb supplementation. 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels Panel (A) shows the log2 average abundance of transcripts in counts per million mapped reads (log CPM) on the x-axis and the log2 fold-change (log FC) on the y-axis. Non-signiﬁcant genes are represented by grey dots. Over- and under-expressed genes (FC > 1.25) with unadjusted signiﬁcant diﬀerences (paired t-test p-value < 0.05) are colored in green and red, respectively. Signiﬁcant diﬀerentially expressed genes from paired t-tests (p-value < 0.001) and showing at least a 1.25 FC are labeled with gene names. The dashed lines represent 1.25 FC. Panel (B) shows the top diﬀerentially expressed genes between pre- and post-supplementation states in the Rb group. Box and whisker plots show median, ﬁrst, and third quartiles, and maximum and minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementation. The three transcripts, which exhibited the most signiﬁcant (p-value < 0.001) over- and under-expression derived from paired t-tests (post vs. pre), are shown on the top and bottom rows, respectively. Green and red lines stand for increasing or decreasing gene expression levels between pre- and post-supplementation states within individual paired samples. Panel (C) shows network plots of enriched terms following the Rb supplementation. Network plots depict the linkages among diﬀerentially regulated gene clusters and functional enriched terms in the Gene Ontology Biological Processes (GO-BP) (left) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (right) pathway databases. The size of the grey dots is proportional to the number of genes in the enriched pathway (from 7 to 13 genes), and the red-to-green color gradient of gene dots represents the direction of the gene expression fold-change following the Rb supplementation. 13 of 20 g the Rb Nutrients 2020, 12, 3858 g of gene dots rep supplementation. Figure 4. Impact of Rb supplementation on plasma metabolite levels. Panel (A) shows a volcano of paired comparisons between metabolite plasma levels in pre- and post-supplementation grou On the x-axis, a count of significant sample pairs is shown. On the y-axis, the minus logarithm paired t-test p-values is shown. Metabolites showing statistically significant changes following the supplementation (p-value < 0.05 and fold change > 1.25) are depicted as blue dots on the right (incre and left (decrease) top corners. Top-three significantly different metabolites of both increasing decreasing values are labeled. Panel (B) shows top metabolites showing significant changes follow Rb supplementation. 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels Box and whisker plots show median, first, and third quartiles, and maxim and minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementat The three metabolites, which exhibited the most significant decrease and increase following supplementation, are shown on the top and bottom rows, respectively. Green and red lines stand ncreasing or decreasing plasma metabolite levels between pre- and post-supplementation st within individual paired samples. Panel (C) shows a bi-dimensional score plot depicting the dist plasma metabolomic profile between pre- (red dots) and post-supplementation (green dots) pai participants. The two principal components of the smPLS-DA model along with their correspond variance in group discrimination are shown on y- and x-axis, respectively. Panel (D) shows a load plot representing the top 10 metabolites selected on the first component of the smPLS-DA mo Most important metabolites in group discrimination are ordered according to their loading weig Figure 4. Impact of Rb supplementation on plasma metabolite levels. Panel (A) shows a volcano plot of paired comparisons between metabolite plasma levels in pre- and post-supplementation groups. On the x-axis, a count of signiﬁcant sample pairs is shown. On the y-axis, the minus logarithm of paired t-test p-values is shown. Metabolites showing statistically signiﬁcant changes following the Rb supplementation (p-value < 0.05 and fold change > 1.25) are depicted as blue dots on the right (increase) and left (decrease) top corners. Top-three signiﬁcantly diﬀerent metabolites of both increasing and decreasing values are labeled. Panel (B) shows top metabolites showing signiﬁcant changes following Rb supplementation. Box and whisker plots show median, ﬁrst, and third quartiles, and maximum and minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementation. The three metabolites, which exhibited the most signiﬁcant decrease and increase following the supplementation, are shown on the top and bottom rows, respectively. Green and red lines stand for increasing or decreasing plasma metabolite levels between pre- and post-supplementation states within individual paired samples. Panel (C) shows a bi-dimensional score plot depicting the distinct plasma metabolomic proﬁle between pre- (red dots) and post-supplementation (green dots) paired participants. The two principal components of the smPLS-DA model along with their corresponding variance in group discrimination are shown on y- and x-axis, respectively. Panel (D) shows a loading plot representing the top 10 metabolites selected on the ﬁrst component of the smPLS-DA model. 4. Discussion To our knowledge, this is the ﬁrst randomized controlled trial to investigate immune-metabolic changes in response to Rb supplementation over such a long timescale. Brieﬂy, the present intervention resulted in the modulation of the expression of numerous genes and circulating levels of some plasma metabolites being potential biomarkers of metabolic syndrome. Despite the eﬀect of Rb supplementation at transcriptomic and metabolomic levels, its impact on traditional metabolic syndrome features was relatively modest, with no signiﬁcant eﬀects observed on relevant metabolic outcomes. However, very few clinical trials have looked at the health eﬀects of Rb, and most of them were based on acute postprandial interventions with small numbers of subjects. That way, ﬁndings from two recent studies indicated that acute Rb supplementation, with daily consumptions of 125 and 250 g, may lower postprandial hyperglycemia, hypertriglyceridemia, and inﬂammatory response (IL-6 and TNF-α), as well as SBP when extended for four weeks, in diabetic or prediabetic individuals [20,21]. By contrast, two other acute postprandial studies conducted on healthy subjects oﬀered less conclusive results, with no lowering eﬀects on glycemic and insulinemic responses [23,24]. Overall, results from clinical trials suggest that Rb consumption can acutely attenuate metabolic syndrome features in subjects with a pre-existing metabolic condition, lowering postprandial glucose, plasma TG and inﬂammatory biomarkers levels. The mild clinical impact of the present Rb supplementation is, therefore, not surprising and may be attributable to the fact that participants were at risk of developing metabolic syndrome but displayed limited metabolic alterations. Thus, it appears that Rb eﬀects may be clinically noticeable on individuals whose homeostatic set points have shifted to altered states. However, the signiﬁcant increase in glucose and fructose intake observed in the Rb group, and attributable to the reported increase in fruit servings, might have masked the eﬀects of Rb on cardiometabolic health. We further used transcriptomics and metabolomics to reveal the eﬀects of Rb at a molecular level. Two of the most signiﬁcantly down-regulated genes, ZNF683 and ADGRG1, are both linked to immune system functioning. ZNF683 encodes for a key regulatory transcription factor of natural killer (NK) cell diﬀerentiation [44] that also drives the expression of ADGRG1 [45]. Furthermore, ADGRG1 is down-regulated upon NK cell activation, controls NK cell eﬀector functions, and is thus closely associated with the production of cytolytic proteins, such as perforin and granzyme, which are major players of the cytolytic process of NK cells and T lymphocytes [45]. 3.4.2. Eﬀects of Rb Supplementation on Metabolite Levels Box and whisker plots show median, ﬁrst, and third quartiles, and maximum and minimum values for the 24 sample pairs before (Pre) and after (Post) the Rb supplementation. The three metabolites, which exhibited the most signiﬁcant decrease and increase following the supplementation, are shown on the top and bottom rows, respectively. Green and red lines stand for increasing or decreasing plasma metabolite levels between pre- and post-supplementation states within individual paired samples. Panel (C) shows a bi-dimensional score plot depicting the distinct plasma metabolomic proﬁle between pre- (red dots) and post-supplementation (green dots) paired participants. The two principal components of the smPLS-DA model along with their corresponding variance in group discrimination are shown on y- and x-axis, respectively. Panel (D) shows a loading plot representing the top 10 metabolites selected on the ﬁrst component of the smPLS-DA model. Most important metabolites in group discrimination are ordered according to their loading weights (horizontal bars), from bottom to top. Bar color indicates the group for which the mean value is the highest for each feature (orange and green stand for pre- and post-supplementation groups, respectively). 14 of 20 Nutrients 2020, 12, 3858 Table 6. List of the plasma metabolites showing signiﬁcant changes in plasma levels in Rb group after 8 weeks of Rb supplementation. Metabolite Name Common Name Super Pathway p-Value HMDB beta-Ala β-Alanine Biogenic Amines 0.005 HMDB0000056 TMAO Trimethylamine N-oxide Amine Oxides 0.008 HMDB0000925 GDCA Deoxycholic acid glycine conjugate Bile Acids 0.01 HMDB00631 CE15:0 Cholesterol 1-pentadecanoate Cholesterol Esters 0.01 HMDB0060057 TG (22:6/32:0) 1-Palmitoyl-2-palmitoyl- 3-docosahexaenoyl-glycerol Triacylglycerols 0.01 HMDB10418 TG (16:1/32:1) 1-Octadecanoyl-2- (9Z-hexadecenoyl)-3- (9Z-tetradecenoyl)-glycerol Triacylglycerols 0.02 HMDB0044888 TG (18:2/32:2) 1-Palmitoleoyl-2-palmitoleoyl- 3-linoleoyl-glycerol Triacylglycerols 0.02 HMDB05435 HexCer(d18:1/24:1) Hexosylceramide Glucosylceramides 0.02 - PC aa C42:5 1-Arachidonyl-2-docosapentaenoyl- sn-glycero-3-phosphocholine Glycerophospholipids 0.04 HMDB0008287 TG (18:0/32:0) 1-Octadecanoyl-2-octadecanoyl- 3-(9Z-tetradecenoyl)-glycerol Triacylglycerols 0.04 HMDB0044753 p-values stand for paired t-tests. HMDB, Human Metabolome Database. 4. Discussion Accordingly, PRF1, GZMH, GNLY, FGFBP2, and two transcript variants of NKG7, which code, respectively, for the perforin-1 protein, granzyme H, granulysin, ﬁbroblast growth factor-binding protein 2, and NK cell granule protein 7 were 15 of 20 15 of 20 Nutrients 2020, 12, 3858 down-regulated. In addition, the gene encoding for the sphingosine-1-phosphate receptor 5 (S1PR5), which is implicated in monocyte traﬃcking and the exit of mature NK cells from the bone marrow and lymph nodes into blood and lymph circulation, was also signiﬁcantly downregulated [46,47]. S1PR5 belongs to the G protein-coupled receptor family S1PR1-5, known to mediate the eﬀects of sphingosine-1-phosphate (S1P), a bioactive phospholipid, which exerts pleiotropic eﬀects. S1P stems from the phosphorylation by sphingosine kinases of sphingosine that is derived from the hydrolysis of ceramide species [48]. With the class of hexosylceramides (HexCer) being involved in the extensive network of sphingolipids metabolism [49], it is worth highlighting the potential connection between S1P and HexCer(d18:1/24:1), a ceramide whose plasma levels signiﬁcantly increased in response to the intervention. Similarly, GDCA, another metabolite undergoing a signiﬁcant increase following the intervention, has been shown, amongst other conjugated bile acids, to activate S1PR2, which belongs to the receptor family mentioned above [50]. Several signaling pathways related to IL-1β and IL-6 production, as well as to Ras, Rho, and MAPK protein signal transduction, were found to be signiﬁcantly enriched in response to Rb supplementation. Interestingly, Ras, Rho, and MAPK are part of the signal transduction pathways on which S1PR1-5 action relies [51], suggesting a link with the downregulation of S1PR5, as well as with the increase of plasma HexCer(d18:1/24:1) and GDCA. Taken together, this points toward a slight modulation of the “inside-out signaling” process of immune cells. Of interest, animal studies have demonstrated beneﬁcial eﬀects of black raspberry on colorectal and pancreatic cancer, in conjunction with enhanced inﬁltration of NK cells into the colon and modulations of cytotoxic NK and T cell subsets [16,52]. Part of Rb eﬀects has been linked to its richness in ellagitannins and anthocyanins. Previous research has shown that ellagitannins can modulate mice and human leucocytes activity and cytokine production [14,53]. Based on these ﬁndings, part of the transcriptional variations observed herein could be attributed to the high ellagitannins content of Rb. Amongst metabolites tested, β-alanine exhibited the largest increase following Rb supplementation. 4. Discussion It is the intermediate between glycine and gamma-aminobutyric acid (GABA), and acts as a partial GABA receptor antagonist. GABAergic signaling is known to play a role in gastrointestinal motility through vagus nerve stimulation [54]. Components of the GABA signaling machinery are also expressed in immune cells and are involved in the regulation of cytokine production, immune cell proliferation, and chemotaxis [55]. Moreover, we found that GABBR1, which is coding for the subunit 1 of GABA type B receptor, was the most upregulated gene in response to Rb. Interestingly, an agonist of the GABAB receptor has been shown to reduce the secretion of cytokines, including TNF-α and IL-6, in blood mononuclear cells and to interfere with their chemotaxis [56]. Based on these ﬁndings, one may speculate that the signiﬁcant increase in plasma β-alanine concentration is causally related to GABBR1 upregulation, as well as to IL-6 pathways enrichment. Surprisingly, TMAO, a gut microbiota-derived metabolite from dietary choline, choline-containing phospholipids, betaine, and carnitine, also underwent a large increase. It has been previously found that TMAO increases monocyte recruitment and activation, leading to the upregulation of inﬂammatory cytokine genes, monocyte adhesion, and to the formation of foam cells [57]. TMAO is then considered as a pro-atherogenic metabolite associated with an increased risk of cardiovascular disease. This increase in TMAO may be owed to an increase in animal protein consumption of the participants, which was not the case in the present study. More startling is that the reverse trend is generally observed with small fruit extract supplementation, which suggests the eﬀect of an unknown factor that remains to be identiﬁed [58,59]. Finally, in addition to GDCA and HexCer (d18:1/24:1), other lipids belonging to cholesterol esters and glycerophospholipids pathways signiﬁcantly increased, whilst triacylglycerols increased for some and decreased for others. Similarly, in a randomized controlled trial conducted on individuals with metabolic syndrome who consumed 300 g/day of berries comprising 100 g of Rb for 8 weeks, serum lipidomic proﬁling revealed several lipids discriminating berry consumers from controls, notably cholesterol esters, phosphatidylethanolamines, phosphatidylcholines, and TGs [24]. Of note here, are the shared biochemical pathways undergoing modulations following both interventions, 16 of 20 16 of 20 Nutrients 2020, 12, 3858 ﬁrst the cholesterol ester and triacylglycerols pathways, but also the functional connections between the phosphatidylcholines, TMAO, sphingosine and hexosylceramides. Our ﬁndings, together with those of Puupponen-Pimiä et al. [24], point toward sphingolipids and choline metabolism. Author Contributions: Conceptualization, V.G. (Véronique Garneau), D.R., C.C., A.M., and M.-C.V.; data curation, J.d.T.-M. and V.G. (Véronique Garneau); formal analysis, M.F. and J.d.T.-M.; funding acquisition, D.R., C.C., A.M., and M.C.-V.; investigation, M.F., V.G. (Véronique Garneau), V.G. (Valérie Guay), M.K. and P.C.; methodology, J.d.T.-M. and M.C.-V.; project administration, V.G. (Véronique Garneau) and M.C.-V.; resources, M.C.-V.; software, J.d.T.-M.; visualization, M.F. and J.d.T.-M.; writing—original draft, M.F.; writing—review and editing, J.d.T.-M., V.G. (Véronique Garneau), V.G. (Valérie Guay), M.K., G.P., D.R., P.C., C.C., A.M., and M.C.-V., M.F., J.d.T.-M. and M.C.-V. have primary responsibility for ﬁnal content. All authors have read and agreed to the published version of the manuscript. 4. Discussion Accordingly, studies linked sphingolipid metabolism dysregulation to insulin resistance and ceramide accumulation to insulin signaling inhibition [60]. In addition, an interconnection between polyphenols intake and sphingolipid metabolism has been established [61]. Speciﬁcally, anthocyanins were reported to attenuate insulin resistance by modulating sphingomyelin conversion and ceramide de novo synthesis [62], while ellagic acid has been identiﬁed as a potential inhibitor of sphingosine kinase [63]. Taken together, clinical, transcriptional, and metabolomical prisms have shed some light on the potential mechanisms underlying the health eﬀects of Rb. A non-negligible part of these transcriptional results points toward slight modulations in the tight interplay between the cytotoxicity of lymphocytes and immune cell traﬃcking. This phenomenon may be viewed in relation to changes in phospholipid metabolism, S1PR5 downregulation being potentially linked to the modulation of sphingolipid and choline metabolism, reﬂected in the upregulation of HexCer (d18:1/24:1) and TMAO, respectively. In addition, according to the heterogeneity observed in the response of genes and metabolites, these results point to the need for further research on the determinants of the inter-individual variability in response to foods and nutrients. Strengths and Limitations This study has several limitations. First, participants were Caucasians at risk of metabolic syndrome, which limits the generalization of the results. Second, the use of a crossover instead of a parallel design could have reduced the inter-individual variability in baseline characteristics. In addition, freezing small fruits could alter their content in phenolic compounds and, therefore, reduce their eﬀects. For instance, it has been reported that the anthocyanin content of Haskap berries was reduced by nearly 60% after being frozen at −18 ◦C for 6 months [64]. Due to high hemolysis in some plasma samples, which prevents adequate measurement of plasma insulin levels, HOMA-IR and Matsuda indexes have been calculated for only a limited number of subjects in each group. Finally, the free-living design of the present study led to a signiﬁcant increase in fruits consumption in the Rb group. Accordingly, glucose and fructose intakes were signiﬁcantly higher in the Rb group, as compared to the control group, thus limiting the possibilities of clearly delineating the eﬀects of raspberries on cardiometabolic health. Nonetheless, this study has strengths such as its randomization, which prevents selection bias in allocating intervention to participants. Compliance to the treatment was high, with no diﬀerences observed between groups in withdrawals from the study. Thus, although a per protocol analysis was used, exclusion of data are unlikely to have introduced bias. Funding: This research was funded by WA Red Raspberry Commission (WRRC). References 1. Eichelmann, F.; Schwingshackl, L.; Fedirko, V.; Aleksandrova, K. Eﬀect of plant-based diets on obesity-related inﬂammatory proﬁles: A systematic review and meta-analysis of intervention trials. Obes. Rev. 2016, 17, 1067–1079. [CrossRef] [PubMed] 1. Eichelmann, F.; Schwingshackl, L.; Fedirko, V.; Aleksandrova, K. Eﬀect of plant-based diets on obesity-related inﬂammatory proﬁles: A systematic review and meta-analysis of intervention trials. Obes. Rev. 2016, 17, 1067–1079. [CrossRef] [PubMed] 2. Reynolds, A.; Mann, J.; Cummings, J.; Winter, N.; Mete, E.; Morenga, L.T. Carbohydrate quality and human health: A series of systematic reviews and meta-analyses. Lancet 2019, 393, 434–445. [CrossRef] 2. Reynolds, A.; Mann, J.; Cummings, J.; Winter, N.; Mete, E.; Morenga, L.T. Carbohydrate quality and human health: A series of systematic reviews and meta-analyses. Lancet 2019, 393, 434–445. [CrossRef] 3. O’Keefe, S.J. The association between dietary ﬁbre deﬁciency and high-income lifestyle-associated diseases: Burkitt’s hypothesis revisited. Lancet Gastroenterol. Hepatol. 2019, 4, 984–996. [CrossRef] 3. O’Keefe, S.J. The association between dietary ﬁbre deﬁciency and high-income lifestyle-associated d Burkitt’s hypothesis revisited. Lancet Gastroenterol. Hepatol. 2019, 4, 984–996. [CrossRef] yp 4. Alberti, K.; Eckel, R.H.; Grundy, S.M.; Zimmet, P.Z.; Cleeman, J.I.; Donato, K.A.; Fruchart, J.-C.; James, W.P.T.; Loria, C.M.; Smith, S.C. Harmonizing the Metabolic Syndrome. Circulation 2009, 120, 1640–1645. [CrossRef] 4. Alberti, K.; Eckel, R.H.; Grundy, S.M.; Zimmet, P.Z.; Cleeman, J.I.; Donato, K.A.; Fruchart, J.-C.; James, W.P.T.; Loria, C.M.; Smith, S.C. Harmonizing the Metabolic Syndrome. Circulation 2009, 120, 1640–1645. [CrossRef] 5. Health Canada. Canada’s Dietary Guidelines. 2019. Available online: https://food-guide.canada.ca/en/ guidelines (accessed on 31 October 2020) 4. Alberti, K.; Eckel, R.H.; Grundy, S.M.; Zimmet, P.Z.; Cleeman, J.I.; Donato, K.A.; Fruchart, J.-C.; James, W.P.T.; Loria, C.M.; Smith, S.C. Harmonizing the Metabolic Syndrome. Circulation 2009, 120, 1640–1645. [CrossRef] , ; , g y , , [ ] 5. Health Canada. Canada’s Dietary Guidelines. 2019. Available online: https://food-guide.canada.ca/en/ guidelines (accessed on 31 October 2020). 5. Health Canada. Canada’s Dietary Guidelines. 2019. Available online: https://food-guide.canada.ca/en/ guidelines (accessed on 31 October 2020). 6. Shepherd, R. Resistance to Changes in Diet. Proceedings of the Nutrition Society; CABI Publishing: Wallingford, UK, 2002; Volume 61, pp. 267–272. 6. Shepherd, R. Resistance to Changes in Diet. Proceedings of the Nutrition Society; CABI Publishing: Wallingford, UK, 2002; Volume 61, pp. 267–272. 7. Luís, Â.; Domingues, F.; Pereira, L. Association between berries intake and cardiovascular diseases risk factors: A systematic review with meta-analysis and trial sequential analysis of randomized controlled trials. Food Funct. 2018, 9, 740–757. [CrossRef] 8. 5. Conclusions In conclusion, clinical results are in line with those previously reported in human studies [24,25], as it appears that Rb health eﬀects are clinically noticeable only in subjects with advanced metabolic disorders [20,21]. Omics proﬁling provided an overview of Rb health eﬀects at the molecular level. Future investigations should be deepened to increase our understanding of the impact of Rb consumption on phospholipid metabolism and immune system and the links in-between. 17 of 20 17 of 20 Nutrients 2020, 12, 3858 Acknowledgments: The authors thank the study participants for their excellent collaboration and acknowledge the work of the clinical investigation unit staﬀ. M.-C.V. is a Tier 1 Canada Research Chair in Genomics Applied to Nutrition and Metabolic Health. M.F. received a studentship from the Centre Nutrition, Santé et Société (NUTRISS). Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study, data collection and analysis, interpretation of results, decision to publish, or redaction of the manuscript. References Rao, A.V.; Snyder, D.M. Raspberries and Human Health: A Review†. J. Agric. Food Chem. 2010, 58, 3871–3883. [CrossRef] 9. Seeram, N.P.; Adams, L.S.; Zhang, Y.; Lee, R.; Sand, D.; Scheuller, H.S.; Heber, D. Blackberry, Black Raspberry, Blueberry, Cranberry, Red Raspberry, and Strawberry Extracts Inhibit Growth and Stimulate Apoptosis of Human Cancer Cells In Vitro. J. Agric. Food Chem. 2006, 54, 9329–9339. [CrossRef] 10. Wang, P.-W.; Cheng, Y.-C.; Hung, Y.-C.; Lee, C.-H.; Fang, J.-Y.; Li, W.-T.; Wu, Y.-R.; Pan, T.-L. Red Raspberry Extract Protects the Skin against UVB-Induced Damage with Antioxidative and Anti-inﬂammatory Properties. Oxidative Med. Cell. Longev. 2019, 2019, 1–14. [CrossRef] 11. Tu, L.; Sun, H.; Tang, M.; Zhao, J.; Zhang, Z.; Sun, X.; He, S. Red raspberry extract (Rubus idaeus L shrub) intake ameliorates hyperlipidemia in HFD-induced mice through PPAR signaling pathway. Food Chem. Toxicol. 2019, 133, 110796. [CrossRef] 12. Noratto, G.; Chew, B.P.; Atienza, L.M. Red raspberry (Rubus idaeus L.) intake decreases oxidative stress in obese diabetic (db/db) mice. Food Chem. 2017, 227, 305–314. [CrossRef] 13. Kirakosyan, A.; Seymour, E.M.; Kondoleon, N.; Gutierrez-Albanchez, E.; Wolforth, J.; Bolling, S. The intake of red raspberry fruit is inversely related to cardiac risk factors associated with metabolic syndrome. J. Funct. Foods 2018, 41, 83–89. [CrossRef] 14. Allen, C.T.; Peden-Adams, M.M.; Eudaly, J.; Keil, D.E. Subchronic Exposure to Ellagic Acid Impairs Cytotoxic T-Cell Function and Suppresses Humoral Immunity in Mice. Immunopharmacol. Immunotoxicol. 2003, 25, 409–422. [CrossRef] 15. Nowak, A.; Sójka, M.; Klewicka, E.; Lipi´nska, L.; Klewicki, R.; Kołodziejczyk, K. Ellagitannins from Rubus idaeus L. Exert Geno- and Cytotoxic Eﬀects against Human Colon Adenocarcinoma Cell Line Caco-2. J. Agric. Food Chem. 2017, 65, 2947–2955. [CrossRef] [PubMed] 16. Pan, P.; Kang, S.; Wang, Y.; Liu, K.; Oshima, K.; Huang, Y.-W.; Zhang, J.; Yearsley, M.; Yu, J.; Wang, L.-S. Black Raspberries Enhance Natural Killer Cell Inﬁltration into the Colon and Suppress the Progression of Colorectal Cancer. Front. Immunol. 2017, 8, 997. [CrossRef] [PubMed] 17. Knobloch, T.; Ryan, N.M.; Bruschweiler-Li, L.; Wang, C.; Bernier, M.C.; Somogyi, Á.; Yan, P.; Cooperstone, J.L.; Mo, X.; Brüschweiler, R.; et al. Metabolic Regulation of Glycolysis and AMP Activated Protein Kinase Pathways during Black Raspberry-Mediated Oral Cancer Chemoprevention. Metabolites 2019, 9, 140. [CrossRef] [PubMed] 18 of 20 18 of 20 Nutrients 2020, 12, 3858 18. Jeong, H.S.; Kim, S.; Hong, S.J.; Choi, S.C.; Choi, J.-H.; Kim, J.-H.; Park, C.-Y.; Cho, J.Y.; Lee, T.-B.; Kwon, J.-W.; et al. References Black Raspberry Extract Increased Circulating Endothelial Progenitor Cells and Improved Arterial Stiﬀness in Patients with Metabolic Syndrome: A Randomized Controlled Trial. J. Med. Food 2016, 19, 346–352. [CrossRef] 19. Sardo, C.; Kitzmiller, J.P.; Apseloﬀ, G.; Harris, R.B.; Roe, D.J.; Stoner, G.D.; Jacobs, E.T. An Open-Label Randomized Crossover Trial of Lyophilized Black Raspberries on Postprandial Inﬂammation in Older Overweight Males. Am. J. Ther. 2016, 23, e86–e91. [CrossRef] 20. Xiao, D.; Zhu, L.; Edirisinghe, I.; Fareed, J.; Brailovsky, Y.; Burton-Freeman, B.M. Attenuation of Postmeal Metabolic Indices with Red Raspberries in Individuals at Risk for Diabetes: A Randomized Controlled Trial. Obesity 2019, 27, 542–550. [CrossRef] 21. Schell, J.; Betts, N.M.; Lyons, T.J.; Basu, A. Raspberries Improve Postprandial Glucose and Acute and Chronic Inﬂammation in Adults with Type 2 Diabetes. Ann. Nutr. Metab. 2019, 74, 165–174. [CrossRef] 22. Istas, G.; Feliciano, R.P.; Weber, T.; Garcia-Villalba, R.; Tomás-Barberán, F.A.; Heiss, C.; Rodriguez-Mateos, A. Plasma urolithin metabolites correlate with improvements in endothelial function after red raspberry consumption: A double-blind randomized controlled trial. Arch. Biochem. Biophys. 2018, 651, 43–51. [CrossRef] 23. Clegg, M.E.; Pratt, M.; Meade, C.M.; Henry, C.J.K. The addition of raspberries and blueberries to a starc food does not alter the glycaemic response. Br. J. Nutr. 2011, 106, 335–338. [CrossRef] [PubMed] 24. Puupponen-Pimiä, R.; Seppänen-Laakso, T.; Kankainen, M.; Maukonen, J.; Törrönen, R.; Kolehmainen, M.; Leppänen, T.; Moilanen, E.; Nohynek, L.; Aura, A.-M.; et al. Eﬀects of ellagitannin-rich berries on blood lipids, gut microbiota, and urolithin production in human subjects with symptoms of metabolic syndrome. Mol. Nutr. Food Res. 2013, 57, 2258–2263. [CrossRef] [PubMed] 25. Törrönen, R.; Kolehmainen, M.; Sarkkinen, E.; Poutanen, K.; Mykkänen, H.; Niskanen, L. Berries Reduce Postprandial Insulin Responses to Wheat and Rye Breads in Healthy Women. J. Nutr. 2013, 143, 430–436. [CrossRef] 26. Scarsella, C.; Alméras, N.; Mauriège, P.; Blanchet, C.; Sauvé, L.; Dewailly, E.; Bergeron, J.; Després, J.-P. Prevalence of metabolic alterations predictive of cardiovascular disease risk in the Québec population. Can. J. Cardiol. 2003, 19, 51–57. [PubMed] 27. Norgan, N.G. A Review of: Anthropometric Standardization Reference Manual Edited by T. G. LOHMAN, A.F. ROCHE and R. MARTORELL. (Champaign, IL.: Human Kinetics Books, 1988.) ISBN 087322 121 4. Ergonomics 1988, 31, 1493–1494. [CrossRef] g 28. Labonté, M.-È.; Cyr, A.; Baril-Gravel, L.; Royer, M.-M.; Lamarche, B. Validity and reproducibility of a web-based, self-administered food frequency questionnaire. Eur. J. Clin. Nutr. 2011, 66, 166–173. [CrossRef] 28. References Prognostic value of metabolic response in breast cancer patients receiving neoadjuvant chemotherapy. BMC Cancer 2012, 12, 39. [CrossRef] 41. Westerhuis, J.A.; Van Velzen, E.J.J.; Hoefsloot, H.C.J.; Smilde, A.K. Multivariate paired data analysis: Multilevel PLSDA versus OPLSDA. Metabolomics 2010, 6, 119–128. [CrossRef] 42. Le Cao, K.-A.; Boitard, S.; Besse, P. Sparse PLS discriminant analysis: Biologically relevant feature selection and graphical displays for multiclass problems. BMC Bioinform. 2011, 12, 253. [CrossRef] 43. Rohart, F.; Gautier, B.; Singh, A.; Le Cao, K.-A. mixOmics: An R package for ‘omics feature selection and multiple data integration. PLoS Comput. Biol. 2017, 13, e1005752. [CrossRef] [PubMed] 44. Post, M.; Cuapio, A.; Osl, M.; Lehmann, D.; Resch, U.; Davies, D.M.; Bilban, M.; Schlechta, B.; Eppel, W.; Nathwani, A.; et al. The Transcription Factor ZNF683/HOBIT Regulates Human NK-Cell Development. Front. Immunol. 2017, 8, 535. [CrossRef] [PubMed] 45. Chang, G.-W.; Hsiao, C.-C.; Peng, Y.-M.; Braga, F.A.V.; Kragten, N.A.; Remmerswaal, E.B.; Van De Garde, M.D.; Straussberg, R.; König, G.M.; Kostenis, E.; et al. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells. Cell Rep. 2016, 15, 1757–1770. [CrossRef] [PubMed] 46. Debien, E.; Mayol, K.; Biajoux, V.; Daussy, C.; De Agüero, M.G.; Taillardet, M.; Dagany, N.; Brinza, L.; Henry, T.; Dubois, B.; et al. S1PR5 is pivotal for the homeostasis of patrolling monocytes. Eur. J. Immunol. 2013, 43, 1667–1675. [CrossRef] 47. Drouillard, A.; Mathieu, A.-L.; Marçais, A.; Belot, A.; Viel, S.; Mingueneau, M.; Guckian, K.; Walzer, T. S1PR5 is essential for human natural killer cell migration toward sphingosine-1 phosphate. J. Allergy Clin. Immunol. 2018, 141, 2265–2268.e1. [CrossRef] 48. Maceyka, M.; Milstien, S.; Spiegel, S. Sphingosine kinases, sphingosine-1-phosphate and sphingolipi Prostaglandins Other Lipid Mediat. 2005, 77, 15–22. [CrossRef] 49. Iqbal, J.; Walsh, M.T.; Hammad, S.M.; Cuchel, M.; Tarugi, P.; Hegele, R.A.; Davidson, N.O.; Rader, D.J.; Klein, R.L.; Hussain, M.M. Microsomal Triglyceride Transfer Protein Transfers and Determines Plasma Concentrations of Ceramide and Sphingomyelin but Not Glycosylceramide. J. Biol. Chem. 2015, 290, 25863–25875. [CrossRef] 50. Studer, E.; Zhou, X.; Zhao, R.; Wang, Y.; Takabe, K.; Nagahashi, M.; Pandak, W.M.; Dent, P.; Spiegel, S.; Shi, R.; et al. Conjugated bile acids activate the sphingosine-1-phosphate receptor 2 in primary rodent hepatocytes. Hepatology 2011, 55, 267–276. [CrossRef] 51. Bryan, A.M.; Del Poeta, M. Sphingosine-1-phosphate receptors and innate immunity. Cell. Microbiol. 2018, 20, e12836. [CrossRef] 52. References Labonté, M.-È.; Cyr, A.; Baril-Gravel, L.; Royer, M.-M.; Lamarche, B. Validity and reproducibility of a web-based, self-administered food frequency questionnaire. Eur. J. Clin. Nutr. 2011, 66, 166–173. [CrossRef] 29. Taylor, H.L.; Jacobs, D.R.; Schucker, B.; Knudsen, J.; Leon, A.S.; Debacker, G. A questionnaire for the assessment of leisure time physical activities. J. Chronic Dis. 1978, 31, 741–755. [CrossRef] q y q 29. Taylor, H.L.; Jacobs, D.R.; Schucker, B.; Knudsen, J.; Leon, A.S.; Debacker, G. A questionnaire for the assessment of leisure time physical activities. J. Chronic Dis. 1978, 31, 741–755. [CrossRef] 30. Burstein, M.; Samaille, J. Sur un dosage rapide du cholesterol lié aux α-et aux β-lipoprotéines du sérum. Clin. Chim. Acta 1960, 5, 609. [CrossRef] 31. Friedewald, W.T.; Levy, R.I.; Fredrickson, D.S. Estimation of the Concentration of Low-Density Lipoprotein Cholesterol in Plasma, Without Use of the Preparative Ultracentrifuge. Clin. Chem. 1972, 18, 499–502. [CrossRef] 32. LeDue, T.B.; Weiner, D.L.; Sipe, J.D.; Poulin, S.E.; Collins, M.F.; Rifai, N. Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid A and mannose-binding protein in human serum. Ann. Clin. Biochem. Int. J. Lab. Med. 1998, 35, 745–753. [CrossRef] 33. Richterich, R.; Dauwalder, H. [Determination of plasma glucose by hexokinase-glucose-6-phosphate dehydrogenase method]. Schweiz. Med. Wochenschr. 1971, 101, 615–618. [PubMed] 34. Matthews, D.R.; Hosker, J.P.; Rudenski, A.S.; Naylor, B.A.; Treacher, D.F.; Turner, R.C. Homeostasis model assessment: Insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985, 28, 412–419. [CrossRef] [PubMed] 35. Matsuda, M.; DeFronzo, R.A. Insulin sensitivity indices obtained from oral glucose tolerance testing: Comparison with the euglycemic insulin clamp. Diabetes Care 1999, 22, 1462–1470. [CrossRef] [PubMed] 36. Reynés, B.; Priego, T.; Cifre, M.; Oliver, P.; Palou, A. Peripheral Blood Cells, a Transcriptomic Tool in Nutrigenomic and Obesity Studies: Current State of the Art. Compr. Rev. Food Sci. Food Saf. 2018, 17, 1006–1020. [CrossRef] Nutrients 2020, 12, 3858 19 of 20 37. Bray, N.L.; Pimentel, H.; Melsted, P.; Pachter, L. Near-optimal probabilistic RNA-seq quantiﬁcation. Nat. Biotechnol. 2016, 34, 525–527. [CrossRef] 38. McCarthy, D.J.; Chen, Y.; Smyth, G.K. Diﬀerential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012, 40, 4288–4297. [CrossRef] 39. Chong, J.; Xia, J. MetaboAnalystR: An R package for ﬂexible and reproducible analysis of metabolomics data. Bioinformatics 2018, 34, 4313–4314. [CrossRef] [PubMed] 40. Cao, M.D.; Giskeødegård, G.F.; Bathen, T.F.; Sitter, B.; Boﬁn, A.M.; Lønning, P.E.; Lundgren, S.; Gribbestad, I.S. References Pan, P.; Zhu, Z.; Oshima, K.; Aldakkak, M.; Tsai, S.; Huang, Y.-W.; Dong, W.; Zhang, J.; Lin, C.-W.; Wang, Y.; et al. Black raspberries suppress pancreatic cancer through modulation of NKp46 + CD8 +, and CD11b + immune cells. Food Front. 2020, 1, 70–82. [CrossRef] 53. Bessler, H. On the Link between Ellagic Acid and the Immune Balance between Human Mononuclear and Colon Carcinoma Cells. Immunol Curr Res 2017, 1, 1. 54. Wu, F.-S.; Gibbs, T.T.; Farb, D.H. Dual activation of GABAA and glycine receptors by β-alanine: Inverse modulation by progesterone and 5α-pregnan-3α-ol-20-one. Eur. J. Pharmacol. Mol. Pharmacol. 1993, 246, 239–246. [CrossRef] 55. Jin, Z.; Mendu, S.K.; Birnir, B. GABA is an eﬀective immunomodulatory molecule. Amino Acids 2013, 45, 87–94. [CrossRef] [PubMed] 56. Duthey, B.; Hübner, A.; Diehl, S.; Boehncke, S.; Pfeﬀer, J.; Boehncke, W.-H. Anti-inﬂammatory eﬀects of the GABAB receptor agonist baclofen in allergic contact dermatitis. Exp. Dermatol. 2010, 19, 661–666. [CrossRef] y, ; , ; , ; , ; , J ; , y GABAB receptor agonist baclofen in allergic contact dermatitis. Exp. Dermatol. 2010, 19, 661–666. [CrossRef] 57. Geng, J.; Yang, C.; Wang, B.; Zhang, X.; Hu, T.; Gu, Y.; Li, J. Trimethylamine N-oxide promotes atherosclerosis via CD36-dependent MAPK/JNK pathway. Biomed. Pharmacother. 2018, 97, 941–947. [CrossRef] [PubMed] 57. Geng, J.; Yang, C.; Wang, B.; Zhang, X.; Hu, T.; Gu, Y.; Li, J. Trimethylamine N-oxide promotes atherosclerosis via CD36-dependent MAPK/JNK pathway. Biomed. Pharmacother. 2018, 97, 941–947. [CrossRef] [PubMed] 20 of 20 20 of 20 Nutrients 2020, 12, 3858 58. Annunziata, G.; Maisto, M.; Schisano, C.; Ciampaglia, R.; Narciso, V.; Tenore, G.C.; Novellino, E. Eﬀects of Grape Pomace Polyphenolic Extract (Taurisolo®) in Reducing TMAO Serum Levels in Humans: Preliminary Results from a Randomized, Placebo-Controlled, Cross-Over Study. Nutrients 2019, 11, 139. [CrossRef] [PubMed] 59. Bresciani, L.; Dall’Asta, M.; Favari, C.; Calani, L.; Del Rio, D.; Brighenti, F. An in vitro exploratory study of dietary strategies based on polyphenol-rich beverages, fruit juices and oils to control trimethylamine production in the colon. Food Funct. 2018, 9, 6470–6483. [CrossRef] p 60. Mandal, N.; Grambergs, R.; Mondal, K.; Basu, S.K.; Tahia, F.; Dagogo-Jack, S. Role of ceramides in the pathogenesis of diabetes mellitus and its complications. J. Diabetes Complicat. 2020, 10, 107734. [CrossRef] 61. Cas, M.D.; Ghidoni, R. Cancer Prevention and Therapy with Polyphenols: Sphingolipid-Mediated Mechanisms. Nutrients 2018, 10, 940. [CrossRef] 62. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Si, X.; Tian, J.; Shu, C.; Wang, Y.; Gong, E.S.; Zhang, Y.; Zhang, W.; Cui, H.; Li, B. Serum Ceramide Reduction by Blueberry Anthocyanin-Rich Extract Alleviates Insulin Resistance in Hyperlipidemia Mice. J. Agric. Food Chem. 2020, 68, 8185–8194. [CrossRef] 63. Gupta, P.; Mohammad, T.; Khan, P.; Alajmi, M.F.; Hussain, A.; Rehman, T.; Hassan, I. Evaluation of ellagic acid as an inhibitor of sphingosine kinase 1: A targeted approach towards anticancer therapy. Biomed. Pharmacother. 2019, 118, 109245. [CrossRef] [PubMed] 64. Khattab, R.; Celli, G.B.; Ghanem, A.; Brooks, M.S.-L. Eﬀect of frozen storage on polyphenol content and antioxidant activity of haskap berries (Lonicera caerulea L.). J. Berry Res. 2015, 5, 231–242. [CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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/).
W4295872921.txt	https://www.frontiersin.org/articles/10.3389/fpara.2022.984152/pdf	en		Immunomodulatory and biological properties of helminth-derived small molecules: Potential applications in diagnostics and therapeutics	Frontiers in parasitology	2,022	cc-by	19,245	Review 09 September 2022 DOI 10.3389/fpara.2022.984152 TYPE PUBLISHED OPEN ACCESS EDITED BY Javier Sotillo, Instituto de Salud Carlos III (ISCIII), Spain REVIEWED BY Tao Wang, The University of Melbourne, Australia Dalia Ashour, Tanta University, Egypt Alba Cortés, University of Valencia, Spain CORRESPONDENCE Karma Yeshi karma.yeshi@my.jcu.edu.au SPECIALTY SECTION This article was submitted to Parasitology Omics, a section of the journal Frontiers in Parasitology 01 July 2022 23 August 2022 PUBLISHED 09 September 2022 Immunomodulatory and biological properties of helminth-derived small molecules: Potential applications in diagnostics and therapeutics Karma Yeshi, Roland Ruscher, Alex Loukas and Phurpa Wangchuk Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine (AITHM), James Cook University, Cairns, QLD, Australia RECEIVED ACCEPTED CITATION Yeshi K, Ruscher R, Loukas A and Wangchuk P (2022) Immunomodulatory and biological properties of helminth-derived small molecules: Potential applications in diagnostics and therapeutics. Front. Parasitol. 1:984152. doi: 10.3389/fpara.2022.984152 COPYRIGHT © 2022 Yeshi, Ruscher, Loukas and Wangchuk. 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. Frontiers in Parasitology Parasitic helminths secrete and excrete a vast array of molecules known to help skew or suppress the host’s immune response, thereby establishing a niche for sustained parasite maintenance. Indeed, the immunomodulatory potency of helminths is attributed mainly to excretory/secretory products (ESPs). The ESPs of helminths and the identiﬁed small molecules (SM) are reported to have diverse biological and pharmacological properties. The available literature reports only limited metabolites, and the identity of many metabolites remains unknown due to limitations in the identiﬁcation protocols and helminth-speciﬁc compound libraries. Many metabolites are known to be involved in host-parasite interactions and pathogenicity. For example, fatty acids (e.g., stearic acid) detected in the infective stages of helminths are known to have a role in host interaction through facilitating successful penetration and migration inside the host. Moreover, excreted/secreted SM detected in helminth species are found to possess various biological properties, including anti-inﬂammatory activities, suggesting their potential in developing immunomodulatory drugs. For example, helminths-derived somatic tissue extracts and whole crude ESPs showed anti-inﬂammatory properties by inhibiting the secretion of proinﬂammatory cytokines from human peripheral blood mononuclear cells and suppressing the pathology in chemically-induced experimental mice model of colitis. Unlike bigger molecules like proteins, SM are ideal candidates for drug development since they are small structures, malleable, and lack immunogenicity. Future studies should strive toward identifying unknown SM and isolating the under-explored niche of helminth metabolites using the latest metabolomics technologies and associated software, which hold potential keys for ﬁnding new diagnostics and novel therapeutics. 01 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 KEYWORDS helminths, immunomodulatory, small molecules, excretory-secretory products, biological activities, pharmacological properties, metabolomics Introduction administration programs in many countries, individuals in endemic areas are susceptible to re-infection. No vaccine is available for any human helminthiasis, and its development has been confounded by the complex life cycles of parasitic worms. Moreover, we do not fully understand the cues that these parasites require from the host to trigger developmental changes during their complicated lifecycles. It is indisputable that chronic parasitic helminth infections are a major human health burden, but at the same time, epidemiological evidence supports the “Old Friends” hypothesis, notably through the observation that the complete elimination of helminths from deﬁned human populations is associated with an increased incidence of inﬂammatory diseases and immune dysfunction (Rook, 2012; Rook, 2013). Although this link has not yet established cause and effect, there is clear evidence that helminth infections suppress the development of some inﬂammatory conditions, which is discussed in later sections. The “Old Friends” hypothesis (Rook, 2007) describes the requirement for early exposure to organisms like STHs, microbes, and environmental factors to prime essential immune development during childhood and ensure the optimal balance between effector and regulatory arms of the immune system. These immunomodulatory strategies mean that individual parasites can colonise a host for many years while preventing the development of sterilising protective immunity and suppressing the onset of inappropriate immune responses to innocuous antigens (McSorley et al., 2011; McSorley et al., 2013). Indeed, hookworms are one of the most adept infectious agents at driving regulatory responses (Chazot et al., 1994). It is wellknown that the incidence of inﬂammatory diseases, including inﬂammatory bowel disease (IBD), is highest in countries where endemic exposure to helminths is lowest (Weinstock and Elliott, 2009; Rook, 2012; Molodecky et al., 2012). Extensive studies show that as a population urbanises and exposure to helminths is reduced, an increase in the incidence of inﬂammatory disease is observed (Prideaux et al., 2012; Ahuja and Tandon, 2010). The implication of helminths in this process is documented in mass drug administration programs, where an increase in immune dysfunction disorders soon follows the removal of helminths (Flohr et al., 2006). In a study by Kabeerdoss et al. (2011) (Kabeerdoss et al., 2011) of Crohn’s disease patients in India, individuals depleted of hookworms were more likely to have Crohn’s disease than matched infected subjects. In South Africa, childhood exposure to helminths was shown to be protective against IBD (Chu et al., 2013), and in Papua New Guinea, Helminths, the cause of neglected helminthiases, are comprised of two major phyla, namely nematodes (roundworms) and platyhelminths (ﬂatworms) (Hotez et al., 2008). Nematodes include intestinal soil-transmitted helminths (STHs) such as roundworms (e.g., Ascaris lumbricoides), whipworms (e.g., Trichuris trichiura), hookworms (e.g., Ancylostoma spp., and Necator americanus), and tissue or intestinal nematode (e.g., Trichinella spp.); and the ﬁlarial worms that cause lymphatic ﬁlariasis and onchocerciasis. Platyhelminths include trematodes such as blood-ﬂukes (schistosomes) and cestodes such as tapeworms that cause cysticercosis. These parasites have been associated with humans since they were hominids, with an intimate evolutionary relationship that harks back at least two million years, and they are thus considered “heirloom parasites” (Araujo et al., 2013). Intestinal nematodes have long been recognized as a public health burden contributing to anaemia, digestive diseases, and stunted growth (Sanchez et al., 2014). The exact global statistics for STH infections and associated morbidity are still elusive due to the inadequacy of up-to-date epidemiological data and the nonspeciﬁcity of clinical signs of STH infections (De Silva et al., 2003; Brooker, 2010). According to the World Health Organisation (WHO), more than 1.5 billion people (approximately 24% of the world’s population) are infected with STHs alone, mainly affecting sub-Saharan Africa, the Americas, China, and East Asia, with children being the primary victims (WHO, 2020). More than 267 million preschool-age children and 568 million school-age children live in parasite-endemic areas needing treatment and preventive interventions. As per the Global Burden of Disease study conducted in 2010, chronic parasitic worm infections signiﬁcantly contribute to disability-adjusted life years (DALYs) lost, causing health and economic losses to billions of people worldwide (Murray et al., 2012; Vos et al., 2012). As of 2010, STHs have caused 5.2 million DALYs, schistosomiasis (3.3 million), lymphatic ﬁlariasis (2.8 million), and onchocerciasis (0.5 million) (Murray et al., 2012). However, in 2017, the estimated global burden of STHs dropped to 1.9 million DALYS (Kyu et al., 2018). According to Montresor et al. (2020), more than one billion people worldwide are still affected by STHs (Montresor et al., 2020). Currently, only a few WHO-recommended anthelmintic drugs, such as albendazole, mebendazole, and ivermectin, are used to treat STH infections (WHO, 2020). Despite repeated deworming and mass-drug Frontiers in Parasitology 02 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 trials,” “host-parasite interaction,” “biomarkers,” “downregulation,” “up-regulation,” “excretory/secretory products,” and names of different helminth species.” We have conducted a narrative review of the available literature and presented SM identiﬁed from helminths and their reported biological activities, exploring their potential scope in diagnostics and therapeutics development. exposure to intestinal parasites protected infected subjects against the development of allergic rhinitis (Blount et al., 2009). There are also small open-label studies showing disease improvement in adult IBD patients (e.g., Crohn’s disease) when treated with tolerable numbers of live helminths (e.g., human hookworm N. americanus infective larvae) (Croese et al., 2006; Helmby, 2015). Inspired by these beneﬁcial health effects, many patients suffering from autoimmune and metabolic diseases, especially IBD, have resorted to an unregulated and risky selftreatment modality called helminth therapy propagated by the availability of helminths online (Zhang and Gems, 2021). The key to the helminth’s ability to successfully evade the host immune system’s anti-helminth responses and establish a chronic infection while rendering protection to their host against various inﬂammatory conditions lies in their excretory/secretory products (ESPs) (Loukas et al., 2016; Eichenberger et al., 2018; Wangchuk et al., 2019). Helminths actively induce tolerance (both local and systemic) by producing ESPs that interact with the host immune system. The ESPs comprise proteins, peptides, organic acids, lipids, carbohydrates, and nucleic acids (Maizels et al., 2018). Some of these molecules are homologous to host molecules and can skew the host’s immune response by mimicking immunologically relevant proteins or miRNAs that target the host’s gene expression (Buck et al., 2014; Smyth et al., 2018). Other helminth-derived molecules are unique to the parasites and help them initiate tissue damage and establish infection (Periago and Bethony, 2012; Flynn et al., 2019). Such helminth-speciﬁc biomolecules have also been explored widely as biomarkers for developing diagnostics and targets for developing vaccines and drugs (Hamed et al., 2011; Ryan et al., 2020; Whitman et al., 2021). Numerous reviews (McSorley et al., 2013; Maizels and McSorley, 2016; Maizels et al., 2018; Zakeri et al., 2018; Ryan et al., 2020) have substantially discussed the nature and functions of many helminth-derived macromolecules, including secreted proteins, peptides, and extracellular vesicles. Recently, work on helminthderived small molecules (SM) has gained traction (Bobardt et al., 2020). However, to our knowledge, there is no comprehensive review article on the SM components of helminths. Small molecules are chemical compounds whose size/ molecular weights are less than one kilodalton (kDa) (Makurvet, 2021). Herein, we have collected published information on SM (identiﬁed from helminths) and their immunomodulatory potential from PubMed, Scopus, MEDLINE Ovid, Google Scholar Databases, and journal articles and analysed their contents. To retrieve relevant published information, we used the keywords “small molecules,” “helminths small molecules,” “immunomodulatory,” “helminth-derived small molecules,” “host-parasite interaction,” “helminth metabolites,” “lipidomics,” “helminth therapy,” “metabolomics,” “targeted metabolomics,” “untargeted metabolomics,” “inﬂammation,” “inﬂammatory bowel diseases,” “autoimmune disease,” “inﬂammatory cytokines,” “anti-inﬂammatory,” “helminthiases,” “clinical Frontiers in Parasitology Host-parasite interaction and immunomodulation Parasitic helminths are believed to have coevolved with humans over millennia, continuously reﬁning and improving their mechanisms to skew or suppress the host’s immune response to establish a sustained niche inside their host. They do so by secreting or excreting a vast array of molecules, including metabolites. Dendritic cells (DCs) respond to ESPs and also microbiome metabolites, consequently promoting the development of regulatory T cells (Treg), regulatory B cells (Breg), and T helper type 2 (Th2) cells and inhibiting Th1 and Th17 cell responses (Figure 1). For instance, Trichinella spiralis adult and muscle larva-derived ESPs activated bone marrowderived dendritic cells enabled naïve mice to acquire Treg cells differentiation (Sun et al., 2019), suggesting that T. spiralis ESPs might stimulate differentiation of the host’s Treg cells via dendritic cells activation favoring parasite survival inside the host. Helminths residing in the human gut induce tolerogenic DCs, alternatively activated macrophages, and Treg cells, which produce suppressor cytokines that help keep inﬂammatory T cells and their effector molecules in check (Loukas et al., 2016). Helminths-induced type 2 immune response causes profound changes in the host tissue physiology, hyperplasia of goblet cells (Miller and Nawa, 1979), and hypercontractility of smooth muscles (Castro et al., 1976). Additionally, the innate lymphoid cell (ILC) family and T and B lymphocytes are involved in host defence, barrier integrity, and tissue homeostasis and repair (Artis and Spits, 2015). Alarmins such as thymic stromal lymphopoietin (TSLP), IL-33, and IL-25 released by tuft cells (Figure 1) and other epithelial cells following tissue damage act on granulocytes (mast cells, basophils, and eosinophils) and tissue-resident group 2 ILCs (ILCs-2), consequently enhancing the production of type 2 cytokines, including IL-13 (Grencis, 2015; von Moltke et al., 2016). As helminths (both nematodes and platyhelminths) transition from their free-living stage to parasitism, they require long-lasting niches, including intermediate hosts, to complete their life cycle (Hotez et al., 2008). In the course of their journey, they either secrete or excrete many molecules that help them enter their host, invade tissues, shield them from the host’s immune assault and thus, establish chronic infections 03 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 FIGURE 1 Parasite–host interaction: manipulation of the host immune system by helminth excretory/secretory products (ESPs). Helminth infection together with additional events and signals (such as worm ESPs, microbiota metabolites, and alarmins from tissue damage) initiate processes including the differentiation of Th2 cells, Treg expansion and responses, macrophage polarisation, and mucus production. DCs respond to various stimuli (alarmins, ESPs, and metabolites) and promote development of Treg, Breg, and Th2 cells, and inhibit Th1 and Th17 cell responses. Additionally, alarmins (TSLP, IL-33, IL-25) released by tuft cells and other epithelial cells following tissue damage, act on granulocytes (mast cells, basophils, and eosinophils) and ILC2s, consequently enhancing the production of type 2 cytokines. Gut sensory neurons also inﬂuence this immune network upon receiving signals from helminths and microbes by releasing neuropeptides (NMU- neuropeptide neuromedin U and CGRP-calcitonin gene-related peptide) which directly effects the ILC2 responses. APR, aspartic protease; Breg, regulatory B cell; CGRP, calcitonin gene-related protein; DC, dendritic cell; ESP, excretory/secretory product; GST, glutathione-S-transferase; ILC2, type 2 innate lymphoid cell; NMU, neuromedin U; serpin, serine protease inhibitor; Th2, T helper type 2, TLSP, thymic stromal lymphopoietin; Treg, regulatory T cell. peroxide (Kumagai et al., 2009). Surface-bound enolases of schistosomula in both S. japonicum and S. mansoni are associated with a ﬁbrinolytic activity which prevents clot formation in their vicinity (Figueiredo et al., 2015). Glycoproteins such as alpha-1 (also known as IL-4 inducing principle of S. mansoni eggs, IPSE) and omega-1 are known to involve in skewing T cell response in granulomas (Th2 type response) while migrating through the intestine towards the lumen (Everts et al., 2009; Haeberlein et al., 2017). The interaction mechanism with host tissues is complex and hard to dissect at the molecular level, partly due to the lack of suitable animal models (Loukas and Prociv, 2001), and the roles of many bigger molecules, particularly proteins, are yet to be understood. This review highlights known or potential roles of SM either secreted and/or excreted by various helminths in their life cycle (reviewed elsewhere (Maizels et al., 1993; Maizels and Yazdanbakhsh, 2003; Dzik, 2006; Maizels and McSorley, 2016; Ryan et al., 2020)). For instance, skin is the ﬁrst immune barrier Schistosoma mansoni has to pass through. Proteolytic factors, including various serine proteases, are reported to be responsible for skin penetration (Marikovsky et al., 1988; Chavez-Olortegu et al., 1992). Among many serine proteases, the 28/30kDa variant called SmCE (S. mansoni cercarial Elastase) is considered most crucial as inhibition (by inhibitor succinylalanyl-alanyl-prolyl-phenylalanine chloromethyl ketone, AAPFCMK) of this protease reduces skin penetration up to 80% (Salter et al., 2000). After successful skin penetration, one of the mechanisms that favor the survival of lung stage schistosomula is using their surface-associated peroxiredoxin 1 to overcome oxidative stress scavenging surrounding hydrogen Frontiers in Parasitology 04 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 macrophage-mediated change in the gut microbiome (Durack and Lynch, 2019; Su et al., 2020). For instance, targeted removal of the bacterial family Enterobacteriaceae reduced inﬂammation in mice (Zhu et al., 2018). Reviews done by Brussow (2019) (Brussow, 2020), Kupritz et al. (2021) (Kupritz et al., 2021), and Durack and Lynch (2019) have more detailed updates on the helminth-induced human gut microbiome composition and their beneﬁts and therapeutic potential. The human gut microbiome is also said to be inﬂuenced by deworming (the clearance effect) and anthelmintic drugs (the treatment effect) (Kupritz et al., 2021). For instance, a study conducted in a rural Kenyan village observed that the clearance of STHs (e.g., A. lumbricoides and N. americanus) using albendazole therapy led to a signiﬁcant increase in Clostridials and reduction in Enterobacteriales in the gut microbiome of participants when compared with their age-matched controls (Easton et al., 2019). A similar ﬁnding was also reported from the cross-sectional studies between Indonesia and Liberia, where both countries showed a signiﬁcant association of 12 bacterial taxa with STH infections, including Olsenella (associated with reduced gut inﬂammation), which decreases in numbers after deworming (Rosa et al., 2018). It is difﬁcult to discriminate the inﬂuence between the clearance and treatment effect, and further, the change in microbiome composition is mostly signiﬁcant only in treated subjects who remain infected after the treatment (Martin et al., 2018; Martin et al., 2019). Perturbations in the gut microbiome (e.g., the imbalance between harmful and protective bacteria) or dysbiosis characterised by, for instance, reduced diversity of short-chain fatty acids (SCFAs) producers and enriched proinﬂammatory microbes (e.g., H2S producers) is said to be responsible for the pathogenesis of IBD, and it is reviewed elsewhere (Kaur et al., 2011; Sultan et al., 2021). For example, the lack of certain symbiotic species (e.g., Faecalibacterium prausnitzii) is said to be responsible for the recurrence of Crohn’s disease as mice supplemented with this organism experienced reduced inﬂammation after inducing colitis chemically (Sokol et al., 2008). A reduced amount of F. prausnitzii is also linked to relapse of Ulcerative colitis (Werkman et al., 2018). However, the causal effect of dysbiosis on IBD and other diseases is still inconclusive, as dysbiosis is even observed in the fecal samples from healthy relatives of IBD patients (Van de Merwe et al., 1988). There is no cure for IMIDs, and some patients, in desperation and inspired by the above theories, have turned to alternative treatments such as helminth therapy by deliberately self-infecting with live helminths. Only two helminth species, pig whipworm Trichuris suis ova (TSO) and the human hookworm N. americanus are used for human helminth therapy. In recent years, helminth therapy has been assessed in early phase clinical trials using TSO and N. americanus infective larvae against inﬂammatory, autoimmune, and allergic diseases (Summers, 2003; Croese et al., 2006; Blount et al., 2009), yielding both positive and negative outcomes. IBD patients (both Crohn’s stages, including while interacting with their respective hosts (Table 1). Octadecanoic acid secreted by the infective third-stage larvae (L3) of Nippostrongylus brasiliensis (a model organism for human hookworms) is known to help to lyse the host’s red blood cells, and similarly, in Haemonchus contortus (Ward, 1982; Yeshi et al., 2020). Another small molecule, succinic acid, identiﬁed from the ESP of adult N. brasiliensis, can induce intestinal tuft cells to initiate Th2 responses (Wangchuk et al., 2019). Succinic acid is thought to be energy source for helminths during adaptations toward anaerobiosis (Saz, 1981). Succinic acid is also detected as a dominant metabolite from the ESPs of adultstage Trichuris muris (a model organism for the human-dwelling T. trichiura) (Wangchuk et al., 2019). Old friends/hygiene hypothesis: evidence from helminth therapy From an evolutionary viewpoint, the coevolution of humans and helminths may have shaped our metabolism and organ functions (Stilling et al., 2014). Infection with our ‘old friends’ – helminths – is believed to have shaped our immune system to facilitate co-existence (Rook et al., 2014; Versini et al., 2015; Longman and Littman, 2015; Filyk and Osborne, 2016), and there is evidence that helminths can protect us from a broad spectrum of immune-mediated inﬂammatory diseases (IMIDs). These changes to the immune system have often beneﬁted hosts by reducing overall inﬂammation. Moreover, as per the hygiene hypothesis (ﬁrst proposed by Strachan) (Strachan, 1989), reduced contact with infectious agents (particularly those that co-evolved with us) due to improved sanitation, lifestyle changes (including dietary habits) are attributed to the alarming rise in IMIDs, including metabolic syndrome (e.g., obesity, insulin resistance, hypertension, and atherogenic dyslipidemia) (Grundy et al., 2005; Huang, 2009; Rook et al., 2014). Several studies have reported that helminths promote the host’s gut health/inﬂuence microbiome composition (evident from their fecal microbiome) (Lee et al., 2014; Jenkins et al., 2017; Jenkins et al., 2018; Rosa et al., 2018; Schneeberger et al., 2018; Gordon et al., 2020; Mejia et al., 2020), including increased alpha diversity (multispecies-helminth infected individuals with greater diversity than single species-infected individuals). The inﬂuence on microbiome composition is found to be speciﬁc to helminth species and the host’s anatomical niche (e.g., observed strong inﬂuence by helminths in the large intestine such as Enterobius vermicularis and Trichuris trichiura). Studies have also shown that infection with various helminth species can reduce the severity of diseases, including chemically-induced colitis (Fox et al., 2000; Khan et al., 2002; Elliott et al., 2004; Moreels et al., 2004; Hunter et al., 2005) and high fat dietinduced obesity (Su et al., 2018; Su et al., 2020), and such protection is partially attributed to Th2-dependent, M2 Frontiers in Parasitology 05 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 TABLE 1 Reported biological/immunomodulatory properties of MSI level 1 conﬁrmed small molecules from helminths. Small molecules Chemical classes* Helminth species in which SM were detected Reported biological/immunomodulatory properties Acetate Carboxylic acid and A. caninum, N. brasiliensis, derivatives T. muris, T. canis, A. lumbricoides Anti-inﬂammatory (Tedelind et al., 2007) Adenine Imidazopyrimidines N. brasiliensis, N. americanus, T. muris Anti-inﬂammatory (Fukuda et al., 2017) Adenosine Purine nucleosides N. brasiliensis, N. americanus, T. muris Anti-inﬂammatory (Yang et al., 2006) Adenosine 5’monophosphate Purine nucleotides N. brasiliensis, N. americanus, T. muris Anti-inﬂammatory (Sag et al., 2008; Yamaguchi et al., 2014) Arachidonic acid Fatty acyls N. brasiliensis, T. muris, D. caninum, T. canis It regulates blood clotting, including platelet aggregation; helps in smooth muscle contraction, leukocyte chemotaxis, inﬂammatory cytokine production and immune function (Wishart et al., 2018) N. brasiliensis, T. muris Azelaic acid Fatty acyls Betaine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris Neuroprotective (Singhal et al., 2020); improves intestinal barrier function (Wu et al., 2020); hepatoprotective (Khodayar et al., 2020); anti-inﬂammatory (Detopoulou et al., 2008) Anti-inﬂammatory (Mastrofrancesco et al., 2010) Butyrate Faty acyls A. caninum, N. brasiliensis, T. muris, T. canis, A. lumbricoides Anti-inﬂammatory (Andoh et al., 1999; Kovarik et al., 2011) Choline Organonitrogen compounds N. brasiliensis, N. americanus, T. muris Anti-inﬂammatory (Detopoulou et al., 2008) Citric acid Carboxylic acid and N. brasiliensis, T. muris, A. derivatives caninum, D. caninum, T. canis Cardioprotective, antimicrobial (Tang et al., 2013). Deoxyadenosine Purine nucleosides N. brasiliensis, T. muris Cell growth inhibitor and cytotoxic (Ullman et al., 1978) Docosahexaenoic acid Faty acyls N. brasiliensis, T. muris, D. caninum, T. canis Anti-inﬂammatory (Kuda, 2017); cardioprotective (Wishart et al., 2009) Fructose Organooxygen compounds T. canis, T. muris, D. caninum. Associated with hypertension and hepatic disorders (Wishart et al., 2018); pro-inﬂammatory (Glushakova et al., 2008). Glucosamine Organooxygen compounds N. brasiliensis, T. muris, T. canis Used in osteoarthritis treatment (Largo et al., 2003); anti-inﬂammatory (Wishart et al., 2018). Glycerol Organooxygen compounds N. brasiliensis, T. muris, A. caninum, D. caninum, T. canis Anti-inﬂammatory (Szel et al., 2015) g-aminobutyric acid Carboxylic acid and A. caninum, D. caninum, T. derivatives canis Antidiabetic, regulate blood pressure (Wishart et al., 2009); anti-inﬂammatory (wound-healing agent) (Han et al., 2007) Homogentisate Benzene and substituted derivatives Pro-inﬂammatory (Hegedus and Nayak, 1994; Spreaﬁco et al., 2013) Hypoxanthine Imidazopyrimidines N. brasiliensis, N. americanus, T. muris Inosine Purine nucleosides N. brasiliensis, N. americanus, T. muris Anti-inﬂammatory (Hasko et al., 2000; Liaudet et al., 2001) Lactic acid Hydroxy acids and derivatives N. brasiliensis, T. canis, A. caninum, D. caninum. Anti-inﬂammatory and immunosuppressant (Hearps et al., 2017) Lauric acid (dodecanoic acid) Fatty acyls N. brasiliensis, T. muris, A. caninum, D. caninum, T. canis Anti-inﬂammatory, antibacterial (Huang et al., 2014); Facilitate interaction with their hosts (Ferreira et al., 2014) Linoleic acid Fatty acyls N. brasiliensis, T. muris, A. caninum, D. caninum, T. canis Anti-inﬂammatory (Rodrigues et al., 2012); Helps parasites to migrate successfully inside their host (Giera et al., 2018) L-Arginine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris Anti-inﬂammatory (Hnia et al., 2008; Coburn et al., 2012; Wu et al., 2016) L-Alanine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris, A. Anti-inﬂammatory (Naügeli et al., 2014; Raizel et al., 2016; Coqueiro et al., 2017); dissolves kidney stones in experimental animals (Wishart et al., 2009) N. brasiliensis, T. muris Anti-inﬂammatory (Lee et al., 2018) (Continued) Frontiers in Parasitology 06 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 Continued Small molecules Chemical classes* Helminth species in which SM were detected Reported biological/immunomodulatory properties caninum, D. caninum, T. canis L-Aspartate Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris, A. caninum, D. caninum. Anti-inﬂammatory and neuroprotective (Boccella et al., 2015; Afraei et al., 2017); excitatory neurotransmitter (Wishart et al., 2009) L-Carnitine Organonitrogen compounds Anti-inﬂammatory (Lee et al., 2015) and antioxidant (Keskin et al., 2015) L-Citrulline Carboxylic acid and N. brasiliensis, T. muris, N. derivatives americanus Anti-inﬂammatory and antioxidant (Ham et al., 2015; Lee et al., 2018; Darabi et al., 2019) L-Glutamate Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris Antioxidant (Jiao et al., 2015) L-Glutamine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris Anti-inﬂammatory (Naügeli et al., 2014; Raizel et al., 2016; Coqueiro et al., 2017); maintains gut barrier function and reduces the symptoms of IBS (irritable bowel syndrome) (Wishart et al., 2009) L-Histidine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris, A. caninum Anti-inﬂammatory (Hasegawa et al., 2012) L-Leucine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris Analgesic and anti-inﬂammatory (Saxena et al., 1984; Kato et al., 2016) L-Lysine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris, A. caninum Anti-inﬂammatory (He et al., 2018; Han et al., 2018) L-Methionine Carboxylic acid and N. brasiliensis, N. americanus, T. muris, A. derivatives caninum, D. caninum, T. canis Anti-inﬂammatory (Unnikrishnan and Rao, 1990) and antioxidant (Derakhshanfar et al., 2009) L-Phenylalanine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris, A. caninum, D. caninum, T. canis Antidiabetic (Alamshah et al., 2017) L-Proline Carboxylic acid and N. brasiliensis, T. muris, N. derivatives americanus, A. caninum Anti-inﬂammatory (Faure et al., 2006) L-Serine Carboxylic acid and N. brasiliensis, T. muris, N. derivatives americanus, A. caninum Modulates adaptive immunity by controlling T cell proliferative capacity (Ma et al., 2017); colon protection and mucosal healing (Faure et al., 2006) L-Threonine Carboxylic acid and N. brasiliensis, T. muris, N. derivatives americanus, A. caninum Anti-inﬂammatory (Dong et al., 2017; Gaifem et al., 2018) L-Tryptophan Indoles and derivatives Anti-inﬂammatory (Mine and Zhang, 2015; Yue et al., 2017; Islam et al., 2017) L-Valine Carboxylic acid and N. brasiliensis, T. muris, N. derivatives americanus, A. caninum Anti-inﬂammatory (Mine and Zhang, 2015) Maleic acid Carboxylic acid and N. brasiliensis, T. muris derivatives Inﬂammatory/Cytotoxic (Andersen, 2007) Malic acid Hydroxy acids and derivatives N. brasiliensis, T. muris, A. caninum, D. caninum, T. canis Anti-inﬂammatory and anti-aggregant (Tang et al., 2013). Mannitol Organooxygen compounds N. brasiliensis, N. americanus, T. muris, A. caninum, D. caninum, T. canis Anti-edema and diuretic (Cavone et al., 2012). Methionine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris, A. caninum, D. caninum, T. canis N. brasiliensis, N. americanus, T. muris N. brasiliensis, T. muris, N. americanus, A. caninum, T. canis Anti-inﬂammatory (Unnikrishnan and Rao, 1990); antioxidant (Derakhshanfar et al., 2009); associated with intellectual disability in infants, delays motor skills, sluggishness, liver problems and muscle weakness (Wishart et al., 2009) (Continued) Frontiers in Parasitology 07 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 Continued Small molecules myo-inositol Chemical classes* Organooxygen compounds N-Acetylputrescine Carboximidic acids and derivatives Helminth species in which SM were detected Reported biological/immunomodulatory properties N. brasiliensis, T. muris, A. caninum, D. caninum, T. canis Useful for treating polycystic ovary syndrome (Wishart et al., 2009) N. brasiliensis, N. americanus, T. muris Lung cancer biomarker (Liu et al., 2017) N6,N6,N6Trimethyl-L-lysine Carboxylic acid and N. brasiliensis, T. muris, N. derivatives americanus Cardiovascular disease biomarker (Li et al., 2018) Oleic acid Fatty acyls N. brasiliensis, T. muris Anti-inﬂammatory (Rodrigues et al., 2012) Palmitic acid Fatty acyls N. brasiliensis, T. muris, A. caninum, D. caninum, T. canis Anti-inﬂammatory (Aparna et al., 2012) Propionate Carboxylic acid and A. caninum, N. brasiliensis, derivatives T. muris, T. canis, A. lumbricoides Anti-inﬂammatory (Tedelind et al., 2007) Pterin Pteridines and derivatives N. brasiliensis Biomarker of exercise-induced stress (Lindsay and Gieseg, 2020) Scyllo-inositol Organooxygen compounds D. caninum, T. canis Neuroprotective (Wishart et al., 2009) Sorbitol Organooxygen compounds N. brasiliensis, T. canis, A. caninum, D. caninum. Laxatives and irrigating solution in surgeries (Wishart et al., 2009) N. brasiliensis, T. muris, A. caninum, D. caninum, T. canis Anti-inﬂammatory (Ahmad et al., 2015); anticancer and cardioprotective (Chajes et al., 1999; Kris-Etherton et al., 2005); Alters physical properties of the hosts’ red blood cells membrane and causes lysis (Ward, 1982; Yeshi et al., 2020) Fatty acyls Stearic acid (octadecanoic acid) Succinate Carboxylic acid and N. brasiliensis, N. americanus, T. muris, A. derivatives caninum, D. caninum, T. canis Anti-inﬂammatory (Littlewood-Evans et al., 2016; Lei et al., 2018); Induces intestinal tuft cells to initiate Th2 response (Wangchuk et al., 2019) Talose Lactones Cell growth inhibitor (C. elegans) (Sakoguchi et al., 2016) Tyrosine Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris, A. caninum, D. caninum, T. canis Neuroprotective (Wishart et al., 2018). Uridine Pyrimidine nucleosides T. muris, A. caninum Anti-inﬂammatory (Evaldsson et al., 2007) Urocanate Azoles N. brasiliensis, N. americanus, T. muris Chemoattractant (Safer et al., 2007) Xanthine Imidazopyrimidines N. brasiliensis, N. americanus, T. muris Proinﬂammatory (Joshi et al., 2018) 2-Oxoglutarate Keto acids and derivatives N. brasiliensis, N. americanus, T. muris Anti-inﬂammatory (He et al., 2017) and antioxidant (Velvizhi et al., 2002) 4Hydroxybenzoate Benzene and substituted derivatives N. brasiliensis, N. americanus, T. muris Neuroprotective (Winter et al., 2017) 5-Aminolevulinate Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris Anti-inﬂammatory (Fujino et al., 2016) 5-Oxoproline Carboxylic acid and N. brasiliensis, N. derivatives americanus, T. muris, A. caninum, D. caninum, T. canis Neuroprotective (Largo et al., 2003; Pederzolli et al., 2010) N. brasiliensis, T. canis, A. caninum, D. caninum. 2,5Carboxylic acids Dihydroxybenzoate N. brasiliensis Anticancer activity (Kalinowska et al., 2018) 3’,5’-Cyclic AMP N. brasiliensis, N. americanus, T. muris Anti-inﬂammatory (Oldenburger et al., 2012) Purine nucleotides MSI-level-1 conﬁrmed small molecules (SM) are those metabolites/compounds whose structure is fully validated with reference standards based on mass spectrometry, tandem mass spectrometry, and retention time matching. *Chemical classes as described in HMDB and PubMed. Frontiers in Parasitology 08 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 (Kradin et al., 2006). Furthermore, the pathogenicity of Campylobacter jejuni may increase in patients receiving T. suis ova treatment (Shin et al., 2004). These complications and challenges necessitate the investigation of safer molecules to develop alternative therapeutics. In pursuit of a more palatable treatment approach, recent focus has been on ESPs of helminths, constantly secreted by adult worms into the tissues of infected hosts in their various ﬁnal niches (Loukas et al., 2016; Eichenberger et al., 2018; Wangchuk et al., 2019). Studies have identiﬁed several recombinant ESP proteins that can protect mice against inﬂammation and weight loss in two different mouse models of colitis – the T cell-dependent TNBS (2,4,6-Trinitrobenzene sulfonic acid) model (Ruyssers et al., 2008; Buitrago et al., 2021) and the T cell-independent DSS (dextran sodium sulfate) model (Ferreira et al., 2013; Ryan et al., 2020; Wang et al., 2020). Trichinella spiralis 53 kDa glycoprotein (Tsp53) is antiinﬂammatory (induces production of IL-4 and IL-10 in T cells) (Cvetkovic et al., 2016), and its recombinant form rTsp53 confer protection in TNBS-induced colitis in mice (Du e t al., 2011). Moreover, rTsp53 can downregulate proinﬂammatory cytokines and increase IL-10 and TGF-b1 levels via inducing macrophage polarisation (M1 to M2) (Chen et al., 2016). Other T. spiralis-derived molecules, including cystatin-like protein (Ts-CLP) (Liu et al., 2014; Tang et al., 2015), calreticulin (Ts-CRT) (Zhao et al., 2017), cystatin (TsCstN) (Kobpornchai et al., 2020), glutathione-Stransferase (TsGST) (Jin et al., 2019), and paramyosin (TsPmy) (Sun et al., 2015; Guo et al., 2016) are known to possess anti-inﬂammatory and immunomodulatory properties. The main immunomodulatory mechanism shown by these proteins is upregulating Treg cells to produce more IL-10 and TGF-b, thus inhibiting Th1 and Th17-mediated inﬂammation. disease and ulcerative colitis), when given TSO (single dose) in open-label phase I trials, not only tolerated the treatment but also achieved remission, and beneﬁts were maintained through repeated doses (Summers, 2003; Summers et al., 2005). However, in a double-blind, placebo-controlled phase II trial of TSO in adults with active Crohn’s disease (n = 252), ingestion of 2507500 TSO fortnightly over 12 weeks was observed to be safe without any adverse effects, but the treatment did not have an advantage over the placebo in inducing clinical remission and other immunological responses (Scholmerich et al., 2017). Moreover, the study failed to reach its clinical endpoint for both active Crohn’s disease (Scholmerich et al., 2017) and multiple sclerosis (Voldsgaard et al., 2015; Fleming et al., 2019). A meta-analysis on TSO therapy trials (Huang et al., 2018) found that the treatment did not show statistically signiﬁcant beneﬁts for IBD and suggested the need for further studies with larger sample sizes. One of the challenges of using TSO is that frequent dosing is required for human trials as T. suis is not adapted to develop in humans and is rapidly expelled. On the contrary, N. americanus is a human hookworm, and it can survive within the human body for years. Crohn’s disease patients (a few already in remission before the trial started) tolerated a low dose of N. americanus larvae during a phase I open label trial, where larvae were administered percutaneously (Croese et al., 2006). Even at a lower dose (initial 10 larvae, followed by a booster dose of 5 larvae at week 12), N. americanus larvae was partially effective over placebo when tested in 20 HLA-DQ2+ celiac disease patients (autoimmune response to dietary gluten) followed by incremental wheat challenge after 20 weeks. Patients not only tolerated the treatment, but their duodenal biopsies showed reduced inﬂammatory cytokines such as IFN-g and IL-17 (McSorley et al., 2011); however, gluten challenge did not yield any difference in the clinical parameters between the treated and placebo groups (Daveson et al., 2011). In a recent larger randomised controlled trial with the increasing exposure of celiac disease patients to gluten (Croese et al., 2020), N. americanus larvae could not restore tolerance to sustained moderate gluten consumption (2 g/day), but hookworm-infected patients did experience reduced symptoms when gluten was given intermittently at a lower dose. In summary, there are numerous clinical trials involving live helminths, but they remain inconclusive (Elliott and Weinstock, 2017; Ryan et al., 2020). Such drawbacks could be due to difﬁculty controlling the quality and innocuity of the infectious agent, low doses of worms that are insufﬁcient to achieve required immunomodulation, logistical issues with scale-up, patient compliance to the treatments, and inability to deﬁne good manufacturing standards. Moreover, persistent infection can cause various health problems, including anaemia, malnutrition, and cognitive retardation (Albonico et al., 2008), and can sometimes be fatal. For instance, Kradin et al. (2006) found sexually mature male trichurid in the cecum of pediatric Crohn’s disease patients treated with T. suis ova Frontiers in Parasitology Reported biological/ immunomodulatory properties of helminth-derived small molecules Due to their pathogenic and infectious nature, parasitic helminths can produce a wide array of immunomodulatory molecules, including proteins, glycans, nucleic acids, lipids, and SM (Maizels et al., 2018; Kahl et al., 2018). Wangchuk et al. (Wangchuk et al., 2019) reported that crude ESPs from A. caninum could suppress colitis pathology in a mouse model and further inhibit the secretion of inﬂammatory cytokines in primary human leukocytes. Ferreira et al. (Ferreira et al., 2013) also reported a similar reduced inﬂammatory response after administration of A. caninum crude ESPs in a murine colitis model by inducing a type 2 cytokine response. Therefore, we have reviewed bioactivities associated with metabolites (both polar and non-polar) identiﬁed/isolated from helminths, 09 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 derived lipopolysaccharide)-challenged weaned pigs (Zhu et al., 2013). Moreover, this amino acid was reported from infective stages of N. americanus, N. brasiliensis, and T. muris (Yeshi et al., 2020; Wangchuk et al., 2021). Lipid utilization by helminths varies as they develop from free-living to the parasitic stage, and considerable changes occur, including lipid abundance and composition (Wang et al., 2020) (e.g., decreased synthesis of triradylglycerols but increased glycerophospholipids (Phosphatidylcholine, PC and phosphatidylethanolamine, PE in Haemonchus contortus)) (Wang et al., 2018), energy metabolism (Harder, 2016; Mangmee et al., 2020) and membrane fatty acids (FAs) composition (Proudfoot et al., 1990). For instance, the freeliving stage requires more unsaturated FAs in the cell membrane to protect against the effects of low environmental temperatures, but the demand for FAs would be less as they develop to the parasitic stage (Hazel and Williams, 1990; Wang et al., 2018; Mangmee et al., 2020; Wang et al., 2020). Moreover, unlike the free-living infective stage, adults (parasitic stage) metabolise exogenous substrate, relying less on endogenous food stores (Barrett, 1968). In that way, adults use only a few speciﬁc FAs and fat-soluble vitamins as energy (Tielens, 1997; Dalton et al., 2003), and many lipids are eventually stored and excreted (Cheng, 1986) or become food for gametes (Barrett, 1968). We assumed that SCFAs such as propionate and acetate in the ESPs of adult A. caninum, N. brasiliensis, and T. muris might be stored as food reserves for their gametes. Fatty acids also play an essential role in diverse biological processes, and most importantly, FAs such as the cis-form of octadecanoic acid including literature describing metabolites’ role in the development of diagnostics/biomarkers and therapeutics. Polar metabolites, including carbohydrates, amino acids, nucleic acids, and organic acids, are hydrophilic. While non-polar metabolites, mainly lipids, are hydrophobic. From the literature review of helminth-derived SM reported so far, about 65 were associated with various biological activities such as anti-inﬂammatory and antioxidant, anti-microbial, anticancer, anti-diabetic, cardio-, hepato-, and neuroprotective, and maintaining gut-barrier integrity (Table 1). These 65 helminth-derived compounds were identiﬁed using metabolomics standard initiative level I identiﬁcation protocols, the highest identiﬁcation level achieved using standards. Most of these compounds (45 metabolites) are associated with antiinﬂammatory and antioxidant properties, suggesting roles in active immune regulation (Figure 2). For example, major metabolites detected in the infective stages of T. muris and N. brasiliensis, such as adenine, adenosine, betaine, and choline, possess anti-inﬂammatory properties (see Table 1). Betaine and other amino acids such as glutamine and serine are known to help maintain colon homeostasis (Faure et al., 2006; Wishart et al., 2009; Wu et al., 2020). The amino acid L-serine produced by N. americanus, N. brasiliensis, A. caninum, and T. muris has been shown to modulate adaptive immunity by controlling T cell proliferative capacity (Ma et al., 2017) and is involved in colon protection and mucosal healing (Faure et al., 2006). L-arginine in intestinal ischemia animal models aids mucosal healing by reducing tissue damage (Fotiadis et al., 2016), and a similar protective effect of L-arginine was also reported in LPS (bacteria- FIGURE 2 Number of small molecules identiﬁed in helminths with reported biological activities from the literature. Others include analgesics, laxatives, anti-aggregant, immuno-modulatory, cell-growth inhibitors, regulate blood clotting, treatment of osteoarthritis, polycystic ovary syndrome, and disease biomarkers. Frontiers in Parasitology 10 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 succinate (one of the metabolites secreted/excreted by a few helminth species) given ad libitum in the drinking water could induce a Th2 immune response in the mouse intestine through the tuft cell-ILC2 circuit, which could activate its receptor SUCNR1 to initiate succinate-induced IL-13 production, which is responsible for initiating Th2 responses. When Giera et al. (Giera et al., 2018) analyzed the lipid composition of different life cycle stages of S. mansoni, eggs were found enriched with prostaglandins, while the cercaria stage contains mainly resolvins. However, Salafsky et al. (Salafsky et al., 1984) proposed that prostaglandins released by cercariae into the skin after initiating penetration upon stimulation by host skin essential fatty acids might further promote penetration via releasing histamine. Resolvins are well known for their antiinﬂammatory properties (reviewed by Moro et al. (Moro et al., 2016)) as resolvin E1 protects liver ﬁbrosis in mice (pathogenfree Kunming mice) infected with S. japonicum (Qiu et al., 2014). These ﬁndings suggest that prostaglandins and resolvins might be involved in the host immune adjustment. (stearic acid), monoenoic acids (oleic acid and vaccenic acid), and other branched-chain acids are known to help in breaking through the host cell membrane (Ward, 1982). Probably, the presence of stearic acid and oleic acid in the ESPs or somatic tissues of various helminth species, including Brugia malayi, Dipylidium caninum, Dictyocaulus viviparus, N. brasiliensis, Strongyloides ratti, Toxocara canis, and T. muris (Smith et al., 1996; Ferreira et al., 2014; Becker et al., 2017; Wangchuk et al., 2019; Wangchuk et al., 2019; Wangchuk et al., 2020; Yeshi et al., 2020) suggest that these worms might be producing compounds that could help them in invasion and establishing infection in their respective host(s) (Yeshi et al., 2020). Lipids are also known to have important functions in signalling transition. Endogenous steroidal hormones, such as dafachronic acid, have been demonstrated to play essential roles in regulating larval development in parasitic nematodes (e.g., in Ancylostoma ceylanicum, Strongyloides stercolis, and Haemonchus contortus) (Wang et al., 2009; Stoltzfus et al., 2012; Zhi et al., 2012; Albarqi et al., 2016; Ma et al., 2019). Other key nematode species, including Ascaris suum and Toxocara canis also produce dafachronic acid (Ma et al., 2019). Zaiss et al. (2015) reported the immunoregulatory role of an increase in the concentration of bacterial-derived SCFAs induced by the Heligmosomoides polygyrus bakeri infection in mice with allergic inﬂammation (Zaiss et al., 2015). The same study also observed the increased SCFAs in the host-parasite setting by analysing colon contents of pigs infected with Ascaris suum eggs and stools from human volunteers infected with N. americanus infective larvae (Zaiss et al., 2015). Thus, the presence of SCFAs like propionate and acetate in A. caninum, N. brasiliensis, T. muris, T. canis, and A. lumbricoides might have immuno-modulatory roles in their host. Short-chain fatty acids derived from other species (not worms) also possess antiinﬂammatory properties (Andoh et al., 1999; Tedelind et al., 2007; Kovarik et al., 2011), and they are known to promote gut health. Phosphatidylcholines (PCs), reported as major lipids in S. mansoni (Ferreira et al., 2015), are also said to have similar functions. For instance, a glycoprotein ES-62 secreted by Acanthocheilonema viteae can protect against rheumatoid arthritis, systemic lupus erythematosus, and airway hyperresponsiveness is mainly attributed to the covalently-bonded phosphorylcholine moieties (Pineda et al., 2014; Pineda et al., 2015). However, ES-62 did not protect against autoimmune diseases like type I diabetes, multiple sclerosis, and IBD (Doonan et al., 2018). Some schistosome-derived PCs are also responsible for pathogenesis. For instance, when toll-like receptor-2 knockout (TLR2 −/−) mice were given S. mansoni-derived lysophosphatidylcholine (lyso-PC), it induced eosinophil recruitment and cytokine production in a TLR2-dependent manner, suggesting that this mechanism might contribute to the pathogenesis and lethality, particularly during the chronic phase of schistosome infection (Magalhaes et al., 2010). Nadjsombati et al. (Nadjsombati et al., 2018) showed that Frontiers in Parasitology Potential application of small biomarker molecules in diagnostics Currently, there is no 100% sensitive diagnostic tool for detecting STHs infections. The WHO-recommended gold standard microscopy-based techniques (including Kato-Katz thick smear, McMaster, and ﬂotation, which enable to count/ detect parasite eggs in a fecal sample) (Khurana et al., 2021) are not adequately sensitive in low-endemic and near-elimination areas (McCarthy et al., 2012; Werkman et al., 2018). Moreover, diagnostic assays commonly used for detecting many STHs infections are considered invasive and risky as they involve collecting samples such as body ﬂuids (e.g., stool and blood) and tissues through biopsy, requiring both special equipment and specialist, which is often a big challenge in developing countries. There has been continued effort toward developing more sensitive, low-cost, and easy-to-operate molecular diagnostics, which can be used in both endemic and lowendemicity settings, and this topic is reviewed elsewhere (Ullah et al., 2020; Khurana et al., 2021). Recent ﬁndings about the immunomodulatory roles of helminth-derived ESPs, including proteins, microRNAs, and extracellular vesicles (Hoy et al., 2014; Kammoun-Rebai et al., 2016; Eichenberger et al., 2018; Bi et al., 2021), is a paradigm shift in diagnosis/prognosis of helminthiasis and more about proteins/protein microarrays, microRNAs, and extracellular vesicles are available in these reviews (Sotillo et al., 2020; Li et al., 2021; Mu et al., 2021). An alternative potential area of diagnostic development is small biomarker molecules. Recent advances in metabolomics techniques and technologies have enabled the identiﬁcation of small biomarker molecules of infections caused by helminths. 11 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 metabolites (e.g., N-Glycoloylganglioside, adrenochrome, 3succinoylpyridine, 1-Nitro-5,6-dihydroxy-dihydronaphthalene) from individuals with urogenital schistosomiasis (caused by S. haematobium) associated with bladder pathology and are worth further analysis (including MSI level 1 identiﬁcation) to be used as potential biomarkers. Oxidized pterins (e.g., neopterin) are used as urine markers (e.g., for hyperphenylalaninemia) (Longo, 2009). Pterin was detected in the ESPs of the infective L3 of N. brasiliensis (Yeshi et al., 2020). Considering these examples of potential small biomarker molecules and those mentioned in Table 2, investigating other neglected helminthiases may lead to discovering more unique biomarkers to better understand the helminth infections. Comparative analysis of metabolites proﬁles in samples obtained from infected and non-infected individuals/animals can be used to identify diagnostic biomarkers. For instance, Denery et al. (2010) (Denery et al., 2010) identiﬁed 14 potential candidate metabolites biomarkers, including long-chain fatty acids hexacosenoic acid and pentacosenoic acid in the plasma and serum obtained from onchocerciasis positive human volunteers from African countries. Another example of a urine biomarker is 2-methyl pentanoyl carnitine (2-MPC), and 2-MPC could be used as A. lumbricoides-speciﬁc urine biomarker (Table 2) since it gives high accuracy for detecting both infection and infection intensity (Lagatie et al., 2020). Moreover, the 2-MPC level in urine decreased signiﬁcantly when infected individuals were given a single albendazole dose (400 mg) (Lagatie et al., 2020). Due to challenges in biomarkers from human host-parasite interactions, focus has been diverted to study this phenomenon in animal models. There are variations in the metabolite proﬁles in the samples obtained from animals infected with different helminth species (Table 2). For example, urine samples from S. mansoni-infected mice showed decreased levels of SCFAs such as acetate, butyrate, and propionate (Wang et al., 2004) but urine samples from S. japonicum-infected and S. japonicum and N. americanus co-infected hamster had increased levels of acetate and butyrate, and reduction in propionate (Wang et al., 2006; Wu et al., 2010) (Table 2). Such ﬂuctuations in the SM proﬁle could be due to the type of helminth species used for infection or duration of infection and animal models used. Some of the commonly detected increased levels of SM in samples from infected animals are microbial-related metabolites such as pcresol glucuronide, trimethylamine, and phenylacetylglycine (Table 2). The metabolic product of Clostridium difﬁcile (opportunistic mild pathogenic anaerobe), p-cresol, is known to be the source of p-cresol glucuronide (Selmer and Andrei, 2001). Thus, elevation in such metabolites/SM could be related to the impact of infection on gut-microbial homeostasis. Another secretory product of O. volvulus as a potential urine biomarker for river blindness is N-acetyltyramine-O,bglucuronide (NATOG) (Globisch et al., 2013). However, NATOG alone is inadequate to discriminate between infected and control (Lagatie et al., 2016; Hotterbeekx et al., 2020), whereby considering the level of additional urine metabolites like cinnamoyl glycine can enhance the onchocerciasis assessment (Wewer et al., 2021). Wewer et al. (2017) (Wewer et al., 2017) reported etherlinked PC and PE in Onchocerca volvulus, O. ochengi, and Litomosoides sigmodontis through targeted shotgun lipidomics. Their study could not detect parasite-speciﬁc lipids in the infected bovine’s (host) serum, but the ﬂuid taken from O. ochengi nodules from the host contained nematode-derived ether-linked PE species [e.g., PE(e38:4), PE(e40:8), and PE (e40:9)], which could be potentially used to develop diagnostic biomarkers for river blindness (Wewer et al., 2017). Adebayo et al. (2018) (Adebayo et al., 2018) reported few putative urine Frontiers in Parasitology Potential application of small molecules in immunomodulatory drugs Several small molecule immunomodulatory drugs (IMiDs) (e.g., aminosalicylates, azathioprine, 6-mercaptopurine, and methotrexate) have been historically used for the management of inﬂammatory conditions, including IBD (Yeshi et al., 2020). Recent studies demonstrated that hookworm-derived SM extract could suppress pathology in TNBS-induced colitis and inhibit the secretion of key inﬂammatory cytokines from human peripheral blood mononuclear cells (Wangchuk et al., 2019; Wangchuk et al., 2019). Excretory/secretory products from other helminth species also showed similar protection in various experimental colitis models (Ruyssers et al., 2008; Varyani et al., 2017; Bobardt et al., 2020; Wang et al., 2020). Small molecule analogs of Acanthocheilonema viteae secreted immunomodulatory ESP ES-62, namely ES-62-SMAs 11a and 12b, protected ovalbumin-induced Th2 response-mediated airway inﬂammation and lungs eosinophil inﬁltration in mice suggesting potential drug leads to allergies (Rzepecka et al., 2014). Dafachronic acid detected in a few species of parasitic helminths (A. ceylanicum, A. suum, S. stercolis, H. contortus, and T. canis) is another example of a small molecule with therapeutic potential. When methylprednisolone acetated-treated NSG mice undergoing hyperinfection with S. stercolis were administered 10 mM dafachronic acid orally with drinking water, it signiﬁcantly reduced the worm burden in mice (Patton et al., 2018; Wang et al., 2021). Although saturated fatty acids such as palmitic acid and stearic acid were detected in all bioactive extracts, SCFAs such as acetate, propionate, and butyrate could be responsible for the observed protection (Wangchuk et al., 2019) as Tedelind et al. (2007) have reported anti-inﬂammatory properties associated to these SCFAs (Tedelind et al., 2007). Dodecanoic acid produced by the miracidium of S. mansoni is involved in host-parasite interactions, including penetration into its intermediate host (snail) (Ferreira et al., 2014; Wangchuk 12 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 TABLE 2 Reported small molecules as potential diagnostic biomarkers in samples obtained from human and animals. Helminth species infected with/ used for infection and ESP collection Life-cycle stage used for infection/ESP collection Samples in which SM detected Upregulated SM Downregulated SM References (a) From infected human/patients Ascaris lumbricoides Onchocerca volvulus Samples were obtained from already infected patients Schistosoma haematobium Urine 2‐Methyl pentanoyl carnitine NA (Lagatie et al., 2020) Urine N-Acetyltyramine-O,b-glucuronide NA (Lagatie et al., 2016) Urine Cinnamoyl glycine NA (Wewer et al., 2021) Plasma, serum Hexacosenoic acid, Pentacosenoic acid, Hydroxyoctadecenoic acid NA (Denery et al., 2010) Urine, plasma Indolylacryloylglycine, N-Glycoloylganglioside GM2, Adrenochrome O-quinone NA (Adebayo et al., 2018) (b) From infected experimental animal models Echinostoma caproni Fasciola hepatica Metacercariae Metacercariae Mice urine Mannitol, p-Cresol glucuronide, Phenylacetylglycine, Succinate, Trimethylamine, Trimethylamine-N-oxide 2-Ketoisocaproate, Acetate, Creatine, Hippurate, Taurine, Glucose Mice plasma Acetate, Formate Choline, Creatine, Glucose, Isoleucine, Leucine, Valine Mice fecal water 5-Aminovalerate, Isoleucine, Leucine, Uracil, Valine Acetate, Alanine, Butyrate, Glycine, Propionate Mice urine Aminoadipic acid Creatinine, Hippurate Mice plasma 2-Ketoglutarate, Betaine, Glucose, TrimethylamineN-oxide NA Mice fecal water 3-Methyl-2-oxovalerate, Butyrate Glucose (Saric et al., 2008) (Saric et al., 2010) Necator americanus L3 Hamster urine Aminoadipic acid, p-Cresol-glucuronide, Phenylacetylglycine Hippurate, 4-Hydroxyphenylactate, Dimethylamine, 4-Hydroxy-3-methylphenylpropionic acid (Wang et al., 2009) Onchocerca ochengi Sample obtained already infected cattle Bovine nodule ﬂuid PE(e38:4), PE(e40:8), PE(e40:9 NA (Wewer et al., 2017) Schistosoma japonicum Cercariae Hamster urine Acetate, Hippurate, p-Cresol glucuronide, Phenylacetylglycine, Pyruvate, Trimethylamine Dimethylamine, Lactate, Succinate, Propionate (Wang et al., 2006) Mice urine 2-Keto-3-methyl-valerate, 2-Keto-isocaproate, 2Keto-isovalerate, 3-Ureidopropionate, p-Cresol glucuronide, Dimethylamine, N-Acetyl-glycoprotein, Phenylacetylglycine, Trimethylamine, Trimethylamine-N-oxide 2-Oxoglutarate, 2-Hydroxyisobutyrate, 2(4-Hydroxyphenyl) propionic acid, Acetate, Adipate, Citrate, Fumarate, Hippurate, Succinate, Taurine Mice plasma Choline, Creatine, Dihydrothymine, Glycine, Glucose, Glutamate, Glutamine, Lysine, N-Acetylglycoprotein, Scyllo-inositol, Valine Acetone, D-3-Hydroxybutyrate, pyruvate Mice urine Trimethylamine. Phenylacetylglycine, Creatine, bAlanine, p-Cresol glucuronide, Pyruvate, Tryptophan Citrate, Acetate, Hippurate, Butyrate, Malonate, Propionate, Alanine, D-3Hydroxybutyrate, Succinate, 2Oxoglutarate, Taurine, 2-Oxoisocaproate, 2-Oxoisovalerate (Wang et al., 2004) Tryptophan, Creatine, Benzoic acid, Uric acid, Glycolic acid Hippurate, Citrate (Garcia-Perez et al., 2008) Schistosoma mansoni Cercariae Mice urine Hippurate, Phenylacetylglycine, 2-Oxoadipate NA Mice plasma D-3-hydroxybutyrate, Glycerophosphorylcholine NA Mice feces 5-Aminovalerate NA (Wu et al., 2010) (Li et al., 2011) (Continued) Frontiers in Parasitology 13 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 TABLE 2 Continued Helminth species infected with/ used for infection and ESP collection Life-cycle stage used for infection/ESP collection Samples in which SM detected Upregulated SM Downregulated SM References 4-Ethylphenol, Alanine, NAcetylglycoprotein, Betaine, Succinate, 3Hydroxyphenylpropionic acid, 4Hydroxyphenylpropionic acid, Trimethylamine-N-oxide, Hippurate (Wu et al., 2010) S. japonicum, N. americanus (coinfection) Cercariae, L3 Hamster urine Butyrate, Acetone, Creatine, Phenylacetylglycine, pCresol glucuronide Toxocara canis Embryonated T. canis eggs Dog serum Calcitroic acid NA (Zheng et al., 2019) (c) From helminth-derived excretory/secretory products Ancylostoma caninum Adult ESPs Succinic acid (dominant) NA (Wangchuk et al., 2019) Nippostrongylus brasiliensis L3 ESPs Octadecanoic acid (dominant) NA (Yeshi et al., 2020) Succinic acid (dominant) NA (Wangchuk et al., 2019) Adult Schistosoma mansoni Cercariae ESPs Linoleic acid and resolvins NA (Giera et al., 2018) Trichuris muris Adult ESPs Succinic acid (dominant) NA (Wangchuk et al., 2019) ESPs, Excretory/secretory products; L3, Stage 3 infective larva; NA, Not Available; reported metabolites are based on changes in the metabolite abundance (infected vs non-infected/control); ‘dominant’ means are abundant when analysed statistically (e.g., applying multivariate statistics like principal component analysis, PCA); ‘Up- or downregulation’ means increase or decrease in the levels, respectively, when compared with the negative control (e.g., samples from uninfected human or animals). 2017). Therefore, there is an urgent need to enhance efforts to analyse helminth ESPs for bioactive molecules and establishing a helminth-speciﬁc DNA-encoded SM library would help revolutionize the development of novel therapeutics for neglected helminthiases. et al., 2021). Linoleic acid produced by cercariae of S. mansoni, and prostaglandins (PGE2) enriched in their eggs helps cercariae to migrate successfully inside their human hosts (Ramaswamy et al., 2000; Giera et al., 2018). Thus, with many helminthderived SM showing various immunomodulatory/biological properties, including anti-inﬂammatory (Table 1), it has the potential to spur novel anti-inﬂammatory drug and IMiDs development. Unlike bigger molecules like proteins (biologics), SM are considered ideal candidates for drug development since they are small structures, are stable, cheaper to scale up, and lack immunogenicity (Yeshi et al., 2020). Moreover, unlike biologics that typically require parenteral delivery, SM can be developed into oral dosage forms, boosting patient satisfaction and compliance (Olivera et al., 2017). A search for SM in the innovative therapeutic design and development is still in the picture, and as of 2012, the Chemical Universe Database GDB17 has recorded 166 billion SM (Ruddigkeit et al., 2012). Moreover, recently, a Sweden-based biopharmaceutical company, Nuevolution, has revolutionized drug discovery by creating a DNA-encoded library of 40 trillion unique SM (Makurvet, 2021). Such a library with DNA-encoded SM has enabled rapid evaluation and identiﬁcation of bioactive drug-like compounds (Kleiner et al., 2011; Kunig et al., 2018). For example, a benzoxazepinone inhibitor discovered from a DNA-encoded library has become a clinical candidate that can potentially treat multiple inﬂammatory conditions (Harris et al., Frontiers in Parasitology Conclusion Recent studies have reported the protective role of small molecule crude extract obtained from helminth ES products against allergic and various autoimmune diseases, including chemically-induced colitis experimental mice models. Focusing on isolating compounds from such bioactive crude ES products of helminths would likely result in discovering novel drug leads, especially against inﬂammatory diseases. Limited known SM (e.g., amino acids and SCFAs) were reported from many helminths, and these molecules are associated with various biological and pharmacological activities. The available literature reports only limited known metabolites present in the crude extracts, and the identity of many metabolites remain unknown due to limitations in the identiﬁcation protocols and helminth-speciﬁc compound libraries. Many of these unknown metabolites, particularly lipids that remain largely unstudied, may likely have interesting biological and pharmacological properties, which could facilitate the development of novel diagnostic and therapeutic products. 14 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 There is a real need to pursue extensive and comprehensive metabolomics studies to identify such unknown compounds. With the rapid improvement in metabolomics platforms such as high-resolution mass spectrometry (HRMS) and other associated software tools and growing databases, it is expected to accelerate the identiﬁcation of many novel immunomodulatory SM produced by helminths. For example, a new pulsed mass-spectrometry ion generation technique called triboelectric nanogenerator inductive nano-electrospray ionization (TENGi nanoESI) mass spectrometry, introduced in 2020 could analyse volume-limited samples even at nanolitre scale. Such technological advancement is likely to help popularise immuno-metabolomics studies of helminths, where obtaining even modest quantities of ESPs is challenging without a good rodent model. Furthermore, future studies should prioritize those parasitic helminths not studied so far and develop a small molecule database (i.e., helminth-speciﬁc) to allow researchers to dive deep into the treasure trove of helminth metabolites. Funding This work was funded by a James Cook University Postgraduate Research Scholarship (JCUPRS) to KY, the National Health and Medical Research Council (NHMRC) Ideas Grant (APP1183323) to PW, and NHMRC Program Grant (APP1132975) and a Senior Principal Research Fellowship (APP1117504) to AL. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions All authors contributed to the article and approved the submitted version. References Adebayo, A. S., Mundhe, S. D., Awobode, H. O., Onile, O. S., Agunloye, A. M., Isokpehi, R. D., et al. (2018). Metabolite proﬁling for biomarkers in Schistosoma haematobium infection and associated bladder pathologies. PloS Negl. Trop. Dis. 12 (4), 1–17. doi: 10.1371/journal.pntd.0006452 Aparna, V., Dileep, K. V., Mandal, P. K., Karthe, P., Sadasivan, C., and Haridas, M. (2012). Anti-inﬂammatory property of n-hexadecanoic acid: Structural evidence and kinetic assessment. Chem. Biol. Drug Des. 80 (3), 434–439. doi: 10.1111/j.1747-0285.2012.01418.x Afraei, S., D'Aniello, A., Sedaghat, R., Ekhtiari, P., Azizi, G., Tabrizian, N., et al. (2017). Therapeutic effects of d-aspartate in a mouse model of multiple sclerosis. J. Food Drug Anal. 25 (3), 699–708. doi: 10.1016/j.jfda.2016.10.025 Araujo, A., Reinhard, K., Ferreira, L. F., Pucu, E., and Chiefﬁ, P. P. (2013). Paleoparasitology: The origin of human parasites. Arq Neuropsiquiatr 71 (9B), 722–726. doi: 10.1590/0004-282X20130159 Ahmad, M., Baba, W. N., Gani, A., Wani, T. A., Gani, A., Masoodi, F. A., et al. (2015). Effect of extraction time on antioxidants and bioactive volatile components of green tea (Camellia sinensis), using GC/MS. Cogent Food Agric. 1 (1), 1–11. doi: 10.1080/23311932.2015.1106387 Artis, D., and Spits, H. (2015). The biology of innate lymphoid cells. Nature. 517 (7534), 293–301. doi: 10.1038/nature14189 Barrett, J. (1968). Lipids of the infective and parasitic stages of some nematodes. Nature. 218, 1267–1268. doi: 10.1038/2181267a0 Ahuja, V., and Tandon, R. K. (2010). Inﬂammatory bowel disease in the Asiapaciﬁc area: a comparison with developed countries and regional differences. J. Dig Dis. 11 (3), 134–147. doi: 10.1111/j.1751-2980.2010.00429.x Becker, A.-C., Willenberg, I., Springer, A., Schebb, N. H., Steinberg, P., and Strube, C. (2017). Fatty acid composition of free-living and parasitic stages of the bovine lungworm dictyocaulus viviparus. Mol. Biochem. Parasitol. 216, 39–44. doi: 10.1016/j.molbiopara.2017.06.008 Alamshah, A., Spreckley, E., Norton, M., Kinsey-Jones, J. S., Amin, A., Ramgulam, A., et al. (2017). L-phenylalanine modulates gut hormone release and glucose tolerance, and suppresses food intake through the calcium-sensing receptor in rodents. Int. J. Obes. 41 (11), 1693–1701. doi: 10.1038/ijo.2017.164 Bi, N. N., Zhao, S., Zhang, J. F., Cheng, Y., Zuo, C. Y., Yang, G. L., et al. (2021). Proteomics investigations of potential protein biomarkers in sera of rabbits infected with schistosoma japonicum. Front. Cell Infect. Microbiol. 11, 1–13. doi: 10.3389/ fcimb.2021.784279 Albarqi, M. M., Stoltzfus, J. D., Pilgrim, A. A., Nolan, T. J., Wang, Z., Kliewer, S. A., et al. (2016). Regulation of life cycle checkpoints and developmental activation of infective larvae in Strongyloides stercoralis by dafachronic acid. PloS Pathog. 12 (1), 1–20. doi: 10.1371/journal.ppat.1005358 Blount, D., Hooi, D., Feary, J., Venn, A., Telford, G., Brown, A., et al. (2009). Immunologic proﬁles of persons recruited for a randomized, placebo-controlled clinical trial of hookworm infection. Am. J. Trop. Med. Hyg. 81 (5), 911–916. doi: 10.4269/ajtmh.2009.09-0237 Albonico, M., Allen, H., Chitsulo, L., Engels, D., Gabrielli, A. F., and Savioli, L. (2008). Controlling soil-transmitted helminthiasis in pre-school-age children through preventive chemotherapy. PloS Negl. Trop. Dis. 2 (3), 1–11. doi: 10.1371/journal.pntd.0000126 Bobardt, S. D., Dillman, A. R., and Nair, M. G. (2020). The two faces of nematode infection: Virulence and immunomodulatory molecules from nematode parasites of mammals, insects and plants. Front. Microbiol. 11, 1–13. doi: 10.3389/ fmicb.2020.577846 Andersen, F. A. (2007). Final report on the safety assessment of maleic acid. Int. J. Toxicol. 26, 125–130. doi: 10.1080/10915810701351251 Boccella, S., Vacca, V., Errico, F., Marinelli, S., Squillace, M., Guida, F., et al. (2015). D-aspartate modulates nociceptive-speciﬁc neuron activity and pain threshold in inﬂammatory and neuropathic pain condition in mice. BioMed. Res. Int. 2015, 1–10. doi: 10.1155/2015/905906 Andoh, A., Bamba, T., and Sasaki, M. (1999). Physiological and antiinﬂammatory roles of dietary ﬁber and butyrate in intestinal functions. J. Parenter Enteral Nutr. 23 (5), S70–SS3. doi: 10.1177/014860719902300518 Frontiers in Parasitology 15 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 Brooker, S. (2010). Estimating the global distribution and disease burden of intestinal nematode infections: Adding up the numbers–a review. Int. J. Parasitol. 40, 1137–1144. doi: 10.1016/j.ijpara.2010.04.004 Derakhshanfar, A., Bidadkosh, A., and Hashempour Sadeghian, M. (2009). Lmethionine attenuates gentamicin nephrotoxicity in male wistar rat: pathological and biochemical ﬁndings. Iran J. Vet. Res. 10, 323–328. doi: 10.22099/ IJVR.2009.1715 Brussow, H. (2020). Problems with the concept of gut microbiota dysbiosis. Microb. Biotechnol. 13 (2), 423–434. doi: 10.1111/1751-7915.13479 De Silva, N. R., Brooker, S., Hotez, P. J., Montresor, A., Engels, D., and Savioli, L. (2003). Soil-transmitted helminth infections: Updating the global picture. Trends Parasitol. 19, 547–551. doi: 10.1016/j.pt.2003.10.002 Buck, A. H., Coakley, G., Simbari, F., McSorley, H. J., Quintana, J. F., Le Bihan, T., et al. (2014). Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity. Nat. Commun. 5, 1–11. doi: 10.1038/ncomms6488 Detopoulou, P., Panagiotakos, D. B., Antonopoulou, S., Pitsavos, C., and Stefanadis, C. (2008). Dietary choline and betaine intakes in relation to concentrations of inﬂammatory markers in healthy adults: The ATTICA study. Am. J. Clin. Nutr. 87, 424–430. doi: 10.1093/ajcn/87.2.424 Buitrago, G., Pickering, D., Ruscher, R., Cobos Caceres, C., Jones, L., Cooper, M., et al. (2021). A netrin domain-containing protein secreted by the human hookworm Necator americanus protects against CD4 T cell transfer colitis. Transl. Res. 232, 88–102. doi: 10.1016/j.trsl.2021.02.012 Dong, Y. W., Jiang, W. D., Liu, Y., Wu, P., Jiang, J., Kuang, S. Y., et al. (2017). Threonine deﬁciency decreased intestinal immunity and aggravated inﬂammation associated with NF-kappaB and target of rapamycin signalling pathways in juvenile grass carp (Ctenopharyngodon idella) after infection with aeromonas hydrophila. Br. J. Nutr. 118 (2), 92–108. doi: 10.1017/S0007114517001830 Castro, G. A., Badial-Aceves, F., Smith, J. W., Dudrick, S. J., and Weisbrodt, N. W. (1976). Altered small bowel propulsion associated with parasitism. Gastroenterology. 71, 620–625. doi: 10.1016/S0016-5085(76)80553-7 Doonan, J., Thomas, D., Wong, M. H., Ramage, H. J., Al-Riyami, L., Lumb, F. E., et al. (2018). Failure of the anti-inﬂammatory parasitic worm product ES-62 to provide protection in mouse models of type I diabetes, multiple sclerosis, and inﬂammatory bowel disease. Molecules. 23 (10), 1–14. doi: 10.3390/ molecules23102669 Cavone, L., Calosi, L., Cinci, L., Moroni, F., and Chiarugi, A. (2012). Topical mannitol reduces inﬂammatory edema in a rat model of arthritis. Pharmacology 89 (1-2), 18–21. doi: 10.1159/000335094 Chajes, V., Hulten, K., Van Kappel, A.-L., Winkvist, A., Kaaks, R., Hallmans, G., et al. (1999). Fatty-acid composition in serum phospholipids and risk of breast cancer: an incident case-control study in Sweden. Int. J. Cancer. 83, 585–590. doi: 10.1002/(SICI)1097-0215(19991126)83:5<585::AID-IJC2>3.0.CO;2-Z Durack, J., and Lynch, S. V. (2019). The gut microbiome: Relationships with disease and opportunities for therapy. J. Exp. Med. 216 (1), 20–40. doi: 10.1084/ jem.20180448 Chavez-Olortegu, C., Tavares, C. A. P., and Resende, M. (1992). Puriﬁcation and characterization of a 47 kDa protease from Schistosoma mansoni cerearial secretion. Parasitology. 105 (2), 211–218. doi: 10.1017/S0031182000074138 Du, L., Tang, H., Ma, Z., Xu, J., Gao, W., Chen, J., et al. (2011). The protective effect of the recombinant 53-kDa protein of Trichinella spiralis on experimental colitis in mice. Dig Dis. Sci. 56 (10), 2810–2817. doi: 10.1007/s10620-011-1689-8 Chazot, P. L., Coleman, S. K., Cik, M., and Stephenson, F. A. (1994). Molecular characterization of n-methyl-D-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule. J. Biol. Chem. 269 (39), 24403–24409. doi: 10.1016/S0021-9258(19) 51098-5 Dzik, J. M. (2006). Molecules released by helminth parasites involved in host colonization. Acta Biochim. Pol. 53 (1), 33–64. doi: 10.18388/abp.2006_3361 Easton, A. V., Quinones, M., Vujkovic-Cvijin, I., Oliveira, R. G., Kepha, S., Odiere, M. R., et al. (2019). The impact of anthelmintic treatment on human gut microbiota based on cross-sectional and pre- and postdeworming comparisons in Western Kenya. mBio. 10 (2), 1–14. doi: 10.1128/mBio.00519-19 Cheng, T. C. (1986). General parasitology (USA: Academic Press). 2. Chen, Z. B., Tang, H., Liang, Y. B., Yang, W., Wu, J. G., Hu, X. C., et al. (2016). Recombinant Trichinella spiralis 53-kDa protein activates M2 macrophages and attenuates the LPS-induced damage of endotoxemia. Innate Immun. 22, 419–432. doi: 10.1177/1753425916651984 Eichenberger, R. M., Ryan, S., Jones, L., Buitrago, G., Polster, R., Montes de Oca, M., et al. (2018). Hookworm secreted extracellular vesicles interact with host cells and prevent inducible colitis in mice. Front. Immunol. 9, 1–14. doi: 10.3389/ ﬁmmu.2018.00850 Chu, K. M., Watermeyer, G., Shelly, L., Janssen, J., May, T. D., Brink, K., et al. (2013). Childhood helminth exposure is protective against inﬂammatory bowel disease: a case control study in south Africa. Inﬂammation Bowel Dis. 19 (3), 614– 620. doi: 10.1097/MIB.0b013e31827f27f4 Eichenberger, R. M., Talukder, M. H., Field, M. A., Wangchuk, P., Giacomin, P., Loukas, A., et al. (2018). Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host-parasite communication. J. Extracell Vesicles. 7 (1), 1–16. doi: 10.1080/20013078.2018.1428004 Coburn, L. A., Gong, X., Singh, K., Asim, M., Scull, B. P., Allaman, M. M., et al. (2012). L-arginine supplementation improves responses to injury and inﬂammation in dextran sulfate sodium colitis. PloS One 7 (3), 1–18. doi: 10.1371/journal.pone.0033546 Elliott, D. E., Setiawan, T., Metwali, A., Blum, A., Urban, J. F.Jr., and Weinstock, J. V. (2004). Heligmosomoides polygyrus inhibits established colitis in IL-10deﬁcient mice. Eur. J. Immunol. 34 (10), 2690–2698. doi: 10.1002/eji.200324833 Elliott, D. E., and Weinstock, J. V. (2017). Nematodes and human therapeutic trials for inﬂammatory disease. Parasite Immunol. 39 (5), 1–22. doi: 10.1111/ pim.12407 Coqueiro, A. Y., Raizel, R., Hypó lito, T. M., and Tirapegui, J. (2017). Effects of supplementation with l-glutamine and l-alanine in the body composition of rats submitted to resistance exercise. Rev. Bras. Ciências do Esporte. 39 (4), 417–423. doi: 10.1016/j.rbce.2017.06.003 Evaldsson, C., Rydé n, I., and Uppugunduri, S. (2007). Anti-inﬂammatory effects of exogenous uridine in an animal model of lung inﬂammation. Int. Immunopharmacol. 7 (8), 1025–1032. doi: 10.1016/j.intimp.2007.03.008 Croese, J., Miller, G. C., Marquart, L., Llewellyn, S., Gupta, R., Becker, L., et al. (2020). Randomized, placebo controlled trial of experimental hookworm infection for improving gluten tolerance in celiac disease. Clin. Transl. Gastroenterol. 11 (12), 1–11. doi: 10.14309/ctg.0000000000000274 Everts, B., Perona-Wright, G., Smits, H. H., Hokke, C. H., van der Ham, A. J., Fitzsimmons, C. M., et al. (2009). Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses. J. Exp. Med. 206 (8), 1673–1680. doi: 10.1084/ jem.20082460 Croese, J., O'Neil, J., Masson, J., Cooke, S., Melrose, W., Pritchard, D., et al. (2006). A proof of concept study establishing Necator americanus in crohn's patients and reservoir donors. Gut. 55 (1), 136–137. doi: 10.1136/gut.2005.079129 Faure, M., Mettraux, C., Moennoz, D., Godin, J.-P., Vuichoud, J., Rochat, F., et al. (2006). Speciﬁc amino acids increase mucin synthesis and microbiota in dextran sulfate sodium–treated rats. J. Nutr. 136, 1558–1564. doi: 10.1093/jn/136.6.1558 Cvetkovic, J., Sofronic-Milosavljevic, L., Ilic, N., Gnjatovic, M., Nagano, I., and Gruden-Movsesijan, A. (2016). Immunomodulatory potential of particular Trichinella spiralis muscle larvae excretory-secretory components. Int. J. Parasitol. 46, 833–842. doi: 10.1016/j.ijpara.2016.07.008 Ferreira, M. S., de Oliveira, D. N., de Oliveira, R. N., Allegretti, S. M., and Catharino, R. R. (2014). Screening the life cycle of Schistosoma mansoni using highresolution mass spectrometry. Anal. Chim. Acta 845, 62–69. doi: 10.1016/ j.aca.2014.06.013 Dalton, J. P., Skelly, P., and Halton, D. W. (2003). Role of the tegument and gut in nutrient uptake by parasitic platyhelminthes. Can. J. Zool. 82, 211–232. doi: 10.1139/z03-213 Ferreira, M. S., de Oliveira, D. N., de Oliveira, R. N., Allegretti, S. M., Vercesi, A. E., and Catharino, R. R. (2014). Mass spectrometry imaging: a new vision in differentiating Schistosoma mansoni strains. J. Mass Spectrom. 49 (1), 86–92. doi: 10.1002/jms.3308 Darabi, Z., Darand, M., Yari, Z., Hedayati, M., Faghihi, A., Agah, S., et al. (2019). Inﬂammatory markers response to citrulline supplementation in patients with non-alcoholic fatty liver disease: A randomized, double blind, placebo-controlled, clinical trial. BMC Res. Notes. 12 (1), 1–5. doi: 10.1186/s13104-019-4130-6 Ferreira, M. S., de Oliveira, R. N., de Oliveira, D. N., Esteves, C. Z., Allegretti, S. M., and Catharino, R. R. (2015). Revealing praziquantel molecular targets using mass spectrometry imaging: An expeditious approach applied to schistosoma mansoni. Int. J. Parasitol. 45 (6), 385–391. doi: 10.1016/j.ijpara.2014.12.008 Daveson, A. J., Jones, D. M., Gaze, S., McSorley, H., Clouston, A., Pascoe, A., et al. (2011). Effect of hookworm infection on wheat challenge in celiac disease-a randomised double-blinded placebo controlled trial. PloS One 6 (3), 1–9. doi: 10.1371/journal.pone.0017366 Ferreira, I., Smyth, D., Gaze, S., Aziz, A., Giacomin, P., Ruyssers, N., et al. (2013). Hookworm excretory/secretory products induce interleukin-4 (il-4)+ il-10+ cd4+ t cell responses and suppress pathology in a mouse model of colitis. Infect. Immun. 81 (6), 2104–2111. doi: 10.1128/IAI.00563-12 Denery, J. R., Nunes, A. A., Hixon, M. S., Dickerson, T. J., and Janda, K. D. (2010). Metabolomics-based discovery of diagnostic biomarkers for onchocerciasis. PloS Negl. Trop. Dis. 4 (10), 1–9. doi: 10.1371/journal.pntd.0000834 Frontiers in Parasitology 16 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 Figueiredo, B. C., Da’dara, A. A., Oliveira, S. C., and Skelly, P. J. (2015). Schistosomes enhance plasminogen activation: The role of tegumental enolase. PloS Pathog. 11 (12), 1–22. doi: 10.1371/journal.ppat.1005335 Ham, D. J., Gleeson, B. G., Chee, A., Baum, D. M., Caldow, M. K., Lynch, G. S., et al. (2015). L-citrulline protects skeletal muscle cells from cachectic stimuli through an iNOS-dependent mechanism. PloS One 10 (10), 1–17. doi: 10.1371/ journal.pone.0141572 Filyk, H. A., and Osborne, L. C. (2016). The multibiome: the intestinal ecosystem's inﬂuence on immune homeostasis, health, and disease. EBioMedicine. 13, 46–54. doi: 10.1016/j.ebiom.2016.10.007 Han, D., Kim, H. Y., Lee, H. J., Shim, I., and Hahm, D. H. (2007). Wound healing activity of gamma-aminobutyric acid (GABA) in rats. J. Microbiol. Biotechnol. 17, 1661–1669. Fleming, J., Hernandez, G., Hartman, L., Maksimovic, J., Nace, S., Lawler, B., et al. (2019). Safety and efﬁcacy of helminth treatment in relapsing-remitting multiple sclerosis: Results of the HINT 2 clinical trial. Mult Scler. 25 (1), 81–91. doi: 10.1177/1352458517736377 Han, H., Yin, J., Wang, B., Huang, X., Yao, J., Zheng, J., et al. (2018). Effects of dietary lysine restriction on inﬂammatory responses in piglets. Sci. Rep. 8 (1), 1–8. doi: 10.1038/s41598-018-20689-3 Harder, A. (2016). The biochemistry of Haemonchus contortus and other parasitic nematodes. Adv. Parasitol. 93, 69–94. doi: 10.1016/bs.apar.2016.02.010 Flohr, C., Tuyen, L. N., Lewis, S., Quinnell, R., Minh, T. T., Liem, H. T., et al. (2006). Poor sanitation and helminth infection protect against skin sensitization in Vietnamese children: A cross-sectional study. J. Allergy Clin. Immunol. 118 (6), 1305–1311. doi: 10.1016/j.jaci.2006.08.035 Harris, P. A., Berger, S. B., Jeong, J. U., Nagilla, R., Bandyopadhyay, D., Campobasso, N., et al. (2017). Discovery of a ﬁrst-in-class receptor interacting protein 1 (RIP1) kinase speciﬁc clinical candidate (GSK2982772) for the treatment of inﬂammatory diseases. J. Med. Chem. 60 (4), 1247–1261. doi: 10.1021/ acs.jmedchem.6b01751 Flynn, A. F., Joyce, M. G., Taylor, R. T., Bennuru, S., Lindrose, A. R., Sterling, S. L., et al. (2019). Intestinal UDP-glucuronosyltransferase as a potential target for the treatment and prevention of lymphatic ﬁlariasis. PloS Negl. Trop. Dis. 13 (9), 1–25. doi: 10.1371/journal.pntd.0007687 Hasegawa, S., Ichiyama, T., Sonaka, I., Ohsaki, A., Okada, S., Wakiguchi, H., et al. (2012). Cysteine, histidine and glycine exhibit anti-inﬂammatory effects in human coronary arterial endothelial cells. Clin. Exp. Immunol. 167 (2), 269–274. doi: 10.1111/j.1365-2249.2011.04519.x Fotiadis, C., Adamis, S., Misiakos, E. P., Genetzakis, M., Antonakis, P. T., Tsekouras, D. K., et al. (2016). The prophylactic effect of l-arginine in acute ischaemic colitis in a rat model of ischaemia/reperfusion injury. Acta Chirurgica Belgica. 107 (2), 192–200. doi: 10.1080/00015458.2007.11680039 Hasko, G., Kuhel, D. G., Nemeth, Z. H., Mabley, J. G., Stachlewitz, R. F., Virag, L., et al. (2000). Inosine inhibits inﬂammatory cytokine production by a posttranscriptional mechanism and protects against endotoxin-induced shock. J. Immunol. 164 (2), 1013–1019. doi: 10.4049/jimmunol.164.2.1013 Fox, J. G., Beck, P., Dangler, C. A., Whary, M. T., Wang, T. C., Shi, H. N., et al. (2000). Concurrent enteric helminth infection modulates inﬂammation and gastric immune responses and reduces helicobacter-induced gastric atrophy. Nat. Med. 6, 536–542. doi: 10.1038/75015 Hazel, J. R., and Williams, E. E. (1990). The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment. Prog. Lipid Res. 29, 167–227. doi: 10.1016/0163-7827(90)90002-3 Fujino, M., Nishio, Y., Ito, H., Tanaka, T., and Li, X. K. (2016). 5-aminolevulinic acid regulates the inﬂammatory response and alloimmune reaction. Int. Immunopharmacol. 37, 71–78. doi: 10.1016/j.intimp.2015.11.034 Hearps, A. C., Tyssen, D., Srbinovski, D., Bayigga, L., Diaz, D. J. D., Aldunate, M., et al. (2017). Vaginal lactic acid elicits an anti-inﬂammatory response from human cervicovaginal epithelial cells and inhibits production of pro-inﬂammatory mediators associated with HIV acquisition. Mucosal Immunol. 10, 1480–1490. doi: 10.1038/mi.2017.27 Fukuda, T., Majumder, K., Zhang, H., Matsui, T., and Mine, Y. (2017). Adenine has an anti-inﬂammatory effect through the activation of adenine receptor signaling in mouse macrophage. J. Funct. Foods. 28, 235–239. doi: 10.1016/ j.jff.2016.11.013 Hegedus, Z. L., and Nayak, U. (1994). Homogentisic acid and structurally related compounds as intermediates in plasma soluble melanin formation and in tissue toxicities. Arch. Internationalesde Physiologic Biochimie Biophysique 102, 175–181. doi: 10.3109/13813459409007534 Gaifem, J., Goncalves, L. G., Dinis-Oliveira, R. J., Cunha, C., Carvalho, A., Torrado, E., et al. (2018). L-threonine supplementation during colitis onset delays disease recovery. Front. Physiol. 9, 1–7. doi: 10.3389/fphys.2018.01247 Garcia-Perez, I., Whitﬁeld, P., Bartlett, A., Angulo, S., Legido-Quigley, C., Hanna-Brown, M., et al. (2008). Metabolic ﬁngerprinting of Schistosoma mansoni infection in mice urine with capillary electrophoresis. Electrophoresis. 29 (15), 3201–3206. doi: 10.1002/elps.200800031 He, L., Li, H., Huang, N., Zhou, X., Tian, J., Li, T., et al. (2017). Alphaketoglutarate suppresses the NF-kB-mediated inﬂammatory pathway and enhances the PXR-regulated detoxiﬁcation pathway. Oncotarget. 8, 102974– 102988. doi: 10.18632/oncotarget.16875 Giera, M., Kaisar, M. M. M., Derks, R. J. E., Steenvoorden, E., Kruize, Y. C. M., Hokke, C. H., et al. (2018). The Schistosoma mansoni lipidome: Leads for immunomodulation. Anal. Chim. Acta 1037, 107–118. doi: 10.1016/ j.aca.2017.11.058 Helmby, H. (2015). Human helminth therapy to treat inﬂammatory disorders where do we stand? BMC Immunol. 16, 12. doi: 10.1186/s12865-015-0074-3 He, F., Wu, C., Li, P., Li, N., Zhang, D., Zhu, Q., et al. (2018). Functions and signaling pathways of amino acids in intestinal inﬂammation. BioMed. Res. Int. 2018, 1–13. doi: 10.1155/2018/9171905 Globisch, D., Moreno, A. Y., Hixon, M. S., Nunes, A. A., Denery, J. R., Specht, S., et al. (2013). Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness. Proc. Natl. Acad. Sci. 110 (11), 4218–4223. doi: 10.1073/ pnas.1221969110 Hnia, K., Gayraud, J., Hugon, G., Ramonatxo, M., de la Porte, S., Matecki, S., et al. (2008). L-arginine decreases inﬂammation and modulates the nuclear factorkappaB/matrix metalloproteinase cascade in mdx muscle ﬁbers. Am. J. Pathol. 172 (6), 1509–1519. doi: 10.2353/ajpath.2008.071009 Glushakova, O., Kosugi, T., Roncal, C., Mu, W., Heinig, M., Cirillo, P., et al. (2008). Fructose induces the inﬂammatory molecule ICAM-1 in endothelial cells. J. Am. Soc. Nephrol. 19, 1712–1720. doi: 10.1681/ASN.2007121304 Hotez, P. J., Brindley, P. J., Bethony, J. M., King, C. H., Pearce, E. J., and Jacobson, J. (2008). Helminth infections: the great neglected tropical diseases. J. Clin. Invest. 118, 1311–1321. doi: 10.1172/JCI34261 Gordon, C. A., Krause, L., McManus, D. P., Morrison, M., Weerakoon, K. G., Connor, M. C., et al. (2020). Helminths, polyparasitism, and the gut microbiome in the Philippines. Int. J. Parasitol. 50 (3), 217–225. doi: 10.1016/j.ijpara.2019.12.008 Hotterbeekx, A., Dusabimana, A., Mandro, M., Abhafule, G. M., Deogratias, W., Siewe Fodjo, J. N., et al. (2020). Urinary n-acetyltyramine-O,beta-glucuronide in persons with onchocerciasis-associated epilepsy. Pathogens. 9 (3), 1–10. doi: 10.3390/pathogens9030191 Grencis, R. K. (2015). Immunity to helminths: Resistance, regulation, and susceptibility to gastrointestinal nematodes. Annu. Rev. Immunol. 33, 201–225. doi: 10.1146/annurev-immunol-032713-120218 Hoy, A. M., Lundie, R. J., Ivens, A., Quintana, J. F., Nausch, N., Forster, T., et al. (2014). Parasite-derived microRNAs in host serum as novel biomarkers of helminth infection. PloS Negl. Trop. Dis. 8 (2), 1–14. doi: 10.1371/journal.pntd.0002701 Grundy, S. M., Cleeman, J. I., Daniels, S. R., Donato, K. A., Eckel, R. H., Franklin, B. A., et al. (2005). Diagnosis and management of the metabolic syndrome: An American heart Association/National heart, lung, and blood institute scientiﬁc statement. Circulation. 112 (17), 2735–2752. doi: 10.1161/ CIRCULATIONAHA.105.169404 Huang, P. L. (2009). A comprehensive deﬁnition for metabolic syndrome. Dis. Model. Mech. 2 (5-6), 231–237. doi: 10.1242/dmm.001180 Huang, W. C., Tsai, T. H., Chuang, L. T., Li, Y. Y., Zouboulis, C. C., and Tsai, P. J. (2014). Antibacterial and anti-inﬂammatory properties of capric acid against propionibacterium acnes: A comparative study with lauric acid. J. Dermatol. Sci. 73, 232–240. doi: 10.1016/j.jdermsci.2013.10.010 Guo, K., Sun, X., Gu, Y., Wang, Z., Huang, J., and Zhu, X. (2016). Trichinella spiralis paramyosin activates mouse bone marrow-derived dendritic cells and induces regulatory T cells. Parasit Vectors. 9 (1), 1–11. doi: 10.1186/s13071-0161857-y Huang, X., Zeng, L. R., Chen, F. S., Zhu, J. P., and Zhu, M. H. (2018). Trichuris suis ova therapy in inﬂammatory bowel disease: A meta-analysis. Med. (Baltimore). 97 (34), 1–9. doi: 10.1097/MD.0000000000012087 Haeberlein, S., Obieglo, K., Ozir-Fazalalikhan, A., Chayé , M. A. M., Veninga, H., van der Vlugt, L. E. P. M., et al. (2017). Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory b cells. PloS Pathog. 13 (7), 1–28. doi: 10.1371/journal.ppat.1006539 Hunter, M. M., Wang, A., Hirota, C. L., and McKay, D. M. (2005). Neutralizing anti-IL-10 antibody blocks the protective effect of tapeworm infection in a murine model of chemically induced colitis. J. Immunol. 174 (11), 7368–7375. doi: 10.4049/ jimmunol.174.11.7368 Hamed, M. A. A., Ahmed, S. A. A., and Khaled, H. M. (2011). Efﬁciency of diagnostic biomarkers among colonic schistosomiasis Egyptian patients. Mem Inst Oswaldo Cruz. 106 (3), 322–329. doi: 10.1590/S0074-02762011000300011 Frontiers in Parasitology 17 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 Islam, J., Sato, S., Watanabe, K., Watanabe, T., Ardiansyah,, Hirahara, K., et al. (2017). Dietary tryptophan alleviates dextran sodium sulfate-induced colitis through aryl hydrocarbon receptor in mice. J. Nutr. Biochem. 42, 43–50. doi: 10.1016/j.jnutbio.2016.12.019 Kuda, O. (2017). Bioactive metabolites of docosahexaenoic acid. Biochimie. 136, 12–20. doi: 10.1016/j.biochi.2017.01.002 Kumagai, T., Osada, Y., Ohta, N., and Kanazawa, T. (2009). Peroxiredoxin-1 from Schistosoma japonicum functions as a scavenger against hydrogen peroxide but not nitric oxide. Mol. Biochem. Parasitol. 164 (1), 26–31. doi: 10.1016/ j.molbiopara.2008.11.002 Jenkins, T. P., Formenti, F., Castro, C., Piubelli, C., Perandin, F., Buonfrate, D., et al. (2018). A comprehensive analysis of the faecal microbiome and metabolome of Strongyloides stercoralis infected volunteers from a non-endemic area. Sci. Rep. 8 (1), 1–13. doi: 10.1038/s41598-018-33937-3 Kunig, V., Potowski, M., Gohla, A., and Brunschweiger, A. (2018). DNAEncoded libraries - an efﬁcient small molecule discovery technology for the biomedical sciences. Biol. Chem. 399 (7), 691–710. doi: 10.1515/hsz-2018-0119 Jenkins, T. P., Rathnayaka, Y., Perera, P. K., Peachey, L. E., Nolan, M. J., Krause, L., et al. (2017). Infections by human gastrointestinal helminths are associated with changes in faecal microbiota diversity and composition. PloS One 12 (9), 1–18. doi: 10.1371/journal.pone.0184719 Kupritz, J., Angelova, A., Nutman, T. B., and Gazzinelli-Guimaraesa, P. H. (2021). Helminth-induced human gastrointestinal dysbiosis: A systematic review and meta-analysis reveals insights into altered taxon diversity and microbial gradient collapse. mBio. 12, 1–17. doi: 10.1128/mBio.02890-21 Jiao, N., Wu, Z., Ji, Y., Wang, B., Dai, Z., and Wu, G. (2015). L-glutamate enhances barrier and antioxidative functions in intestinal porcine epithelial cells. J. Nutr. 145 (10), 2258–2264. doi: 10.3945/jn.115.217661 Kyu, H. H., Abate, D., Abate, K. H., Abay, S. M., Abbafati, C., Abbasi, N., et al. (2018). Global, regional, and national disability-adjusted life-years (DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990–2017: A systematic analysis for the global burden of disease study 2017. Lancet 392 (10159), 1859–1922. doi: 10.1016/S0140-6736(18)32335-3 Jin, X., Yang, Y., Liu, X., Shi, H., Cai, X., Luo, X., et al. (2019). Glutathione-stransferase of Trichinella spiralis regulates maturation and function of dendritic cells. Parasitology. 146 (14), 1725–1732. doi: 10.1017/S003118201900115X Lagatie, O., Njumbe Ediage, E., Batsa Debrah, L., Diels, L., Nolten, C., Vinken, P., et al. (2016). Evaluation of the diagnostic potential of urinary n-Acetyltyramine-O, beta-glucuronide (NATOG) as diagnostic biomarker for Onchocerca volvulus infection. Parasit Vectors. 9 (1), 1–10. doi: 10.1186/s13071-016-1582-6 Joshi, A., Kimball, A., Schaller, M., denDekker, A., Burant, C., Kunkel, S. L., et al. (2018). Modulation of xanthine metabolism ameliorates inﬂammation and accelerates diabetic wound healing. J. Immunol. 200 (1):170.13 13. Kabeerdoss, J., Pugazhendhi, S., Subramanian, V., Binder, H. J., and Ramakrishna, B. S. (2011). Exposure to hookworms in patients with crohn's disease: a case-control study. Aliment Pharmacol. Ther. 34 (8), 923–930. doi: 10.1111/j.1365-2036.2011.04824.x Lagatie, O., Verheyen, A., Van Asten, S., Odiere, M. R., Djuardi, Y., Levecke, B., et al. (2020). 2-methyl-pentanoyl-carnitine (2-MPC): A urine biomarker for patent Ascaris lumbricoides infection. Sci. Rep. 10 (1), 1–13. doi: 10.1038/s41598-02072804-y Kahl, J., Brattig, N., and Liebau, E. (2018). The untapped pharmacopeic potential of helminths. Trends Parasitol. 34, 828–842. doi: 10.1016/j.pt.2018.05.011 Largo, R., Alvarez-Soria, M. A., Diez-Ortego, I., Calvo, E., Sá nchez-Pernaute, O., Egido, J., et al. (2003). Glucosamine inhibits IL-1-induced NF-kB activation in human osteoarthritic chondrocytes. Osteoarthr Cartil. 11, 290–298. doi: 10.1016/ S1063-4584(03)00028-1 Kalinowska, M., Mazur, L., Jabłoń ska-Trypuć , A., and Lewandowski, W. (2018). A new calcium 2,5-dihydroxybenzoate: synthesis, characterization and antioxidant studies and stress mediated cytotoxity in MCF-7 cells. J. Saudi Chem. Soc 22 (6), 742–756. doi: 10.1016/j.jscs.2017.12.006 Lee, B. J., Lin, J. S., Lin, Y. C., and Lin, P. T. (2015). Antiinﬂammatory effects of lcarnitine supplementation (1000 mg/d) in coronary artery disease patients. Nutr. 31 (3), 475–479. doi: 10.1016/j.nut.2014.10.001 Kammoun-Rebai, W., Naouar, I., Libri, V., Albert, M., Louzir, H., MeddebGarnaoui, A., et al. (2016). Protein biomarkers discriminate Leishmania majorinfected and non-infected individuals in areas endemic for cutaneous leishmaniasis. BMC Infect. Dis. 16, 1–10. doi: 10.1186/s12879-016-1458-6 Lee, Y. C., Su, Y. T., Liu, T. Y., Tsai, C. M., Chang, C. H., and Yu, H. R. (2018). Larginine and l-citrulline supplementation have different programming effect on regulatory T-cells function of infantile rats. Front. Immunol. 9, 1–11. doi: 10.3389/ ﬁmmu.2018.02911 Kato, H., Miura, K., Nakano, S., Suzuki, K., Bannai, M., and Inoue, Y. (2016). Leucine-enriched essential amino acids attenuate inﬂammation in rat muscle and enhance muscle repair after eccentric contraction. Amino Acids 48 (9), 2145–2155. doi: 10.1007/s00726-016-2240-1 Lee, S. C., Tang, M. S., Lim, Y. A., Choy, S. H., Kurtz, Z. D., Cox, L. M., et al. (2014). Helminth colonization is associated with increased diversity of the gut microbiota. PloS Negl. Trop. Dis. 8 (5), 1–15. doi: 10.1371/journal.pntd.0002880 Kaur, N., Chen, C. C., Luther, J., and Kao, J. Y. (2011). Intestinal dysbiosis in inﬂammatory bowel disease. Gut Microbes 2 (4), 211–216. doi: 10.4161/ gmic.2.4.17863 Lee, J. S., Wang, R. X., Alexeev, E. E., Lanis, J. M., Battista, K. D., Glover, L. E., et al. (2018). Hypoxanthine is a checkpoint stress metabolite in colonic epithelial energy modulation and barrier function. J. Biol. Chem. 293 (16), 6039–6051. doi: 10.1074/jbc.RA117.000269 Keskin, E., Uluisik, D., and Altin, M. (2015). Antioxidant effect of l-carnitine in rats fed cholesterol rich diet. Anim. Vet. Sci. 3 (4), 113–116. doi: 10.11648/ j.avs.20150304.13 Lei, W., Ren, W., Ohmoto, M., Urban, J. F.Jr., Matsumoto, I., Margolskee, R. F., et al. (2018). Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine. Proc. Natl. Acad. Sci. 115 (21), 5552–5557. doi: 10.1073/pnas.1720758115 Khan, W. I., Blennerhasset, P. A., Varghese, A. K., Chowdhury, S. K., Omsted, P., Deng, Y., et al. (2002). Intestinal nematode infection ameliorates experimental colitis in mice. Infect. Immun. 70 (11), 5931–5937. doi: 10.1128/IAI.70.11.59315937.2002 Liaudet, L., Mabley, J. G., Soriano, F. G., Pacher, P., Marton, A., Hasko, G., et al. (2001). Inosine reduces systemic inﬂammation and improves survival in septic shock induced by cecal ligation and puncture. Am. J. Respir. Crit. Care Med. 164, 1213–1220. doi: 10.1164/ajrccm.164.7.2101013 Khodayar, M. J., Kalantari, H., Khorsandi, L., Rashno, M., and Zeidooni, L. (2020). Upregulation of Nrf2- related cytoprotective genes expression by acetaminophen-induced acute hepatotoxicity in mice and the protective role of betaine. Hum. Exp. Toxicol., 39(7), 948–959. doi: 10.1177/0960327120905962 Lindsay, A., and Gieseg, S. P. (2020). Pterins as diagnostic markers of exerciseinduced stress: a systematic review. J. Sci. Med. Sport. 23 (1), 53–62. doi: 10.1016/ j.jsams.2019.08.018 Khurana, S., Singh, S., and Mewara, A. (2021). Diagnostic techniques for soiltransmitted helminths-recent advances. Res. Rep. Trop. Med. 12, 181–196. doi: 10.2147/RRTM.S278140 Li, J. V., Saric, J., Wang, Y., Keiser, J., Utzinger, J., and Holmes, E. (2011). Chemometric analysis of bioﬂuids from mice experimentally infected with schistosoma mansoni. Parasit Vectors 4, 179:1–17916. doi: 10.1186/1756-3305-4179 Kleiner, R. E., Dumelin, C. E., and Liu, D. R. (2011). Small-molecule discovery from DNA-encoded chemical libraries. Chem. Soc. Rev. 40 (12), 5707–5717. doi: 10.1039/c1cs15076f Li, S., Song, G., Bai, Y., Song, N., Zhao, J., Liu, J., et al. (2021). Applications of protein microarrays in biomarker discovery for autoimmune diseases. Front. Immunol. 12, 1–13. doi: 10.3389/ﬁmmu.2021.645632 Kobpornchai, P., Flynn, R. J., Reamtong, O., Rittisoonthorn, N., Kosoltanapiwat, N., Boonnak, K., et al. (2020). A novel cystatin derived from Trichinella spiralis suppresses macrophage-mediated inﬂammatory responses. PloS Negl. Trop. Dis. 14 (4), 1–24. doi: 10.1371/journal.pntd.0008192 Littlewood-Evans, A., Sarret, S., Apfel, V., Loesle, P., Dawson, J., Zhang, J., et al. (2016). GPR91 senses extracellular succinate released from inﬂammatory macrophages and exacerbates rheumatoid arthritis. J. Exp. Med. 213 (9), 1655– 1662. doi: 10.1084/jem.20160061 Kovarik, J. J., Tillinger, W., Hofer, J., Holzl, M. A., Heinzl, H., Saemann, M. D., et al. (2011). Impaired anti-inﬂammatory efﬁcacy of n-butyrate in patients with IBD. Eur. J. Clin. Invest. 41 (3), 291–298. doi: 10.1111/j.1365-2362.2010.02407.x Kradin, R. L., Badizadegan, K., Auluck, P., Korzenik, J., and Lauwers, G. Y. (2006). Iatrogenic Trichuris suis infection in a patient with crohn disease. Arch. Pathol. Lab. Med. 130 (5), 718–720. doi: 10.5858/2006-130-718-ITSIIA Liu, R., Li, P., Bi, W. C., Ma, R., Yin, Y., Bi, K., et al. (2017). Plasma nacetylputrescine, cadaverine and 1,3-diaminopropane: Potential biomarkers of lung cancer used to evaluate the ef cacy of anticancer drugs. Oncotarget. 8, 88575–88585. doi: 10.18632/oncotarget.19304 Kris-Etherton, P. M., Griel, A. E., Psota, T. L., Gebaur, S. K., Zhang, J., and Etherton, T. D. (2005). Dietary stearic acid and risk of cardiovascular disease: intake, sources, digestion, and absorption. Lipids. 40, 1193–1200. doi: 10.1007/ s11745-005-1485-y Liu, X. D., Wang, X. L., Bai, X., Liu, X. L., Wu, X. P., Zhao, Y., et al. (2014). Oral administration with attenuated salmonella encoding a Trichinella cystatin-like protein elicited host immunity. Exp. Parasitol. 141, 1–11. doi: 10.1016/ j.exppara.2014.03.015 Frontiers in Parasitology 18 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 Li, X. S., Wang, Z., Cajka, T., Buffa, J. A., Nemet, I., Hurd, A. G., et al. (2018). Untargeted metabolomics identiﬁes trimethyllysine, a TMAO-producing nutrient precursor, as a predictor of incident cardiovascular disease risk. JCI Insight 3 (6), 1– 18. doi: 10.1172/jci.insight.99096 Mejia, R., Damania, A., Jeun, R., Bryan, P. E., Vargas, P., Juarez, M., et al. (2020). Impact of intestinal parasites on microbiota and cobalamin gene sequences: A pilot study. Parasit Vectors. 13 (1), 1–9. doi: 10.1186/s13071-020-04073-7 Miller, H. R., and Nawa, Y. (1979). Immune regulation of intestinal goblet cell differentiation. speciﬁc induction of nonspeciﬁc protection against helminths? Nouv Rev. Fr Hematol. 21 (1), 31–45. Longman, R. S., and Littman, D. R. (2015). The functional impact of the intestinal microbiome on mucosal immunity and systemic autoimmunity. Curr. Opin. Rheumatol. 27, 381–387. doi: 10.1097/BOR.0000000000000190 Mine, Y., and Zhang, H. (2015). Calcium-sensing receptor (CaSR)-mediated anti-inﬂammatory effects of l-amino acids in intestinal epithelial cells. J. Agric. Food Chem. 63 (45), 9987–9995. doi: 10.1021/acs.jafc.5b03749 Longo, N. (2009). Disorders of biopterin metabolism. J. Inherit Metab. Dis. 32, 333–342. doi: 10.1007/s10545-009-1067-2 Molodecky, N. A., Soon, I. S., Rabi, D. M., Ghali, W. A., Ferris, M., Chernoff, G., et al. (2012). Increasing incidence and prevalence of the inﬂammatory bowel diseases with time, based on systematic review. Gastroenterology. 142 (1), 46– 54.e42. doi: 10.1053/j.gastro.2011.10.001 Loukas, A., Hotez, P. J., Diemert, D., Yazdanbakhsh, M., McCarthy, J. S., CorreaOliveira, R., et al. (2016). Hookworm infection. Nat. Rev. Dis. Primers. 2, 1–18. doi: 10.1038/nrdp.2016.88 Loukas, A., and Prociv, P. (2001). Immune responses in hookworm infections. Clin. Microbiol. Rev. 14 (4), 689–703. doi: 10.1128/CMR.14.4.689-703.2001 Ma, E. H., Bantug, G., Griss, T., Condotta, S., Johnson, R. M., Samborska, B., et al. (2017). Serine is an essential metabolite for effector T cell expansion. Cell Metab. 25 (2), 345–357. doi: 10.1016/j.cmet.2016.12.011 Montresor, A., Mupfasoni, D., Mikhailov, A., Mwinzi, P., Lucianez, A., Jamsheed, M., et al. (2020). The global progress of soil-transmitted helminthiases control in 2020 and world health organization targets for 2030. PloS Negl. Trop. Dis. 14 (8), 1–17. doi: 10.1371/journal.pntd.0008505 Magalhaes, K., Almeida, P. E., Atella, G., Maya-Monteiro, C. M., Castro-FariaNeto, H., Pelajo-Machado, M., et al. (2010). Schistosomal-derived lysophosphatidylcholine are involved in eosinophil activation and recruitment through toll-like receptor-2-dependent mechanisms. J. Infect. Dis. 202 (9), 1369– 1379. doi: 10.1086/656477 Moreels, T. G., Nieuwendijk, R. J., De Man, J. G., De Winter, B. Y., Herman, A. G., Van Marck, E. A., et al. (2004). Concurrent infection with Schistosoma mansoni attenuates inﬂammation induced changes in colonic morphology, cytokine levels, and smooth muscle contractility of trinitrobenzene sulphonic acid induced colitis in rats. Gut. 53, 99–107. doi: 10.1136/gut.53.1.99 Maizels, R. M., Bundy, D. A. P., Selkirk, M. E., Smith, D. F., and Anderson, R. M. (1993). Immunological modulation and evasion by helminth parasites in human populations. Nature. 365, 797–805. doi: 10.1038/365797a0 Moro, K., Nagahashi, M., Ramanathan, R., Takabe, K., and Wakai, T. (2016). Resolvins and omega three polyunsaturated fatty acids: Clinical implications in inﬂammatory diseases and cancer. World J. Clin. Cases. 4 (7), 155–164. doi: 10.12998/wjcc.v4.i7.155 Maizels, R. M., and McSorley, H. J. (2016). Regulation of the host immune system by helminth parasites. J. Allergy Clin. Immunol. 138, 666–675. doi: 10.1016/j.jaci.2016.07.007 Mu, Y., McManus, D. P., Gordon, C. A., and Cai, P. (2021). Parasitic helminthderived microRNAs and extracellular vesicle cargos as biomarkers for helminthic infections. Front. Cell Infect. Microbiol. 11, 1–8. doi: 10.3389/fcimb.2021.708952 Maizels, R. M., Smits, H. H., and McSorley, H. J. (2018). Modulation of host immunity by helminths: the expanding repertoire of parasite effector molecules. Immunity. 49, 801–818. doi: 10.1016/j.immuni.2018.10.016 Murray, C. J. L., Vos, T., Lozano, R., Naghavi, M., Flaxman, A. D., Michaud, C., et al. (2012). Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the global burden of disease study 2010. Lancet 380 (9859), 2197–2223. doi: 10.1016/S0140-6736(12)61689-4 Maizels, R. M., and Yazdanbakhsh, M. (2003). Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat. Rev. Immunol. 3 (9), 733–744. doi: 10.1038/nri1183 Naügeli, M., Fasshauer, M., Sommerfeld, J., Fendel, A., Brandi, G., and Stover, J. F. (2014). Prolonged continuous intravenous infusion of the dipeptide l-alanine- lglutamine signiﬁcantly increases plasma glutamine and alanine without elevating brain glutamate in patients with severe traumatic brain injury. Crit. Care 18, R139:1–R13912. doi: 10.1186/cc13962. Makurvet, F. D. (2021). Biologics vs. small molecules: Drug costs and patient access. Med. Drug Discovery 9, 1–8. doi: 10.1016/j.medidd.2020.100075 Mangmee, S., Adisakwattana, P., Tipthara, P., Simanon, N., Sonthayanon, P., and Reamtong, O. (2020). Lipid proﬁle of Trichinella papuae muscle-stage larvae. Sci. Rep. 10 (1), 1–11. doi: 10.1038/s41598-020-67297-8 Marikovsky, M., Fishelson, Z., and Arnon, R. (1988). Puriﬁcation and characterization of proteases secreted by transforming schistosomula of schistosoma mansoni. Mol. Biochem. Parasitol. 30 (1), 45–54. doi: 10.1016/0166-6851(88)90131-4 Nadjsombati, M. S., McGinty, J. W., Lyons-Cohen, M. R., Jaffe, J. B., DiPeso, L., Schneider, C., et al. (2018). Detection of succinate by intestinal tuft cells triggers a type 2 innate immune circuit. Immunity. 49 (1), 33–41.e7. doi: 10.1016/ j.immuni.2018.06.016 Martin, I., Djuardi, Y., Sartono, E., Rosa, B. A., Supali, T., Mitreva, M., et al. (2018). Dynamic changes in human-gut microbiome in relation to a placebocontrolled anthelminthic trial in Indonesia. PloS Negl. Trop. Dis. 12 (8), 1–19. doi: 10.1371/journal.pntd.0006620 Oldenburger, A., Roscioni, S. S., Jansen, E., Menzen, M. H., Halayko, A. J., Timens, W., et al. (2012). Anti-inﬂammatory role of the cAMP effectors epac and PKA: implications in chronic obstructive pulmonary disease. PloS One 7 (2), 1–12. doi: 10.1371/journal.pone.0031574 Martin, I., Kaisar, M. M. M., Wiria, A. E., Hamid, F., Djuardi, Y., Sartono, E., et al. (2019). The effect of gut microbiome composition on human immune responses: An exploration of interference by helminth infections. Front. Genet. 10, 1–10. doi: 10.3389/fgene.2019.01028 Olivera, P., Danese, S., and Peyrin-Biroulet, L. (2017). Next generation of small molecules in inﬂammatory bowel disease. Gut. 66 (2), 199–209. doi: 10.1136/ gutjnl-2016-312912 Patton, J. B., Bonne-Annee, S., Deckman, J., Hess, J. A., Torigian, A., Nolan, T. J., et al. (2018). Methylprednisolone acetate induces, and Delta7-dafachronic acid suppresses, Strongyloides stercoralis hyperinfection in NSG mice. Proc. Natl. Acad. Sci. 115 (1), 204–209. doi: 10.1073/pnas.1712235114 Mastrofrancesco, A., Ottaviani, M., Aspite, N., Cardinali, G., Izzo, E., Graupe, K., et al. (2010). Azelaic acid modulates the inﬂammatory response in normal human keratinocytes through PPARgamma activation. Exp. Dermatol. 19 (9), 813–820. doi: 10.1111/j.1600-0625.2010.01107.x Pederzolli, C. D., Mescka, C. P., Zandona, B. R., de Moura Coelho, D., Sgaravatti, A. M., Sgarbi, M. B., et al. (2010). Acute administration of 5-oxoproline induces oxidative damage to lipids and proteins and impairs antioxidant defenses in cerebral cortex and cerebellum of young rats. Metab. Brain Dis. 25 (2), 145–154. doi: 10.1007/s11011-010-9190-1 Ma, G., Wang, T., Korhonen, P. K., Nie, S., Reid, G. E., Stroehlein, A. J., et al. (2019). Comparative bioinformatic analysis suggests that speciﬁc dauer-like signalling pathway components regulate Toxocara canis development and migration in the mammalian host. Parasit Vectors. 12 (1), 1–10. doi: 10.1186/ s13071-018-3265-y Periago, M. V., and Bethony, J. M. (2012). Hookworm virulence factors: making the most of the host. Microbes Infect. 14, 1451–1464. doi: 10.1016/ j.micinf.2012.09.002 Ma, G., Wang, T., Korhonen, P. K., Young, N. D., Nie, S., Ang, C. S., et al. (2019). Dafachronic acid promotes larval development in Haemonchus contortus by modulating dauer signalling and lipid metabolism. PloS Pathog. 15 (7), 1–20. doi: 10.1371/journal.ppat.1007960 Pineda, M. A., Eason, R. J., Harnett, M. M., and Harnett, W. (2015). From the worm to the pill, the parasitic worm product ES-62 raises new horizons in the treatment of rheumatoid arthritis. Lupus 24 (4-5), 400–411. doi: 10.1177/ 0961203314560004 McCarthy, J. S., Lustigman, S., Yang, G. J., Barakat, R. M., Garcia, H. H., Sripa, B., et al. (2012). A research agenda for helminth diseases of humans: diagnostics for control and elimination programmes. PloS Negl. Trop. Dis. 6 (4), 1–13. doi: 10.1371/journal.pntd.0001601 Pineda, M. A., Lumb, F., Harnett, M. M., and Harnett, W. (2014). ES-62, a therapeutic anti-inﬂammatory agent evolved by the ﬁlarial nematode Acanthocheilonema viteae. Mol. Biochem. Parasitol. 194 (1-2), 1–8. doi: 10.1016/ j.molbiopara.2014.03.003 McSorley, H. J., Gaze, S., Daveson, J., Jones, D., Anderson, R. P., Clouston, A., et al. (2011). Suppression of inﬂammatory immune responses in celiac disease by experimental hookworm infection. PloS One 6 (9), 1–7. doi: 10.1371/ journal.pone.0024092 Prideaux, L., Kamm, M. A., De Cruz, P. P., Chan, F. K., and Ng, S. C. (2012). Inﬂammatory bowel disease in Asia: a systematic review. J. Gastroenterol. Hepatol. 27 (8), 1266–1280. doi: 10.1111/j.1440-1746.2012.07150.x McSorley, H. J., Hewitson, J. P., and Maizels, R. M. (2013). Immunomodulation by helminth parasites: Deﬁning mechanisms and mediators. Int. J. Parasitol. 43, 301–310. doi: 10.1016/j.ijpara.2012.11.011 Frontiers in Parasitology Proudfoot, L., Kusel, J. R., Smith, H. V., Harnett, W., Worms, M. J., and Kennedy, M. W. (1990). The surface lipid of parasitic nematodes: Organization, 19 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 and modiﬁcations during transition to the mammalian host environment. Acta Trop. 47, 323–330. doi: 10.1016/0001-706X(90)90033-V biomarker discovery. PloS Negl. Trop. Dis. 2 (7), 1–15. doi: 10.1371/ journal.pntd.0000254 Qiu, W., Guo, K., Yi, L., Gong, Y., Huang, L., and Zhong, W. (2014). Resolvin E1 reduces hepatic ﬁbrosis in mice with Schistosoma japonicum infection. Exp. Ther. Med. 7 (6), 1481–1485. doi: 10.3892/etm.2014.1641 Saxena, R. N., Pendse, V. K., and Khanna, N. K. (1984). Anti-inﬂammatory and analgesic properties of four amino-acids. Indian J. Physiol. Pharmacol. 28, 299–305. Saz, H. J. (1981). Energy metabolisms of parasitic helminths: adaptations to parasitism. Annu. Rev. Physiol. 43, 323–341. doi: 10.1146/ annurev.ph.43.030181.001543 Raizel, R., Leite, J. S., Hypolito, T. M., Coqueiro, A. Y., Newsholme, P., Cruzat, V. F., et al. (2016). Determination of the anti-inﬂammatory and cytoprotective effects of l-glutamine and l-alanine, or dipeptide, supplementation in rats submitted to resistance exercise. Br. J. Nutr. 116 (3), 470–479. doi: 10.1017/S0007114516001999 Schneeberger, P. H. H., Coulibaly, J. T., Panic, G., Daubenberger, C., Gueuning, M., Frey, J. E., et al. (2018). Investigations on the interplays between Schistosoma mansoni, praziquantel and the gut microbiome. Parasit Vectors. 11 (1), 1–12. doi: 10.1186/s13071-018-2739-2 Ramaswamy, K., Kumar, P., and He, Y. X. (2000). A role for parasite-induced PGE2 in IL-10-mediated host immunoregulation by skin stage schistosomula of schistosoma mansoni. J. Immunol. 165 (8), 4567–4574. doi: 10.4049/ jimmunol.165.8.4567 Scholmerich, J., Fellermann, K., Seibold, F. W., Rogler, G., Langhorst, J., Howaldt, S., et al. (2017). A randomised, double-blind, placebo-controlled trial of Trichuris suis ova in active crohn's disease. J. Crohns Colitis. 11(4), 390–399. doi: 10.1093/ecco-jcc/jjw184 Rodrigues, H. G., Vinolo, M. A. R., Magdalon, J., Vitzel, K., Nachbar, R. T., Pessoa, A. F. M., et al. (2012). Oral administration of oleic or linoleic acid accelerates the inﬂammatory phase of wound healing. J. Invest. Dermatol. 132 (1), 208–215. doi: 10.1038/jid.2011.265 Selmer, T., and Andrei, P. I. (2001). P-hydroxyphenylacetate decarboxylase from Clostridium difﬁcile–a novel glycyl radical enzyme catalysing the formation of pcresol. Eur. J. Biochem. 268, 1363–1372. doi: 10.1046/j.1432-1327.2001.02001.x Rook, G. A. (2007). The hygiene hypothesis and the increasing prevalence of chronic inﬂammatory disorders. Trans. R Soc. Trop. Med. Hyg. 101 (11), 1072– 1074. doi: 10.1016/j.trstmh.2007.05.014 Shin, J. L., Gardiner, G. W., Deitel, W., and Kandel, G. (2004). Does whipworm increase the pathogenicity of Campylobacter jejuni? Can. J. Gastroenterol. Hepatol. 18(3), 175–177. doi: 10.1155/2004/298064 Rook, G. A. (2012). Hygiene hypothesis and autoimmune diseases. Clin. Rev. Allergy Immunol. 42 (1), 5–15. doi: 10.1007/s12016-011-8285-8 Rook, G. A. (2013). Regulation of the immune system by biodiversity from the natural environment: an ecosystem service essential to health. Proc. Natl. Acad. Sci. 110 (46), 18360–18367. doi: 10.1073/pnas.1313731110 Singhal, N. K., Sternbach, S., Fleming, S., Alkhayer, K., Shelestak, J., Popescu, D., et al. (2020). Betaine restores epigenetic control and supports neuronal mitochondria in the cuprizone mouse model of multiple sclerosis. Epigenetics, 15 (8), 871–886. doi: 10.1080/15592294.2020.1735075 Rook, G. A., Raison, C. L., and Lowry, C. A. (2014). Microbial 'old friends', immunoregulation and socioeconomic status. Clin. Exp. Immunol. 177 (1), 1–12. doi: 10.1111/cei.12269 Smith, V. P., Selkirk, M. E., and Gounaris, K. (1996). Identiﬁcation and composition of lipid classes in surface and somatic preparationss of adult brugia malayi. Mol. Biochem. Parasitol. 78, 105–116. doi: 10.1016/S0166-6851(96)02615-1 Rosa, B. A., Supali, T., Gankpala, L., Djuardi, Y., Sartono, E., Zhou, Y., et al. (2018). Differential human gut microbiome assemblages during soil-transmitted helminth infections in Indonesia and Liberia. Microbiome. 6 (1), 1–19. doi: 10.1186/s40168-018-0416-5 Smyth, D. J., Harcus, Y., White, M. P. J., Gregory, W. F., Nahler, J., Stephens, I., et al. (2018). TGF-beta mimic proteins form an extended gene family in the murine parasite heligmosomoides polygyrus. Int. J. Parasitol. 48 (5), 379–385. doi: 10.1016/ j.ijpara.2017.12.004 Ruddigkeit, L., van Deursen, R., Blum, L. C., and Reymond, J. L. (2012). Enumeration of 166 billion organic small molecules in the chemical universe database GDB-17. J. Chem. Inf Model. 52 (11), 2864–2875. doi: 10.1021/ci300415d Sokol, H., Pigneur, B., Watterlot, L., Lakhdari, O., Bermudez-Humaran, L. G., Gratadoux, J. J., et al. (2008). Faecalibacterium prausnitzii is an anti-inﬂammatory commensal bacterium identiﬁed by gut microbiota analysis of crohn disease patients. Proc. Natl. Acad. Sci. 105 (43), 16731–16736. doi: 10.1073/ pnas.0804812105 Ruyssers, N. E., De Winter, B. Y., De Man, J. G., Loukas, A., Herman, A. G., Pelckmans, P. A., et al. (2008). Worms and the treatment of inﬂammatory bowel disease: are molecules the answer? Clin. Dev. Immunol. 2008, 1–7. doi: 10.1155/ 2008/567314 Sotillo, J., Robinson, M. W., Kimber, M. J., Cucher, M., Ancarola, M. E., Nejsum, P., et al. (2020). The protein and microRNA cargo of extracellular vesicles from parasitic helminths - current status and research priorities. Int. J. Parasitol. 50 (9), 635–645. doi: 10.1016/j.ijpara.2020.04.010 Ryan, S. M., Eichenberger, R. M., Ruscher, R., Giacomin, P. R., and Loukas, A. (2020). Harnessing helminth-driven immunoregulation in the search for novel therapeutic modalities. PloS Pathog. 16 (5), 1–20. doi: 10.1371/ journal.ppat.1008508 Spreaﬁco, A., Millucci, L., Ghezzi, L., Geminiani, M., Braconi, D., Amato, L., et al. (2013). Antioxidants inhibit SAA formation and pro-inﬂammatory cytokine release in a human cell model of alkaptonuria. Rheumatology. 52 (9), 1667–1673. doi: 10.1093/rheumatology/ket185 Rzepecka, J., Coates, M. L., Saggar, M., Al-Riyami, L., Coltherd, J., Tay, H. K., et al. (2014). Small molecule analogues of the immunomodulatory parasitic helminth product ES-62 have anti-allergy properties. Int. J. Parasitol. 44 (9), 669–674. doi: 10.1016/j.ijpara.2014.05.001 Stilling, R. M., Bordenstein, S. R., Dinan, T. G., and Cryan, J. F. (2014). Friends with social beneﬁts: host-microbe interactions as a driver of brain evolution and development? Front. Cell Infect. Microbiol. 4, 1–17. doi: 10.3389/fcimb.2014.00147 Safer, D., Brenes, M., Dunipace, S., and Schad, S. (2007). Urocanic acid is a major chemoattractant for the skin-penetrating parasitic nematode strongyloides stercoralis. Proc. Natl. Acad. Sci. 104, 1627–1630. doi: 10.1073/pnas.0610193104 Stoltzfus, J. D., Minot, S., Berriman, M., Nolan, T. J., and Lok, J. B. (2012). RNAseq analysis of the parasitic nematode Strongyloides stercoralis reveals divergent regulation of canonical dauer pathways. PloS Negl. Trop. Dis. 6 (10), 1–24. doi: 10.1371/journal.pntd.0001854 Sag, D., Carling, D., Stout, R. D., and Suttles, J. (2008). Adenosine 5'monophosphate-activated protein kinase promotes macrophage polarization to an anti-inﬂammatory functional phenotype. J. Immunol. 181 (12), 8633–8641. doi: 10.4049/jimmunol.181.12.8633 Strachan, D. P. (1989). Hay fever, hygiene, and household size. Br. Med. J. 299, 1259–1260. doi: 10.1136/bmj.299.6710.1259 Sakoguchi, H., Yoshihara, A., Izumori, K., and Sato, M. (2016). Screening of biologically active monosaccharides: Growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode caenorhabditis elegans. Biosci. Biotechnol. Biochem. 80, 1058–1061. doi: 10.1080/09168451.2016.1146069 Su, C. W., Chen, C. Y., Jiao, L., Long, S. R., Mao, T., Ji, Q., et al. (2020). Helminth-induced and Th2-dependent alterations of the gut microbiota attenuate obesity caused by high-fat diet. Cell Mol. Gastroenterol. Hepatol. 10 (4), 763–778. doi: 10.1016/j.jcmgh.2020.06.010 Salafsky, B., Wang, Y.-S., Kevin, M. B., Hill, H., and Fusco, A. C. (1984). The role of prostaglandins in cercarial (Schistosoma mansoni) response to free fatty acids. J. Parasitol. 70 (4), 584–591. doi: 10.2307/3281410 Su, C. W., Chen, C. Y., Li, Y., Long, S. R., Massey, W., Kumar, D. V., et al. (2018). Helminth infection protects against high fat diet-induced obesity via induction of alternatively activated macrophages. Sci. Rep. 8 (1), 1–14. doi: 10.1038/s41598-01822920-7 Salter, J. P., Lim, K. C., Hansell, E., Hsieh, I., and McKerrow, J. H. (2000). Schistosome invasion of human skin and degradation of dermal elastin are mediated by a single serine protease. J. Biol. Chem. 275 (49), 38667–38673. doi: 10.1074/jbc.M006997200 Sultan, S., El-Mowafy, M., Elgaml, A., Ahmed, T. A. E., Hassan, H., and Mottawea, W. (2021). Metabolic inﬂuences of gut microbiota dysbiosis on inﬂammatory bowel disease. Front. Physiol. 12, 1–27. doi: 10.3389/ fphys.2021.715506 Sanchez, A. L., Gabrie, J. A., Rueda, M. M., Mejia, R. E., Bottazzi, M. E., and Canales, M. (2014). A scoping review and prevalence analysis of soil-transmitted helminth infections in Honduras. PloS Negl. Trop. Dis. 8 (1), 1–15. doi: 10.1371/ journal.pntd.0002653 Summers, R. (2003). Trichuris suis seems to be safe and possibly effective in the treatment of inﬂammatory bowel disease. Am. J. Gastroenterol. 98 (9), 2034–2041. doi: 10.1111/j.1572-0241.2003.07660.x Saric, J., Li, J. V., Utzinger, J., Wang, Y., Keiser, J., Dirnhofer, S., et al. (2010). Systems parasitology: effects of Fasciola hepatica on the neurochemical proﬁle in the rat brain. Mol. Syst. Biol. 6, 1–10. doi: 10.1038/msb.2010.49 Summers, R. W., Elliott, D. E., Urban, J. F.Jr., Thompson, R., and Weinstock, J. V. (2005). Trichuris suis therapy in crohn's disease. Gut. 54 (1), 87–90. doi: 10.1136/ gut.2004.041749 Saric, J., Li, J. V., Wang, Y., Keiser, J., Bundy, J. G., Holmes, E., et al. (2008). Metabolic proﬁling of an Echinostoma caproni infection in the mouse for Sun, X. M., Guo, K., Hao, C. Y., Zhan, B., Huang, J. J., and Zhu, X. (2019). Trichinella spiralis excretory-secretory products stimulate host regulatory T cell Frontiers in Parasitology 20 frontiersin.org 10.3389/fpara.2022.984152 Yeshi et al. differentiation through activating dendritic cells. Cells. 8 (11), 1–19. doi: 10.3390/ cells8111404 Wangchuk, P., Lavers, O., Wishart, D. S., and Loukas, A. (2020). Excretory/ secretory metabolome of the zoonotic roundworm parasite toxocara canis. Biomolecules 10, 1–18. doi: 10.3390/biom10081157 Sun, R., Zhao, X., Wang, Z., Yang, J., Zhao, L., Zhan, B., et al. (2015). Trichinella spiralis paramyosin binds human complement C1q and inhibits classical complement activation. PloS Negl. Trop. Dis. 9 (12), 1–14. doi: 10.1371/ journal.pntd.0004310 Wangchuk, P., Shepherd, C., Constantinoiu, C., Ryan, R. Y. M., Kouremenos, K. A., Becker, L., et al. (2019). Hookworm-derived metabolites suppress pathology in a mouse model of colitis and inhibit secretion of key inﬂammatory cytokines in primary human leukocytes. Infect. Immun. 87 (4), 1–19. doi: 10.1128/IAI.00851-18 Szel, E., Polyanka, H., Szabo, K., Hartmann, P., Degovics, D., Balazs, B., et al. (2015). Anti-irritant and anti-inﬂammatory effects of glycerol and xylitol in sodium lauryl sulphate-induced acute irritation. J. Eur. Acad. Dermatol. Venereol. 29 (12), 2333–2341. doi: 10.1111/jdv.13225 Wang, Z., Hao, C., Zhuang, Q., Zhan, B., Sun, X., Huang, J., et al. (2020). Excretory/secretory products from Trichinella spiralis adult worms attenuated DSS-induced colitis in mice by driving PD-1-mediated M2 acrophage polarization. Front. Immunol. 11, 1–12. doi: 10.3389/ﬁmmu.2020.563784 Tang, X., Liu, J., Dong, W., Li, P., Li, L., Lin, C., et al. (2013). The cardioprotective effects of citric acid and l-malic acid on myocardial ischemia/ reperfusion injury. Evid Based Complement Altern. Med. 2013, 1–11. doi: 10.1155/ 2013/820695 Wang, Y., Holmes, E., Nicholson, J. K., Cloarec, O., Chollet, J., Tanner, M., et al. (2004). Metabonomic investigations in mice infected with Schistosoma mansoni: an approach for biomarker identiﬁcation. Proc. Natl. Acad. Sci. 101 (34), 12676– 12681. doi: 10.1073/pnas.0404878101 Tang, B., Liu, M., Wang, L., Yu, S., Shi, H., Boireau, P., et al. (2015). Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage. Parasit Vectors. 8, 1–12. doi: 10.1186/s13071-015-0689-5 Wang, T., Nie, S., Ma, G., Korhonen, P. K., Koehler, A. V., Ang, C. S., et al. (2018). The developmental lipidome of haemonchus contortus. Int. J. Parasitol. 48 (12), 887–895. doi: 10.1016/j.ijpara.2018.06.002 Tedelind, S., Westberg, F., Kjerrulf, M., and Vidal, A. (2007). Anti-inﬂammatory properties of the short-chain fatty acids acetate and propionate: A study with relevance to inﬂammatory bowel disease. World J. Gastroenterol. 13 (20), 2826– 2832. doi: 10.3748/wjg.v13.i20.2826 Wang, T., Nie, S., Ma, G., Vlaminck, J., Geldhof, P., Williamson, N. A., et al. (2020). Quantitative lipidomic analysis of ascaris suum. PloS Negl. Trop. Dis. 14 (12), 1–19. doi: 10.1371/journal.pntd.0008848 Tielens, A. G. M. (1997). Biochemistry of trematodes. 1st ed (Boca Raton, Florida: CRC Press). Wang, Y., Utzinger, J., Xiao, S. H., Xue, J., Nicholson, J. K., Tanner, M., et al. (2006). System level metabolic effects of a Schistosoma japonicum infection in the Syrian hamster. Mol. Biochem. Parasitol. 146 (1), 1–9. doi: 10.1016/j.molbiopara.2005.10.010 Ullah, H., Qadeer, A., Rashid, M., Rashid, M. I., and Cheng, G. (2020). Recent advances in nucleic acid-based methods for detection of helminth infections and the perspective of biosensors for future development. Parasitology. 147 (4), 383– 392. doi: 10.1017/S0031182019001665 Wang, Y., Xiao, S.-H., Xue, J., Singer, B. H., Utzinger, J., and Holmes, E. (2009). Systems metabolic effects of a Necator americanus infection in Syrian hamster. J. Proteome Res. 8, 5442–5450. doi: 10.1021/pr900711j Wang, Z., Zhou, X. E., Motola, D. L., Gao, X., Suino-Powell, K., Conneely, A., et al. (2009). Identiﬁcation of the nuclear receptor DAF-12 as a therapeutic target in parasitic nematodes. Proc. Natl. Acad. Sci. 106 (23), 9138–9143. doi: 10.1073/pnas.0904064106 Ullman, B., Gudas, L. J., Cohen, A., and Martin, D. W. (1978). Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: A model for immunodeﬁciency disease. Cell. 14, 365–375. doi: 10.1016/0092-8674(78)90122-8 Ward, P. F. (1982). Aspects of helminth metabolism. Parasitology. 84, 177–194. doi: 10.1017/S0031182000051775 Unnikrishnan, M. K., and Rao, M. N. A. (1990). Anti-inﬂammatory activity of methionine, methionine sulfoxide and methionine sulfone. Agents Actions. 31, 110–112. doi: 10.1007/BF02003229 Weinstock, J. V., and Elliott, D. E. (2009). Helminths and the IBD hygiene hypothesis. Inﬂammation Bowel Dis. 15 (1), 128–133. doi: 10.1002/ibd.20633 Van de Merwe, J. P., Schroder, A. M., Wensinck, F., and Hazenberg, M. P. (1988). The obligate anaerobic faecal ﬂora of patients with crohn’s disease and their ﬁrst-degree relatives. Scand. J. Gastroenterol. 23, 1125–1131. doi: 10.3109/ 00365528809090179 Werkman, M., Wright, J. E., Truscott, J. E., Easton, A. V., Oliveira, R. G., Toor, J., et al. (2018). Testing for soil-transmitted helminth transmission elimination: Analysing the impact of the sensitivity of different diagnostic tools. PloS Negl. Trop. Dis. 12 (1), 1–20. doi: 10.1371/journal.pntd.0006114 Varyani, F., Fleming, J. O., and Maizels, R. M. (2017). Helminths in the gastrointestinal tract as modulators of immunity and pathology. Am. J. Physiol. Gastrointest Liver Physiol. 312 (6), G537–GG49. doi: 10.1152/ajpgi.00024.2017 Wewer, V., Makepeace, B. L., Tanya, V. N., Peisker, H., Pfarr, K., Hoerauf, A., et al. (2017). Lipid proﬁling of the ﬁlarial nematodes Onchocerca volvulus, Onchocerca ochengi and Litomosoides sigmodontis reveals the accumulation of nematode-speciﬁc ether phospholipids in the host. Int. J. Parasitol. 47 (14), 903– 912. doi: 10.1016/j.ijpara.2017.06.001 Velvizhi, S., Nagalashmi, T., Essa, M. M., Dakshayani, K. B., and Subramanian, P. (2002). Effects of alpha-ketoglutarate on lipid peroxidation and antioxidant status during chronic ethnol administration in wistar rats. Pol. J. Pharmacol. 54, 231–236. Wewer, V., Peisker, H., Gutbrod, K., Al-Bahra, M., Menche, D., Amambo, N. G., et al. (2021). Urine metabolites for the identiﬁcation of Onchocerca volvulus infections in patients from Cameroon. Parasit Vectors. 14 (1), 1–11. doi: 10.1186/s13071-021-04893-1 Versini, M., Jeandel, P. Y., Bashi, T., Bizzaro, G., Blank, M., and Shoenfeld, Y. (2015). Unraveling the hygiene hypothesis of helminthes and autoimmunity: origins, pathophysiology, and clinical applications. BMC Med. 13, 1–16. doi: 10.1186/s12916-015-0306-7 Whitman, J. D., Sakanari, J. A., and Mitreva, M. (2021). Areas of metabolomic exploration for helminth infections. ACS Infect. Dis. 7 (2), 206–214. doi: 10.1021/ acsinfecdis.0c00658 Voldsgaard, A., Bager, P., Garde, E., Akeson, P., Leffers, A. M., Madsen, C. G., et al. (2015). Trichuris suis ova therapy in relapsing multiple sclerosis is safe but without signals of beneﬁcial effect. Mult Scler. 21 (13), 1723–1729. doi: 10.1177/ 1352458514568173 WHO (2020). Available at: https://www.who.int/news-room/fact-sheets/detail/ soil-transmitted-helminth-infections. Winter, A. N., Brenner, M. C., Punessen, N., Snodgrass, M., Byars, C., Arora, Y., et al. (2017). Comparison of the neuroprotective and anti-inﬂammatory effects of the anthocyanin metabolites, protocatechuic acid and 4-hydroxybenzoic acid. Oxid. Med. Cell Longev. 2017, 1–13. doi: 10.1155/2017/6297080 von Moltke, J., Ji, M., Liang, H. E., and Locksley, R. M. (2016). Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit. Nature. 529 (7585), 221–225. doi: 10.1038/nature16161 Vos, T., Flaxman, A. D., Naghavi, M., Lozano, R., Michaud, C., Ezzati, M., et al. (2012). Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990–2010: a systematic analysis for the global burden of disease study 2010. Lancet 380 (9859), 2163–2196. doi: 10.1016/S0140-6736(12)61729-2 Wishart, D. S., Feunang, Y. D., Marcu, A., Guo, A. C., Liang, K., Vazquez-Fresno, R., et al. (2018). HMDB 4.0: The human metabolome database for 2018. Nucleic Acids Res. 46 (D1), D608–DD17. doi: 10.1093/nar/gkx1089 Wang, Z., Cheong, M. C., Tsien, J., Deng, H., Qin, T., Stoltzfus, J. D., et al. (2021). Characterization of the endogenous DAF-12 ligand and its use as an anthelmintic agent in strongyloides stercoralis. Elife 10, 1–25. doi: 10.7554/eLife.73535 Wishart, D. S., Knox, C., Guo, A. C., Eisner, R., Young, N., Gautam, B., et al. (2009). HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res. 37 (Database issue), D603–D610. doi: 10.1093/nar/gkn810 Wangchuk, P., Anderson, D., Yeshi, K., and Loukas, A. (2021). Identiﬁcation of small molecules of the infective stage of human hookworm using LCMS-based metabolomics and lipidomics protocols. ACS Infect. Dis. 7 (12), 3264–3276. doi: 10.1021/acsinfecdis.1c00428 Wu, J., He, C., Bu, J., Luo, Y., Yang, S., Ye, C., et al. (2020). Betaine attenuates LPS-induced downregulation of occludin and claudin-1 and restores intestinal barrier function. BMC Vet. Res. 16 (1), 1–8. doi: 10.1186/s12917-020-02298-3 Wangchuk, P., Constantinoiu, C., Eichenberger, R. M., Field, M., and Loukas, A. (2019). Characterization of tapeworm metabolites and their reported biological activities. Molecules. 24 (8), 1–13. doi: 10.3390/molecules24081480 Wu, J. F., Holmes, E., Xue, J., Xiao, S. H., Singer, B. H., Tang, H. R., et al. (2010). Metabolic alterations in the hamster co-infected with Schistosoma japonicum and necator americanus. Int. J. Parasitol. 40 (6), 695–703. doi: 10.1016/ j.ijpara.2009.11.003 Wangchuk, P., Kouremenos, K., Eichenberger, R. M., Pearson, M., Susianto, A., Wishart, D. S., et al. (2019). Metabolomic proﬁling of the excretory-secretory products of hookworm and whipworm. Metabolomics. 15 (7), 1–15. doi: 10.1007/ s11306-019-1561-y Wu, T., Wang, C., Ding, L., Shen, Y., Cui, H., Wang, M., et al. (2016). Arginine relieves the inﬂammatory response and enhances the casein expression in bovine mammary epithelial cells induced by lipopolysaccharide. Mediators Inﬂamm. 2016, 1–10. doi: 10.1155/2016/8696481 Frontiers in Parasitology 21 frontiersin.org Yeshi et al. 10.3389/fpara.2022.984152 Wu, J., Xu, W., Ming, Z., Dong, H., Tang, H., and Wang, Y. (2010). Metabolic changes reveal the development of schistosomiasis in mice. PloS Negl. Trop. Dis. 4 (8), 1–11. doi: 10.1371/journal.pntd.0000807 modulate allergic inﬂammation. Immunity. 43 (5), 998–1010. doi: 10.1016/ j.immuni.2015.09.012 Yamaguchi, H., Maruyama, T., Urade, Y., and Nagata, S. (2014). Immunosuppression via F adenosine receptor activation by adenosine monophosphate released from apoptotic cells. elife. 3, 1–15. doi: 10.7554/ eLife.02172 Zakeri, A., Hansen, E. P., Andersen, S. D., Williams, A. R., and Nejsum, P. (2018). Immunomodulation by helminths: Intracellular pathways and extracellular vesicles. Front. Immunol. 9, 1–21. doi: 10.3389/ﬁmmu.2018.02349 Zhang, B., and Gems, D. (2021). Gross ways to live long: Parasitic worms as an anti-inﬂammaging therapy? Elife 10, 1–12. doi: 10.7554/eLife.65180 Yang, D., Zhang, Y., Nguyen, H. G., Koupenova, M., Chauhan, A. K., Makitalo, M., et al. (2006). The A2B adenosine receptor protects against inﬂammation and excessive vascular adhesion. J. Clin. Invest. 116 (7), 1913–1923. doi: 10.1172/ JCI27933 Zhao, L., Shao, S., Chen, Y., Sun, X., Sun, R., Huang, J., et al. (2017). Trichinella spiralis calreticulin binds human complement C1q as an immune evasion strategy. Front. Immunol. 8, 1–15. doi: 10.3389/ﬁmmu.2017.00636 Zheng, W. B., Zou, Y., Elsheikha, H. M., Liu, G. H., Hu, M. H., Wang, S. L., et al. (2019). Serum metabolomic alterations in beagle dogs experimentally infected with toxocara canis. Parasit Vectors. 12 (1), 1–10. doi: 10.1186/s13071-019-3703-5 Yeshi, K., Creek, D. J., Anderson, D., Ritmejerytė, E., Becker, L., Loukas, A., et al. (2020). Metabolomes and lipidomes of the infective stages of the gastrointestinal nematodes, Nippostrongylus brasiliensis and trichuris muris. Metabolites 10 (11), 1– 39. doi: 10.3390/jcm9051273 Zhi, X., Zhou, X. E., Melcher, K., Motola, D. L., Gelmedin, V., Hawdon, J., et al. (2012). Structural conservation of ligand binding reveals a bile acid-like signaling pathway in nematodes. J. Biol. Chem. 287 (7), 4894–4903. doi: 10.1074/ jbc.M111.315242 Yeshi, K., Ruscher, R., Hunter, L., Daly, N. L., Loukas, A., and Wangchuk, P. (2020). Revisiting inﬂammatory bowel disease: Pathology, treatments, challenges and emerging therapeutics including drug leads from natural products. J. Clin. Med. 9 (5), 1-39. doi: 10.3390/jcm9051273 Zhu, H. L., Liu, Y. L., Xie, X. L., Huang, J. J., and Hou, Y. Q. (2013). Effect of larginine on intestinal mucosal immune barrier function in weaned pigs after Escherichia coli LPS challenge. Innate Immun. 19, 242–252. doi: 10.1177/ 1753425912456223 Yue, Y., Guo, Y., and Yang, Y. (2017). Effects of dietary l-tryptophan supplementation on intestinal response to chronic unpredictable stress in broilers. Amino Acids 49 (7), 1227–1236. doi: 10.1007/s00726-017-2424-3 Zhu, W., Winter, M. G., Byndloss, M. X., Spiga, L., Duerkop, B. A., Hughes, E. R., et al. (2018). Precision editing of the gut microbiota ameliorates colitis. Nature. 553 (7687), 208–211. doi: 10.1038/nature25172 Zaiss, M. M., Rapin, A., Lebon, L., Dubey, L. K., Mosconi, I., Sarter, K., et al. (2015). The intestinal microbiota contributes to the ability of helminths to Frontiers in Parasitology 22 frontiersin.org
https://openalex.org/W2121824037	https://pure.rug.nl/ws/files/39471625/20140588.full.pdf	English	null	Optimal orientation in flows: providing a benchmark for animal movement strategies	Journal of the Royal Society interface	2,014	cc-by	11,503	University of Groningen University of Groningen Optimal orientation in flows McLaren, James D.; Shamoun-Baranes, Judy; Dokter, Adriaan M.; Klaassen, Raymond H. G.; Bouten, Willem Published in: Journal of the Royal Society Interface DOI: 10.1098/rsif.2014.0588 DOI: 10.1098/rsif.2014.0588 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Document Version Publisher's PDF, also known as Version of record Link to publication in University of Groningen/UMCG research database Citation for published version (APA): McLaren, J. D., Shamoun-Baranes, J., Dokter, A. M., Klaassen, R. H. G., & Bouten, W. (2014). Optimal orientation in flows: Providing a benchmark for animal movement strategies. Journal of the Royal Society Interface, 11(99), Article 20140588. https://doi.org/10.1098/rsif.2014.0588 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Citation for published version (APA): McLaren, J. D., Shamoun-Baranes, J., Dokter, A. M., Klaassen, R. H. G., & Bouten, W. (2014). Optimal orientation in flows: Providing a benchmark for animal movement strategies. Journal of the Royal Society Interface, 11(99), Article 20140588. https://doi.org/10.1098/rsif.2014.0588 Subject Areas: biogeography, computational biology, environmental science Research 2Dutch Centre for Avian Migration and Demography, Department of Animal Ecology, Netherlands Institute for Ecology (NIOO-KNAW), Wageningen, The Netherlands Cite this article: McLaren JD, Shamoun- Baranes J, Dokter AM, Klaassen RHG, Bouten W. 2014 Optimal orientation in flows: providing a benchmark for animal movement strategies. J. R. Soc. Interface 11: 20140588. http://dx.doi.org/10.1098/rsif.2014.0588 3Dutch Montagu’s Harrier Foundation, Animal Ecology Group, University of Groningen, Groningen, The Netherlands Animal movements in air and water can be strongly affected by experienced flow. While various flow-orientation strategies have been proposed and observed, their performance in variable flow conditions remains unclear. We apply control theory to establish a benchmark for time-minimizing (opti- mal) orientation. We then define optimal orientation for movement in steady flow patterns and, using dynamic wind data, for short-distance mass move- ments of thrushes (Turdus sp.) and 6000 km non-stop migratory flights by great snipes, Gallinago media. Relative to the optimal benchmark, we assess the efficiency (travel speed) and reliability (success rate) of three generic orientation strategies: full compensation for lateral drift, vector orienta- tion (single-heading movement) and goal orientation (continually heading towards the goal). Optimal orientation is characterized by detours to regions of high flow support, especially when flow speeds approach and exceed the animal’s self-propelled speed. In strong predictable flow (short distance thrush flights), vector orientation adjusted to flow on departure is nearly opti- mal, whereas for unpredictable flow (inter-continental snipe flights), only goal orientation was near-optimally reliable and efficient. Optimal orientation pro- vides a benchmark for assessing efficiency of responses to complex flow conditions, thereby offering insight into adaptive flow-orientation across taxa in the light of flow strength, predictability and navigation capacity. Received: 3 June 2014 Accepted: 30 June 2014 Received: 3 June 2014 Accepted: 30 June 2014 Received: 3 June 2014 Accepted: 30 June 2014 Research Cite this article: McLaren JD, Shamoun- Baranes J, Dokter AM, Klaassen RHG, Bouten W. 2014 Optimal orientation in flows: providing a benchmark for animal movement strategies. J. R. Soc. Interface 11: 20140588. http://dx.doi.org/10.1098/rsif.2014.0588 1Computational Geo-Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands Copyright strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of th or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from Keywords: flow orientation, animal navigation, migration, lateral drift, optimization, movement ecology James D. McLaren1, Judy Shamoun-Baranes1, Adriaan M. Dokter1,2, Raymond H. G. Klaassen2,3 and Willem Bouten1 Research Cite this article: McLaren JD, Shamoun- Baranes J, Dokter AM, Klaassen RHG, Bouten W. 2014 Optimal orientation in flows: providing a benchmark for animal movement strategies. J. R. Soc. Interface 11: 20140588. http://dx.doi.org/10.1098/rsif.2014.0588 rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 2 We first determined optimal orientation and assessed each generic orientation strategy in two commonly occurring flow patterns: (i) a gradient in lateral flow along the journey (hereafter, shear flow), emulating, for example, the transition between trade winds and westerly winds with increasing lati- tude for a migratory bird [2] and (ii) purely rotational flow, emulating mid-latitude (anti-)cyclones [39]. For each strategy, we determined the resulting flow support (proportional gain in travel speed due to flow) for movement within each pat- tern with various flow strengths, and assessed the resultant reliability and efficiency. For simplicity, we assumed steady (time-invariant) flow. Our primary aim is to quantify optimal orientation to specified goals assuming constant self-speeds, as a bench- mark for studying the efficiency of animal movement in horizontal flow regimes. While ignoring vertical structure is inappropriate for soaring and buoyant taxa [15–17], long- distance movements among some flapping [18–20] and swimming [21,22] taxa seem to be largely horizontal once selection of appropriate vertical strata is made (but see e.g. [23,24]). For simplicity, we also assume constant self- speeds and purely horizontal movement. We solve optimal orientation using results from optimal control theory, which reduces the seemingly incalculable problem of solving opti- mal headings at every potential point in space and time to an initial-value problem in which only the initial heading needs to be solved [25]. The origins of optimal control theory can be traced from Bernoulli in the seventeenth cen- tury [26] to twentieth century aviation and pursuit studies [27–29]. Somewhat counterintuitively, time-optimal orien- tation in horizontal flow involves continual alteration of headings to steer towards flow which is less supportive of the current travel direction [27,28,30]. Though seldom applied in ecology, optimal control was recently used to esti- mate minimum wind speeds required for albatrosses to maintain dynamic soaring in vertical wind shear [31]. In addition to these steady flow patterns, we also determi- ned optimal orientation and assessed the generic strategies in time-varying flow for two avian migration systems using an individual-based model [36] together with publically available global wind data [40,41]. First, we simulated 14 mass nocturnal migration events across the North Sea which included large numbers of Eurasian redwings (Turdus iliacus) and song thrushes (Turdus philomelos) [42] of Scandinavian origin. rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 2 Analy- sis of radar tracking revealed these events involved a flexible reaction to wind facilitating arrival in The Netherlands [42], which according to ring-recovery data is a preferred autumn destination for these thrush populations [43,44]. In reconstruct- ing these events, we therefore assumed that these migrants aimed to arrive on land within 100 km of a specific goal location in The Netherlands (538 N 68 W), and that flow was predictable to the extent that vector-orienting migrants adjusted their head- ings on departure to ensure arrival. Secondly, we simulated 33 seasons of long and fast non-stop flights by great snipes (Gallinago media) from Scandinavia to within 250 km of a location in a prevalent stopover area in West Africa ([45] and RHG Klaassen 2011-2012, unpublished data). We assumed that great snipes could not predict flow conditions over conti- nental distances, so we chose a single vector-orienting heading for the entire 33-year period which maximized the resultant overall success rate (cf. [37]). Over larger distances, we expect that animals will not be capable of achieving or perhaps even approaching the omnis- cience required to optimally orient. Moreover, the ability to gauge or predict flow (e.g. [32,33]) remains challenging to assess, as does the accuracy and hierarchy among navigational mechanisms, i.e. when and to what extent these are used during movement [34,35]. Nonetheless by comparing the extent to which other strategies approach the time-efficiency of optimal orientation, we can gain insight into the adaptive benefit of flow information and more sophisticated orientation strategies. Our second aim is therefore to demonstrate how optimal orien- tation can be used as a benchmark to assess the absolute and relative efficacy of proposed animal orientation strategies. We evaluate the robustness to flow variability of three generic flow-orientation strategies, assessed by reliability (proportional arrival over varying conditions, hereafter success rate, pA) and efficiency (travel speed relative to optimal orientation, 1): In summary, we quantify optimal orientation in horizon- tal flow for a time-minimizing animal travelling to a specific destination, providing a benchmark to evaluate different possible orientation strategies, and demonstrate its use in eval- uating generic orientation strategies in steady flow patterns and contrasting migration systems in time-varying flow. 2. Material and methods (1) Full compensation: based on continual adjustment of heading to maintain a constant track direction (great circles or rhumblines on a sphere). 1. Introduction Author for correspondence: James D. McLaren e-mail: j.d.mclaren@uva.nl Advection by the surrounding flow can be of paramount importance to an animal moving in water or air [1–3]. By adjusting its body orientation (heading) or self-propelled speed relative to the moving flow (self-speed), an animal can influence its resultant track direction and speed of travel in relation to the ground (ground speed; see Glossary for terms relevant to this study) [3,4]. Hence an organism’s response to flow conditions, or lack thereof, will influence its travel duration, route, total energy expenditure, and whether a destination (goal) can actually be reached [5,6]. Not surprisingly, animals across taxa have evolved mechanisms to gauge and react to the surrounding flow [2,7–10]. In nature, flow conditions often vary unpredictably, especially at longer spatial and temporal scales [11]. This can present a formidable challenge to an animal aiming to minimize its duration of travel to a specific goal [12]. Suc- cessful arrival requires adjustment of headings to compensate for any cumulative lateral drift. This can be accomplished either by gauging and com- pensating for currently experienced drift or by using a map sense to reorient towards the goal [12]. The time-minimizing orientation strategy to reach a & 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. u(t) ¼ uw(x, y, t) þ sin c(t) (2:1) and v(t) ¼ vw(x, y, t) þ cos c(t), (2:2) on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from These strategies and their relevance to animal movement are described in more detail in appendix A (see also Glossary and [3,4]). They are by no means exhaustive, necessarily attainable or expected to be favourable in given flow scenarios. While more sophisticated responses to horizon- tal flow have been proposed [12,38], underlying animal orientation strategies remain to be quantified. goal in unpredictable flow has been proposed to involve minimizing the remaining distance to the goal in a sequence of steps, resulting in an increased degree of compensation on approach to the goal [12,13]. However, in order to determine how reliable and efficient different flow strategies are in given flow conditions, it is helpful to have a benchmark for the absolute fastest, i.e. time-minimizing response. This response (hereafter, optimal orientation) assumes that an animal has perfect information about flow conditions at any time and location, analogous to the omniscient forager in optimal foraging theory [14]. 2 on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from the rates of change in longitude Ø ¼ Ø(t) and latitude u ¼ u(t): xc(t) ¼ x0 þ t cos cc 0 W y0t þ t2 2 sin cc 0 , yc(t) ¼ y0 þ t sin cc 0 , x(t) ¼ 1 2W ln Wt þ cot c 0 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ (Wt þ cot c 0)2 q cot c 0 1= sin c 0 0 @ 1 A t 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ (Wt þ cot c 0)2 q þ 1 2W sin c 0 (Wt þ cot c 0) and y(t) ¼ 1 2W ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ (Wt þ cot c 0)2 q 1 sin c 0 : xc(t) ¼ x0 þ t cos cc 0 W y0t þ t2 2 sin cc 0 , yc(t) ¼ y0 þ t sin cc 0 , xc(t) ¼ x0 þ t cos cc 0 W y0t þ t2 2 sin cc 0 , d dt ¼ Va sin c þ Uw(, u, t) Re cos u and du dt ¼ Va cos c þ Vw(, u, t) Re , yc(t) ¼ y0 þ t sin cc 0 , x(t) ¼ 1 2W ln Wt þ cot c 0 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ (Wt þ cot c 0)2 q cot c 0 1= sin c 0 0 @ 1 A t 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ (Wt þ cot c 0)2 q þ 1 2W sin c 0 (Wt þ cot c 0) and y(t) ¼ 1 2W ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ (Wt þ cot c 0)2 q 1 sin c 0 : where Uw and Vw are the (dimensional) eastward and northward flow velocities, Re is the Earth’s radius, and the heading c is measured clockwise from geographic north. on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 3 Optimal headings change linearly in time in rotational flow c(t) ¼ c 0 Wt, and optimal trajectories follow (via eqn 30 in [30]): x(t) ¼ x0 cos Wt y0 sin Wt þ t sin (c 0 Wt) and y(t) ¼ x0 sin Wt þ y0 cos Wt þ t cos (c 0 Wt): Optimal headings change linearly in time in rotational flow c(t) ¼ c 0 Wt, and optimal trajectories follow (via eqn 30 in [30]): where uw and vw are the x and y flow velocity components, respectively, and c ¼ c(t) is the migrant’s heading clockwise from the y-axis. Equations (2.1) and (2.2) are scaled to the self- speed Va, i.e. spatially to a scale of flow, L and to the travel time in the absence of flow, L/Va. In this scaling, the initial goal distance D is also the travel duration in the absence of flow. Using calculus of variations or optimal control theory, it can be shown that optimal, i.e. time-minimizing, headings follow the classic Zermelo solution [27]: 3 x(t) ¼ x0 cos Wt y0 sin Wt þ t sin (c 0 Wt) and y(t) ¼ x0 sin Wt þ y0 cos Wt þ t cos (c 0 Wt): For both patterns, trajectories with goal orientation (xG(t), yG(t)) and full compensation (xF(t), yF(t)) were computed numerically using equations (2.1)–(2.2) and (2.4)–(2.5) with dimensionless time steps of 1025, and according to the current heading. Goal-oriented headings cG(t) were updated to the current goal direction dc dt ¼ duw dx dvw dy cos c sin c þ duw dy sin2c dvw dx cos2c, dc dt ¼ duw dx dvw dy cos c sin c þ duw dy sin2c g J. R. Soc. Interface 11: 20140588 cG(t) ¼ tan1 xf xG(t) yf yG(t) (2:3) and headings during full compensation cF(t) were updated to counteract the current lateral flow (in [27,30], headings are defined anticlockwise from the x-axis, resulting in the right-hand side of equation (2.3) being of oppo- site sign). 2.1. Orientation in steady flow patterns y p An animal’s trajectory on a horizontal x–y plane over time (t) can be determined via its velocity components relative to the ground, u(t) and v(t), respectively. Each velocity component is a vector sum of the flow velocity and the animal’s self-propelled (self-speed) velocity components: (2) Vector orientation: based on a single heading, set on departure and ignoring drift thereafter (arrival is possi- ble to the extent that the heading can be adjusted to compensate for any cumulative lateral drift, see [36,37]). (3) Goal orientation: based on continually heading towards the goal using a map sense, i.e. ignoring instantaneous lateral drift. u(t) ¼ uw(x, y, t) þ sin c(t) (2:1) and v(t) ¼ vw(x, y, t) þ cos c(t), (2:2) (2:1) (2:2) on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from From equation (2.3), it can be shown that once initial headings are chosen, optimally orienting migrants steer continu- ally away from whichever side has higher flow support relative to the current travel direction [28,30]. Note that in uniform flow, the right-hand side of equation (2.3) is zero, demonstrating (cf. [13]) that full compensation is in this case time-optimal (being the only way to arrive at the goal with a constant heading). cF(t) ¼ sin1(uw(xF(t), yF(t))) ¼ sin1( WyF(t)): Travel durations for vector-orienting Tc and fully compensat- ing TF individuals could be determined analytically as a function of the maximum flow speed W. In shear flow, these are Tc ¼ 1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 W2=4 p , W , 2 and TF ¼ 1 W sin1W, W , 1, Shear flow is characterized by a gradient in lateral flow along the initial goal direction and TF ¼ 1 W sin1W, W , 1, (uw, vw) ¼ (Wy, 0) (2:4) and for travel directly through rotational flow and for travel directly through rotational flow (2:4) Tc ¼ 1 W tan1W and TF ¼ 1 W sin1W, W , 1: and rotational flow by radially increasing flow speed (as modelled for cyclones and anticyclones, see [39]) and rotational flow by radially increasing flow speed (as modelled for cyclones and anticyclones, see [39]) and TF ¼ 1 W sin1W, W , 1: (uw, vw) ¼ (Wy, Wx), (2:5) (2:5) We also simulated movement wholly contained within rotational flow, varying the departure location at regularly spaced intervals within one quadrant (shaded region in figure 3a,b, Dx0 ¼ Dy0 ¼ 0.01, N ¼ 12 959), and setting the initial goal distance to one radius distant on the opposite side of the flow pattern (i.e. (xf, yf) ¼ (x0, y0 2 1)). For these locations, initial headings and trajectories were solved numerically for vector and optimal orientation, as were trajectories for goal orientation and full compensation. where W is the flow strength (maximum flow speed relative to the animal’s self-speed). We scaled the shear flow case to the initial goal distance and the radius of flow in the rotational case, i.e. the dimensionless initial goal distance is D ¼ 1 for shear flow and D ¼ 2 for movement through rotational flow. on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from Since the initial goal direction is along the y-axis, the initial and goal locations are (x0, y0) ¼ (0, 0) and (xf, yf) ¼ (0, 1) for movement through shear flow and (x0, y0) ¼ (0, 21) and (xf, yf) ¼ (0, 1) through rotational flow. For these configurations, analytical formulae for trajectories were derived for vector orientation (xc(t), yc(t)) and optimal orien- tation (x(t), y(t)) via eqn 32 in [30]. This facilitated solving initial headings cc(t ¼ 0) ¼ cc 0 and c(t ¼ 0) ¼ c 0 with vector and optimal orientation, respectively. For each candidate’s initial head- ing, the closest approach to the goal was determined using MATLAB’s minimizing routine fminbnd. Vector-orienting and optimal trajectories in shear flow are where W is the flow strength (maximum flow speed relative to the animal’s self-speed). We scaled the shear flow case to the initial goal distance and the radius of flow in the rotational case, i.e. the dimensionless initial goal distance is D ¼ 1 for shear flow and D ¼ 2 for movement through rotational flow. Since the initial goal direction is along the y-axis, the initial and goal locations are (x0, y0) ¼ (0, 0) and (xf, yf) ¼ (0, 1) for movement through shear flow and (x0, y0) ¼ (0, 21) and (xf, yf) ¼ (0, 1) through rotational flow. For these configurations, analytical formulae for trajectories were derived for vector orientation (xc(t), yc(t)) and optimal orien- tation (x(t), y(t)) via eqn 32 in [30]. This facilitated solving initial headings cc(t ¼ 0) ¼ cc 0 and c(t ¼ 0) ¼ c 0 with vector and optimal orientation, respectively. For each candidate’s initial head- ing, the closest approach to the goal was determined using MATLAB’s minimizing routine fminbnd. Vector-orienting and optimal trajectories in shear flow are 2.2. Simulating avian migration in time-varying winds To simulate long-distance migration, we resolved motion on a spherical surface, i.e. the rates of change in longitude Ø ¼ Ø(t) and latitude u ¼ u(t): 2.2. Simulating avian migration in time-varying winds To simulate long-distance migration, we resolved motion on a spherical surface, i.e. on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from Zermelo’s solution on the sphere becomes dc dt ¼ 1 cosu dUw d dVw du coscsinc þ dUw du sin2c cos2c cosu dVw d þ tanusinc Re (Va þ Uw sinc þ Vw cosc), ( ) For the rotational case, vector-orienting trajectories (xc(t), yc(t)) are readily solved analytically (2:6) (2:6) xc(t) ¼ x0 cos Wt y0 sin Wt þ 1 W ( cos (Wt cc 0) cos cc 0) and yc(t) ¼ x0 sin Wt þ y0 cos Wt þ 1 W ( sin cc 0 þ sin (Wt cc 0)): where the last term accounts for the Earth’s curvature [25]. Here, we have neglected the vertical motion required to maintain alti- tude over the spherical Earth [28]. Finally, we note that with all on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from 0.2 –1.0 –0.5 0 0.5 1.0 0.5 (c) (a) (b) 1 flow strength, W flow support 2 5 10 0.2 0.5 (d) 1 flow strength, W efficiency 2 5 100 0.5 1.0 Figure 1. Orientation in shear flow. For movement through shear flow, trajectories to goals (marked with an O) with optimal orientation (cyan lines with black arrows representing optimal headings), vector orientation (dashed blue lines), goal orientation (dot-dashed green lines) and full compensation (dotted red lines) in moderately flow, W ¼ 0.8, (a) and strong flow, W ¼ 1.7, (b). (c) Flow support (proportional gain in travel speed) and efficiency (travel speed relative to optimal speed) in relation to flow strength W for each strategy. Note that all results are independent of spatial scale and that the y-axis points downwards. (a) ( (a) 0.2 –1.0 –0.5 0 0.5 1.0 0.5 (c) 1 flow strength, W flow support 2 5 10 ( 0.2 0.5 d) 1 flow strength, W efficiency 2 5 100 0.5 1.0 (d) Figure 1. Orientation in shear flow. For movement through shear flow, trajectories to goals (marked with an O) with optimal orientation (cyan lines with black arrows representing optimal headings), vector orientation (dashed blue lines), goal orientation (dot-dashed green lines) and full compensation (dotted red lines) in moderately flow, W ¼ 0.8, (a) and strong flow, W ¼ 1.7, (b). (c) Flow support (proportional gain in travel speed) and efficiency (travel speed relative to optimal speed) in relation to flow strength W for each strategy. on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from Note that all results are independent of spatial scale and that the y-axis points downwards. strategies, the Coriolis effect [46] is assumed to be either insignificant or adjusted for by the animal in question. routine (MATLAB’s fminbnd) and the overall time-minimal initial heading chosen among all intervals. For 25 of the 495 simulated great snipe flights, even smaller intervals were required; these were resolved iteratively by visually comparing time-minimizing trajectories in successively smaller intervals until convergence was achieved. s g ca t o adjusted o by t e a a quest o . To simulate optimal orientation in non-stop flight in time-vary- ing winds, we modified the individual-based model in [36] to solve equation (2.6). Initial and goal locations, departure dates, times and flight characteristics were chosen to match each migration system. Departures in all simulations took place at 1 h following civil dusk, and we furtherassumed sufficient fuel loads to reach goal des- tinations. Non-stop flights of Turdus thrushes across the North Sea were simulated using wind data from the NCEP-NCAR reanalysis dataset [40] for 14 known mass migration events September– November 2006–2008 between Norway and The Netherlands [42]. We assumed departures from inland Norway (608 N 8.58 E) to within 100 km of a 794 km distant goal in The Netherlands (538 N 68 E), flight at 925 mb pressure level (ca 800 m above mean sea level (AMSL)) and self-speeds of 12 m s21, appropriate for Turdus thrushes [47]. Simulated great snipes departed 16–30 August 1979–2011 from a location in Scandinavia (638 N, 128 E) to within 250 km of a 5909 km distant goal (108 N, 88 E) in a prevalent stopover region in West Africa, as evidenced by great snipes tracked using geolocation ([45] and RHG Klaassen 2011-2012, unpublished data). Based on these data, we assumed constant self-speeds (20 m s21) and flight at 700 mb (ca 3000 m AMSL). To ensure unbiased comparisons between strategies, including unsuccessful dates, differences in efficiency between strategies were assessed using Wilcoxon’s non-parametric two-way signed rank test. 3.1. Movement in steady flow patterns We first present results of simulated movement through shear and rotational flow patterns and then, to account for variabil- ity in potential flow support en route, of movements from various departure locations within rotational flow patterns. For all simulations, the flow strength W (maximal flow speed relative to self-speed) was varied between 0 and 10, i.e. up to about twice the range encountered among taxa in fluid media [3]. Trajectories are shown for movement in flows of moderate strength (W ¼ 0.8) and flows exceeding self-speeds (W ¼ 1.7), spanning typical maximal values encountered by birds and by full-grown fish and turtles (cf. fig. 2 in [3]). Figures 1–3 graphically summarize results for each strategy for movement through shear (figure 1) and rotational flow patterns (figure 2) and from various departure locations within rotational flow patterns (figure 3). Headings for full compensation and goal orientation were obtained in relation to orthodromic (great circle) directions at each time step [48]. Since optimal orientation theoretically involves altering headings according to exact spatial derivatives of flow (equation (2.6)) we linearly interpolated the wind data spatially and temporally at each time step. Solutions can further- more be very sensitive to small errors in calculated headings (e.g. [25,49]), so we used time steps of 2 min for thrush simulations and 1 min for snipe simulations. While these resolutions go far beyond that of the original wind data (ca 18 and 1 h, [40]), they enabled precise calculations of benchmarks and accurate assess- ment of the generic strategies. To avoid convergence to local minima, initial headings were therefore determined within small intervals for each departure date (18 for thrush simulations and 0.058 for great snipe simulations) using a standard search Trajectories through weak to moderate flow (e.g. W ¼ 0.8, figure 1a) differ much less between strategies than in strong flow (e.g. W ¼ 1.7, figure 1b). Optimal orientation (cyan lines) takes advantage of the strong flow near the goal by effectively over-compensating for the initially weak lateral drift, resulting in upstream travel, and altering headings (depicted by black arrows in figure 1a,b) to reduce compen- sation on approach to the goal. The resultant detour contrasts with both the straight path resulting from full compensation (dotted red lines) and the initial downstream drift resulting from goal orientation (dot-dashed green lines). 3.1. Movement in steady flow patterns Flow-adjusted vector orientation (dashed blue lines) on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from 0.2 –1 0 1 2 3 0.5 (c) (a) (b) 1 flow strength, W flow support 2 5 10 0.2 0.5 (d) 1 flow strength, W efficiency 2 5 100 0.5 1.0 Figure 2. Orientation through rotational flow. For movement through rotational flow, trajectories in moderate (a) and strong flow (b), and flow support (c) and efficiency (d) as a function of flow strength. Details otherwise as in figure 1. y yp g g 5 (a) ( b) (d) Figure 2. Orientation through rotational flow. For movement through rotational flow, trajectories in moderate (a) and strong flow (b), and flow support (c) and efficiency (d) as a function of flow strength. Details otherwise as in figure 1. 0.2 0 0.25 0.75 0.50 1.00 0.5 (c) (a) (b) 1 flow strength, W success rate 2 5 10 0.2 0.5 (d) 1 flow strength, W efficiency 2 5 100 0.5 1.0 Figure 3. Orientation within rotational flow. For movement from different departure locations within rotational flow (shaded grey areas in (a,b)), trajectories in moderate (a) and strong flow (b) and for each strategy, success rates (c) and median efficiency among departure locations (d), with shaded area representing quartile range. Details otherwise as in figure 1. b) (a) (b) (a) 0.2 0 0.25 0.75 0.50 1.00 0.5 (c) 1 flow strength, W success rate 2 5 10 ( 0.2 0.5 d) 1 flow strength, W efficiency 2 5 100 0.5 1.0 (d) efficiency Figure 3. Orientation within rotational flow. For movement from different departure locations within rotational flow (shaded grey areas in (a,b)), trajectories in moderate (a) and strong flow (b) and for each strategy, success rates (c) and median efficiency among departure locations (d), with shaded area representing quartile range. Details otherwise as in figure 1. compensating for a purely lateral flow of equal strength become stationary. Vector orientation remains feasible as long as W , 2 since the mean lateral flow strength is W/2. compensating for a purely lateral flow of equal strength become stationary. Vector orientation remains feasible as long as W , 2 since the mean lateral flow strength is W/2. With movement through rotational flow, trajectories dif- fered between strategies in both moderately strong flow (e.g. W ¼ 0.8, figure 2a) and in strong flow (e.g. compensating for a purely lateral flow of equal strength become stationary. Vector orientation remains feasible as long as W , 2 since the mean lateral flow strength is W/2. With movement through rotational flow, trajectories dif- fered between strategies in both moderately strong flow (e.g. W ¼ 0.8, figure 2a) and in strong flow (e.g. W ¼ 1.7, figure 2b). Here, trajectories with both optimal orientation and vector orientation follow the counter-clockwise rotation of the flow, drifting far from the direct route with full compensation (figure 2a,b). Optimal orientation involves over-drift, i.e. heading partly towards the lateral flow, 3.1. Movement in steady flow patterns W ¼ 1.7, figure 2b). Here, trajectories with both optimal orientation and vector orientation follow the counter-clockwise rotation of the flow, drifting far from the direct route with full compensation (figure 2a,b). Optimal orientation involves over-drift, i.e. heading partly towards the lateral flow, resembles optimal orientation in over-compensating on departure and initial upstream travel. Movement in pure shear flow remains unsupportive regardless of strategy, as illustrated by the increasingly negative flow support (pro- portional gain in travel speed due to flow) with increasing flow strength (figure 1c). This is also evident from the effi- ciency of each generic strategy (figure 1d), being nearly optimally efficient (1 ﬃ1) in weak flow (W , 0.5), but decreasing rapidly to zero in moderate to strong flow. Arrival with full compensation or goal orientation becomes infea- sible at the point where W ¼ 1, since individuals fully With movement through rotational flow, trajectories dif- fered between strategies in both moderately strong flow (e.g. W ¼ 0.8, figure 2a) and in strong flow (e.g. W ¼ 1.7, figure 2b). Here, trajectories with both optimal orientation and vector orientation follow the counter-clockwise rotation of the flow, drifting far from the direct route with full compensation (figure 2a,b). Optimal orientation involves over-drift, i.e. heading partly towards the lateral flow, on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from 1.0 0.8 0.6 0.4 0.2 FC (b) (a) (c) success rate efficiency VO GO FC VO GO 0 1.0 0.8 5° W 50° N 55° N 60° N 31 Oct, 17 Z 1 Nov, 00 Z 65° N 5° E 10° E 0° 0.6 0.4 0.2 0 Figure 4. Simulated songbird migration across the North Sea. Trajectories of simulated Turdus thrushes (a) departing Norway on 31 October 2006 to land within a 100 km radius (outlined in red) of a goal located in The Netherlands (red cross), and considering 14 mass North Sea crossing events (September–November 2006– 2007), success rates (b) and boxplots of efficiency (c) for each strategy: optimal orientation (cyan lines), vector orientation (VO, dashed blue lines), goal orientation (GO, dot-dashed green lines) and full compensation (FC, dotted red lines). Wind quivers (grey arrows) depicting wind speed and direction are scaled to 26 m s21 and synchronized with optimally orienting migrants at the same latitude (see time stamps on right of maps). rsif.royalsocietypublishing.org J. R. Soc. 3.1. Movement in steady flow patterns Interface 11: 20140588 6 (a) ) 5° W 50° N 55° N 60° N 31 Oct, 17 Z 1 Nov, 00 Z 65° N 5° E 10° E 0° 1.0 0.8 0.6 0.4 0.2 FC (b) success rate VO GO 0 (c) c) efficiency FC VO GO 1.0 0.8 0.6 0.4 0.2 0 Figure 4. Simulated songbird migration across the North Sea. Trajectories of simulated Turdus thrushes (a) departing Norway on 31 October 2006 to land within a 100 km radius (outlined in red) of a goal located in The Netherlands (red cross), and considering 14 mass North Sea crossing events (September–November 2006– 2007), success rates (b) and boxplots of efficiency (c) for each strategy: optimal orientation (cyan lines), vector orientation (VO, dashed blue lines), goal orientation (GO, dot-dashed green lines) and full compensation (FC, dotted red lines). Wind quivers (grey arrows) depicting wind speed and direction are scaled to 26 m s21 and synchronized with optimally orienting migrants at the same latitude (see time stamps on right of maps). orientation the only feasible generic strategy for W ¼ 1.7). Figure 3c depicts success rates over all departure locations, showing that arrival is not always possible in very strong flow (W . 2, at least without first exiting the rotational system). In strong flow, optimal orientation always has the highest success rate, followed by vector orientation, then goal orientation, whereas full compensation is infeasible. Efficien- cies for each strategy (figure 3d) further indicate that vector orientation is the most reliable and efficient among the tested generic strategies. The apparent increase in efficiency with very strong flow (W . 1) reflects that only highly supportive routes remain feasible. whereas vector orientation is equivalent to full drift in this particular example since flow is balanced en route. In strong flow (figure 2b), a fully compensating animal cannot move forward on departure, and a goal-orienting animal winds up spiralling inwards towards a stationary point remote from the goal. This is reflected in the dependence of flow support on flow strength (figure 2c): flow support with vector orientation and optimal orientation is always positive and increases with increasing flow strength, but is always negative with full compensation and goal orienta- tion. Vector orientation, by taking advantage of the inherent balance in rotational flow patterns, remains feasible and efficient (1 . 0.9) regardless of flow strength (figure 2d). 3.1. Movement in steady flow patterns For all strategies, varying the departure location in rotational flow (the shaded region in figure 3a,b) affects both the feasibility and efficiency of travel within the flow pattern. Sample trajectories are shown in moderate (W ¼ 0.8, figure 3a) and strong flow (W ¼ 1.7, figure 3b). Trajectories in supportive flow (e.g. the left-hand trajectories in figure 3a,b) differ much less with flow strength or between strategies compared to trajec- tories in opposing flow (e.g. right-hand trajectories, with goal 3.2. Migration simulations Simulated trajectories and trajectories inferred from geolocator studies of great snipes departing Scandinavia on 30 August 2010 (a) to within a 250 km radius (outlined in red) of a goal located in West Africa (red cross), and considering 33 seasons of simulations (16–30 August 1979–2011), success rates (b) and boxplots of efficiency (c) for each strategy: optimal orientation (cyan lines), vector orientation (VO, dashed blue lines), goal orientation (GO, dot-dashed green lines) and full compensation (FC, dotted red lines). Great snipe trajectories inferred from geolocator data are indicated with pink lines, with flight durations of 72 h (filled squares) and 84 h (open circles). Wind quivers (grey arrows) depict wind speed and direction are scaled to 26 m s21 and synchronized with optimally orienting migrants at the same latitude (see time stamps on right of maps). seasons of simulated flights, success rate (figure 5b) and boxplots of efficiency (figure 5c) for each strategy. With the exception of full compensation, simulated trajectories, flight durations (and efficiencies) for this departure date were similar: 77 h (1.0) with optimal orientation, 80 h (0.96) with vector orientation and 84 h (0.92) with goal orientation. Full compensation took con- siderably more time 107 h (1 ¼ 0.72). Two great snipes tracked with geolocators on this date (pink tracks with symbols in figure 5a) were even faster (more efficient) than optimal simu- lations (1 ¼ 1.04 and 1.10). This is presumably attributable to higher airspeeds and/or superior altitude selectivity by the tagged individuals. The estimated track of the faster tagged indi- vidual (with filled square markers) was nonetheless tantalizing similar to that with optimal orientation. Considering all 33 sea- sons of simulated flights, vector orientation (with constant flow-adjusted heading of 190.68) was slightly but significantly more efficient (median efficiency ~1 ¼ 0:96) than both full com- pensation (~1 ¼ 0:81, Wilcoxon’s signed rank 4, p , 10223) and goal orientation (~1 ¼ 0:93, Wilcoxon’s signed rank 1435, p , 10210), but also the least reliable in arriving within 250 km (figure 2b): simulated flight durations (and efficiencies) for this date were 8.7 h (1.0) with optimal orientation, 9.0 h (0.97) with vector orientation, 39 h (0.22) with goal orien- tation and with fully compensating birds failing to arrive (simulations were terminated when wind speeds exceeded airspeeds). 3.2. Migration simulations Results for simulated North Sea crossings by thrushes are summarized in figure 4, with trajectories for each strategy in strong winds on 31 October 2006 (W ¼ 2.16, figure 4a) and, considering all 14 mass migration events, success rate (figure 4b) and boxplots of efficiency (figure 4c). Trajecto- ries are reminiscent of movement in strong rotational flow on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from 1.0 0.8 0.6 0.4 0.2 FC (b) (c) (a) success rate efficiency VO GO FC VO GO 0 1.0 0.8 20° N 0° 0° 20° E 40° E 40° N 23 Aug, 21 Z 25 Aug, 06 Z 27 Aug, 23 Z 60° N 0.6 0.4 0.2 0 igure 5. Simulated non-stop great snipe migration to Africa. Simulated trajectories and trajectories inferred from geolocator studies of great snipes departing candinavia on 30 August 2010 (a) to within a 250 km radius (outlined in red) of a goal located in West Africa (red cross), and considering 33 seasons of simulations 6–30 August 1979–2011), success rates (b) and boxplots of efficiency (c) for each strategy: optimal orientation (cyan lines), vector orientation (VO, dashed blue nes), goal orientation (GO, dot-dashed green lines) and full compensation (FC, dotted red lines). Great snipe trajectories inferred from geolocator data are indicated with pink lines, with flight durations of 72 h (filled squares) and 84 h (open circles). Wind quivers (grey arrows) depict wind speed and direction are scaled to 6 m s21 and synchronized with optimally orienting migrants at the same latitude (see time stamps on right of maps). rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 7 (a) 20° N 0° 0° 20° E 40° E 40° N 23 Aug, 21 Z 25 Aug, 06 Z 27 Aug, 23 Z 60° N (a) (c) efficiency FC VO GO 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 FC (b) success rate VO GO 0 (c) Figure 5. Simulated non-stop great snipe migration to Africa. 4. Discussion In this study, we have introduced optimal orientation as a benchmark for evaluating the performance of orientation strategies in given flow conditions. With optimal orienta- tion, trajectories are longest yet travel duration is minimized by steering through regions of relatively high flow support. The illustrated trajectories (figures 1a,b and 2a,b) demon- strate that optimal orientation in horizontal flow does not always involve increased compensation on approach to the goal (cf. [12,13]), can involve either over-drift or over- compensation en route (cf. [23,38]) and is not always equivalent to full drift in ‘balanced’ flows (contra [12]). Our results provide insight into the value of information an animal may have about flow conditions. In flow that is weak compared to self-speeds (W 0.5), flow prediction is not essential as full compensation and goal orientation are all nearly as reliable and efficient as flow-adjusted vector orientation and optimal orientation (figures 1d, 2d and 3d). This finding is consistent with evidence of selectivity for low flow speeds among migratory taxa [6,50–52]. In stronger flow, animals have much more to gain from explicit or intrin- sic information about flow patterns, as evidenced by the sometimes dramatic differences in performance between on the one hand optimal orientation and flow-adjusted vector orientation, and on the other hand full compensation and goal orientation. Vector orientation involving pro-active adjustment of headings was shown to be more efficient than either goal orientation (i.e. strictly following a map sense) or full compensation in strong and variable flow, and performed nearly as well as optimal orientation. Natu- rally, if flow cannot be predicted a priori, adjusting headings en route can be advantageous in avoiding unnecessary barriers [36,53], reorienting following unantici- pated drift [54,55] or to avoid becoming trapped in strong rotational flow (see figure 3b and cf. [42,56]). Assessing optimal orientation in flows remains computa- tionally challenging and further limited by availability and accuracy of flow data (cf. [62]) and by uncertainties regarding effects of vertical movements, variable self-speed and energy considerations. Results from this study suggest that in weak flows (e.g. overall less than half the self-speed), the optimal benchmark could be approximated by calculating flow- adjusted vector orientation. Selection of vertical layers is obviously also relevant [19,23,24], but for example among migrating birds, altitudes with the highest instantaneous flow support are not always selected [20]. rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 8 Therefore, the extent to which taxa can approach optimal orientation will depend both on the scale and strength of flow relative to motion and navigation capacities [3,57] and on abilities to gauge and predict flow. Although assessing limitations of and transitions between navigational cues goes beyond the scope of this study, it is relevant to note that long-distance migration is generally thought to require differ- ent navigational cues [34] at various spatial scales to ensure arrival [35]. In this context, the simplicity, near-optimal effi- ciency and relatively close approach to the goal (median 500 km) of the great snipe simulations based on endogenous headings support the notion that vector orientation can pro- vide a basis for long-distance movements, as proposed for Nearctic–Neotropical landbird migration over the Atlantic Ocean [37] and monarch butterfly Danaus plexippus migra- tion [58]. Regarding abilities to gauge and predict flow, swimming animals may be particularly constrained [6], and ocean currents may in fact be even less predictable than in the atmosphere (travel distances being similar [3] but synoptic scales shorter [59,60]). Consistent with such flow unpredictability, migration routes to foraging grounds by loggerhead turtles (Caretta caretta) were recently found to match goal-orienting more closely than time-optimal routes [61]. Acknowledgements. Thanks to Lourens Veen and Emiel van Loon for advice on simulations and statistics, and to three anonymous 3.2. Migration simulations Considering all 14 events, simulated flights were always successful ( pA ¼ 1.0) except with full compensation which was unsuccessful on three of 14 nights ( pA ¼ 0.79). Vector orientation was the most efficient strategy (median efficiency ~1 ¼ 0:98 versus 0.95 with full compensation and 0.88 with goal orientation, Wilcoxon’s signed rank ¼ 4, p , 0.001). This was even more apparent on simulations of the six nights where mean wind speeds exceeded the modelled self-speed, with full compensation resulting in suc- cessful arrival on only three nights, and poor efficiency with goal orientation (~1 ¼ 0:50 versus 0.95 and 0.96 with full compensation and vector orientation, respectively). Results for great snipe simulations are summarized in figure 5, with simulated and tracked great snipe trajectories departing 23 August 2009 (W ¼ 0.6, figure 5a) and, for 33 on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from of the goal (Pa ¼ 0.28 versus 0.92 and 1.0 with full compensation and goal orientation, respectively). Simulated vector-orienting great snipes did however typically pass relatively close to the goal, with a median (and quartile range) in closest approach among departure dates of 500 km (220–800 km, i.e. 3–14% of the initial goal distance). tracked with geolocators apparently curved to the east similar to optimally orientating trajectories, although more detailed and accurate tracks, including information about headings, would be required to unravel the orientation and navigation behaviour of these birds during their astonishing flights. 8 References 1. Gaspar P, Georges J-Y, Fossette S, Lenoble A, Ferraroli S, Le Maho Y. 2006 Marine animal behaviour: neglecting ocean currents can lead us up the wrong track. Proc. R. Soc. B 273, 2697–2702. (doi:10.1098/rspb.2006.3623) 10. Hockley FA, Wilson CAME, Brew A, Cable J. 2014 Fish responses to flow velocity and turbulence in relation to size, sex and parasite load. J. R. Soc. Interface 11, 20130814. (doi:10.1098/rsif.2013.0814) 20. Kemp MU, Shamoun-Baranes J, Dokter AM, van Loon E, Bouten W. 2013 The influence of weather on the flight altitude of nocturnal migrants in mid- latitudes. Ibis 155, 734–749. (doi:10.1111/ibi. 12064) 11. Palmer T, Hagedorn R. 2006 Predictability of weather and climate. Cambridge, UK: Cambridge University Press. 21. Ream RR, Sterling JT, Loughlin TR. 2005 Oceanographic features related to northern fur seal migratory movements. Deep-Sea Res. II 52, 823–843. (doi:10.1016/j.dsr2.2004.12.021) 2. Liechti F. 2006 Birds: blowin’ by the wind? J. Ornithol. 147, 202–211. (doi:10.1007/s10336- 006-0061-9) 12. Alerstam T. 2011 Optimal bird migration revisited. J. Ornithol. 152(Suppl. 1), S5–S23. (doi:10.1007/ s10336-011-0694-1) 3. Chapman JW, Klaassen RHG, Drake VA, Fossette S, Hays GC, Metcalfe JD, Reynolds AM, Reynolds DR, Alerstam T. 2011 Animal orientation strategies for movement in flows. Curr. Biol. 21, R861–R870. (doi:10.1016/j.cub.2011.08.014) 22. Weng KC, Boustany AM, Pyle P, Anderson SD, Brown A, Block BA. 2007 Migration and habitat of white sharks (Carcharodon carcharias) in the eastern Pacific Ocean. Mar. Biol. 152, 877–894. (doi:10. 1007/s00227-007-0739-4) 13. Alerstam T. 1979 Wind as selective agent in bird migration. Ornis Scand. 10, 76–93. (doi:10.2307/ 3676347) 4. Kemp MU, Shamoun-Baranes J, van Loon EE, McLaren JD, Dokter AM, Bouten W. 2012 Quantifying flow-assistance and implications for movement research. J. Theore. Biol. 308, 56–67. (doi:10.1016/j.jtbi.2012.05.026) 14. Anderson DJ. 1983 Optimal foraging and the traveling salesman. Theor. Popul. Biol. 24, 145–159. (doi:10.1016/0040-5809(83)90038-2) 23. Alerstam T. 1979 Optimal use of wind by migrating birds: combined drift and overcompensation. J. Theor. Biol. 79, 341–353. (doi:10.1016/0022- 5193(79)90351-5) 15. Sachs G, Traugott J, Nesterova AP, Dell’Omo G, Ku¨mmeth F, Heidrich H, Vyssotski AL, Bonadonna F. 2012 Flying at no mechanical energy cost: disclosing the secret of wandering albatrosses. PLoS ONE 7, e41449. (doi:10.1371/journal.pone.0041449) 5. Shamoun-Baranes J, Bouten W, van Loon EE. 2010 Integrating meteorology into research on migration. Integr. Comp. Biol. 50, 280–292. (doi:10.1093/icb/ icq011) 24. Narazaki T, Sato K, Abernathy KJ, Marshall GJ, Miyazaki N. 2009 Sea turtles compensate deflection of heading at the sea surface during directional travel. J. Exp. rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 9 reviewers for constructive comments and suggestions. Our model simulations are facilitated by The Netherlands eScience Center (http://esciencecenter.com), project 660.011.305 and BiG Grid infrastructure for e-Science (http://www.biggrid.nl). 9 Appendix A. Generic orientation strategies Full compensation involves heading into the flow to such an extent that the track and goal directions are aligned, thus instantly negating any lateral drift [4]. Foraging fish in swift rivers have for example been shown to fully compensate over short-distances [71]. However, full compensation is only possible if flow speeds do not exceed self-speeds (in this case, simulations were terminated). Vector orientation involves choosing a single heading on departure, based on actual (or prevailing) flow conditions, to ensure (or maximize the likeli- hood of) successful arrival. This is an extension of vector orientation as typically proposed, i.e. as an orientation strategy of juvenile migrants with no or limited map sense [53] and g J. R. Soc. Interface 11: 20140588 4. Discussion Adjustment of self-speed to horizontal flow can be of considerable energetic importance [12,51], but the degree of such adjustment remains obscure [63,64]. The adaptive benefit of adjusting self-speed will depend on trade-offs between time and energy expenditure [65], but may furthermore be physiologi- cally constrained [66,67] or superseded by the selection of favourable flow conditions [36,68]. The comparison between two natural migration systems reveals interesting contrasts in the effect of spatio-temporal scales on the performance of orientation strategies. With the relatively short thrush flights, during which we could expect wind conditions to be somewhat predictable, vector orientation is nearly time-optimal and clearly outperforms full compensation and goal orientation strategies, especially under strong flow conditions. Interestingly, at the much larger spatio-temporal scales of the great snipe flights, during which flow conditions throughout the flight are unli- kely to be predictable, goal orientation is nearly optimally efficient. This probably stems from two factors: (i) the high airspeeds of great snipes (ca 20 m s21 [45]) diminishing the effect of lateral winds and (ii) reduced spatial coherency of experienced flow, limiting the potential advantage of flow predictability. This further suggests that to the extent that great snipe have continual access to navigational cues, they can travel over large distances reliably and nearly optimally fast by heading towards the goal. The routes of the snipes The myriad of animal movement data made available through modern tracking technology offers great potential for understanding the role of flow-orientation in animal movement but also raises a challenge to interpretation [63,69]. The highly contrasting patterns of compensation that can arise in various flow patterns (figures 1–3) empha- size the importance of not only assessing reaction to flow and flow support instantaneously in relation to track direc- tions or implied preferred directions [4,64,70], but also over the entire scale of movement. Comparing optimal and observed orientation over entire routes allows estimation of the potential and realized flow support and potential insight into the navigational capacities and flow-predictive abilities among swimming and flying migrants. on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from being insufficient to reach specific goals [72,73]. However, by choosing a heading that compensates for the cumulative drift over the resulting route an animal could reach a goal des- tination. For example, Neotropical migrants have been proposed to take advantage of easterly trade winds to reach South America via eastern North America on constant head- ings [37]. A special case of vector orientation is in balanced flow, i.e. where simply heading in the initial goal direction results in no cumulative lateral drift, in which case it is equiv- alent to full drift. The third strategy, goal orientation, involves heading continually towards the goal regardless of displace- ment by drift. This is equivalent to a full drift strategy with continually updated preferred direction [4] but is also essen- tially equivalent to a strategy that minimizes the goal distance in infinitesimal steps (cf. [13]). Over entire routes, goal orientation has been shown to be suboptimal in strong uniform flow (e.g. [13]), but it may be relevant when gauging drift is unreliable or impossible [6,32]. rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 2013 Flying with the wind: scale dependency of speed and direction measurements in modelling wind support in avian flight. Mov. Ecol. 1, 4. (doi:10.1186/2051-3933-1-4) 36. McLaren JD, Shamoun-Baranes J, Bouten W. 2012 Wind selectivity and partial compensation for wind drift among nocturnally migrating passerines. Behav. Ecol. 23, 1089–1101. (doi:10.1093/beheco/ ars078) 51. Brodersen J, Nilsson PA, Ammitzbøll J, Hansson L-A, Skov C, Bro¨nmark C. 2008 Optimal swimming speed in head currents and effects on distance movement of winter-migrating fish. PLoS ONE 3, e2156. (doi:10.1371/journal.pone.0002156) 65. Vrugt JA, van Belle J, Bouten W. 2007 Pareto front analysis of flight time and energy use in long- distance bird migration. J. Avian Biol. 38, 432–442. (doi:10.1111/j.2007.0908-8857.03909.x) 52. Erni B, Liechti F, Underhill LG, Bruderer B. 2002 Wind and rain govern the intensity of nocturnal bird migration in Central Europe—a log-linear regression analysis. Ardea 90, 155–166. 37. Stoddard PK, Marsden JE, Williams TC. 1983 Computer simulation of autumnal bird migration over the Western North Atlantic. Anim. Behav. 31, 173–180. (doi:10.1016/S0003-3472(83)80186-9) 66. Thomas ALR, Hedenstro¨m A. 1998 The optimum flight speeds of flying animals. J. Avian Biol. 29, 469–477. (doi:10.2307/3677166) 38. Klaassen RHG, Hake M, Strandberg R, Alerstam A. 2010 Geographical and temporal flexibility in the response to crosswinds by migrating raptors. Proc. R. Soc. B 278, 1339–1346. (doi:10.1098/rspb. 2010.2106) 53. Mouritsen H. 2003 Spatiotemporal orientation strategies of long-distance migrants. In Avian migration (eds P Berthold, E Gwinner, E Sonnenschein), pp. 493–513. Berlin, Germany: Springer. 67. Iosilevskii G, Weihs D. 2008 Speed limits on swimming of fishes and cetaceans. J. R. Soc. Interface 5, 329–338. (doi:10.1098/rsif.2007.1073) 39. Patoux J, Yuan X, Li C. 2009 Satellite-based midlatitude cyclone statistics over the Southern Ocean: 1. Scatterometer-derived pressure fields and storm tracking. J. Geophys. Res. 114, D04105. (doi:10.1029/2008JD010873) 54. Fitzgerald T, Taylor PD. 2008 Migratory orientation of juvenile yellow-rumped warblers (Dendroica coronata) following stopover: sources of variation and the importance of geographic origins. Behav. Ecol. Sociobiol. 62, 1499–1508. (doi:10.1007/ s00265-008-0579-3) 68. Alerstam T, Chapman JW, Ba¨ckman J, Smith AD, Nilsson C, Reynolds DR, Klaassen RH, Hill JK. 2011 Convergent patterns of long-distance nocturnal migration in noctuid moths and passerine birds. Proc. R. Soc. B 278, 3074–3080. (doi:10.1098/rspb. 2011.0058) 40. Kalnay E et al. 1996 The NCEP/NCAR 40-year reanalysis project. Bull. Am. Meteor. Soc. 77, 437–471. (doi:10.1175/1520-0477) 69. Laube P, Purvis RS. 2011 How fast is a cow? Cross- scale analysis of movement data. Trans. GIS 15, 401–418. (doi:10.1111/j.1467-9671.2011.01256.x) 55. rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 59. Simmonds I. 2000 Size changes over the life of sea level cyclones in the NCEP reanalysis. Mon. Weather Rev. 128, 4118–4125. (doi:10.1175/1520- 0493(2000)129,4118:SCOTLO.2.0.CO;2) 45. Klaassen RHG, Alerstam A, Carlsson P, Fox JW, Lindstro¨m A˚. 2011 Great flights by great snipes: long and fast migration over benign habitats. Biol. Lett. 7, 833–835. (doi:10.1098/rsbl.2011.0343) 31. Traugott J, Nesterova A, Sachs G. 2013 The nearly effortless flight of the albatross. IEEE Spectr. 50, 46–54. (doi:10.1109/MSPEC.2013.6545122) 60. Chelton DB, Schlax MG, Samelson RM. 2011 Global observations of nonlinear mesoscale eddies. Prog. Oceanogr. 91, 167–216. (doi:10.1016/j.pocean. 2011.01.002) 46. Tatum JB. 1979 The effect of the Coriolis force on the flight of a bird. Auk 97, 99–117. 32. Alerstam T, Pettersson S-G. 1976 Do birds use waves for orientation when migrating across the sea? Nature 259, 205–207. (doi:10.1038/259205a0) g.org J. R. Soc. Interface 11: 20140588 47. Alerstam T, Rose´n M, Ba¨ckman J, Ericson PGP, Hellgren O. 2007 Flight speeds among bird species: allometric and phylogenetic effects. PLoS Biol. 5, e197. (doi:10.1371/journal.pbio.0050197) 61. Hays GC, Christensen A, Fossette S, Schofield G, Talbot J, Mariani P. 2014 Route optimisation and solving Zermelo’s navigation problem during long distance migration in cross flows. Ecol. Lett. 17, 137–143. (doi:10.1111/ele.12219) 33. Yamaguchi N, Arisawa Y, Shimada Y, Higuchi H. 2012 Real-time weather analysis reveals the adaptability of direct sea-crossing by raptors. J. Ethol. 30, 1–10. (doi:10.1007/s10164-011- 0301-1) 48. Gudmundsson GA, Alerstam T. 1998 Optimal map projections for analysing long-distance migration routes. J. Avian Biol. 29, 597–605. (doi:10.2307/ 3677180) 34. Bostro¨m JE, A˚kesson S, Alerstam T. 2012 Where on earth can animals use a geomagnetic bi-coordinate map for navigation? Ecography 35, 1039–1047. (doi:10.1111/j.1600-0587.2012.07507.x) 62. Putnam NF, He R. 2013 Tracking the long-distance dispersal of marine organisms: sensitivity to ocean model resolution. J. R. Soc. Interface 10, 20120979. (doi:10.1098/rsif.2012.0979) 49. Bijlsma S. 2009 Optimal aircraft routing in general wind fields. J. Guidance Control Dyn. 32, 1025–1029. (doi:10.2514/1.42425) 35. Bingman VP, Cheng K. 2005 Mechanisms of animal global navigation: comparative perspectives and enduring challenges. Ethol. Ecol. Evol. 17, 295–318. (doi:10.1080/08927014.2005.9522584) 63. Shamoun-Baranes J, van Loon E, Liechti F, Bouten W. 2007 Analyzing the effect of wind on flight: pitfalls and solutions. J. Exp. Biol. 210, 82–90. (doi:10.1242/jeb.02612) 50. Richardson WJ. 1978 Timing and amount of bird migration in relation to weather: a review. Oikos 30, 224–272. (doi:10.2307/3543482) 64. Safi K et al. References Biol. 212, 4019–4026. (doi:10.1242/ jeb.034637) 16. Mateos-Rodrı´guez M, Liechti F. 2011 How do diurnal long-distance migrants select flight altitude in relation to wind? Behav. Ecol. 23, 403–409. (doi:10.1093/beheco/arr204) 6. Luschi P, Hays GC, Papi F. 2003 A review of long- distance movements by marine turtles, and the possible role of ocean currents. Oikos 103, 293–302. (doi:10.1034/j.1600-0706.2003.12123.x) 25. Jardin MR, Bryson AE. 2012 Methods for computing minimum-time paths in strong winds. J Guidance Control Dyn. 35, 165–171. (doi:10.2514/1.53614) 17. O’Dor RK, Adamo S, Aitken JP, Andrade JF, Janlon RT, Jackson GD. 2002 Currents as environmental constraints on the behavior, energetics and distribution of cuttlefish. Bull. Mar. Sci. 71, 601–617. 26. Sussman HJ, WIllems JC. 1997 300 years of optimal control: from the brachystochrone to the maximum principal. Control Syst. IEEE 17, 32–44. (doi:10. 1109/37.588098) 7. Scholtyssek C, Dacke M, Kro¨ger R, Baird E. 2014 Control of self-motion in dynamic fluids: fish do it differently from bees. Biol. Lett. 10, 20140279. (doi:10.1098/rsbl.2014.0279) 27. Zermelo E. 1931 U¨ber das Navigationsproblem bei ruhender oder vera¨nderlicher Windverteilung. Z. Angew. Math. Mech. 11, 114–124. (doi:10.1002/ zamm.19310110205) 8. Sterbing-D’Angelo S, Chadha M, Chiu C, Falk B, Xian W, Barcelo J, Zook JM, Moss CF. 2011 Bat wing sensors support flight control. Proc. Natl Acad. Sci. USA 108, 11 291–11 296. (doi:10.1073/pnas. 10187440108) 18. Chapman JW, Reynolds DR, Mouritsen H, Hill JK, Riley JR, Sivell D, Smith AD, Woiwood IP. 2008 Wind selection and drift compensation optimize migratory pathways in a high-flying moth. Curr. Biol. 18, 514–518. (doi:10.1016/j.cub.2008.02.080) 28. Arrow KJ. 1949 On the use of winds in flight planning. J Meteorol. 6, 150–159. (doi:10.1175/ 1520-0469(1949)006,0150:OTUOWI.2.0.CO;2) 19. Dokter AM, Shamoun-Baranes J, Kemp MU, Tijm S, Holleman I. 2013 High altitude bird migration at temperate latitudes: a synoptic perspective on wind assistance. PLoS ONE 8, e52300. (doi:10.1371/ journal.pone.0052300) 9. Schmaljohann H, Naef-Daenzer B. 2011 Body condition and wind support initiate the shift of migratory direction and timing of nocturnal departure in a songbird. J. Anim. Ecol. 80, 1115– 1122. (doi:10.1111/j.1365-2656.2011.01867.x) 29. Isaacs R. 1999 Differential games: a mathematical theory with applications to warfare and pursuit, control and optimization. New York, NY: Dover. on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from 30. Techy L. 2011 Optimal navigation in planar time- varying flow: Zermelo’s problem revisited. Intel. Serv. Robot. 4, 271–283. (doi:10.1007/s11370-011- 0092-9) navigators. Proc. Natl Acad. Sci. USA 110, 7348– 7353. (doi:10.1073/pnas.1221701110) Fennoscandinavia, Poland, the Baltic Republics, Russia, Siberia and Iceland. References Ringing and Migration 21, 5–15. (doi:10.1080/03078698.2002.9674271) navigators. Proc. Natl Acad. Sci. USA 110, 7348– 7353. (doi:10.1073/pnas.1221701110) 10 rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 Thorup K, Ortvad TE, Rabøl J, Holland RA, Tøttrup AP, Wikelski M. 2011 Juvenile songbirds compensate for displacement to oceanic islands during autumn migration. PLoS ONE 6, e17903. (doi:10.1371/journal.pone.0017903) 41. Kemp MU, van Loon EE, Shamoun-Baranes J, Bouten W. 2012 RNCEP: global weather and climate data at your fingertips. Methods Ecol. Evol. 3, 65–70. (doi:10.1111/j.2041-210X.2011.00138.x) 70. Green M, Alerstam T. 2002 The problem of estimating wind drift in migrating birds. J. Theor. Biol. 218, 485–496. (doi:10.1016/S0022-5193(02) 93094-8) 56. Galli S, Gaspar P, Fossette S, Calmettes B, Hays GC, Lutjeharms JRE, Luschi P. 2012 Orientation of migrating leatherback turtles in relation to ocean currents. Anim. Behav. 84, 1491–1500. (doi:10. 1016/j.anbehav.2012.09.022) 42. Shamoun-Baranes J, van Gasteren H. 2010 Atmospheric conditions facilitate mass migration events across the North Sea. Anim. Behav. 81, 691–704. (doi:10.1016/j.anbehav.2011.01.003) 71. Krupczynski P, Schuster S. 2008 Fruit-catching fish tune their fast starts to compensate for drift. Curr. Biol. 18, 1961–1965. (doi:10.1016/j.cub.2008.10.066) 72. Alerstam T. 2006 Conflicting evidence about long- distance animal navigation. Science 313, 791–794. (doi:10.1126/science.1129048) 57. Nathan R, Getz WM, Revilla E, Holyoak M, Kadmon R, Saltz D, Smouse PE. 2008 A movement ecology paradigm for unifying organismal movement research. Proc. Natl Acad. Sci. USA 105, 19 052– 19 059. (doi:10.1073/pnas.0800375105) 43. Milwright RDP. 2002 Postbreeding dispersal, breeding site fidelity and migration/wintering areas of migratory populations of song thrush Turdus philomeelos in the Western Palearctic. Ringing and Migration 23, 21–32. (doi:10.1080/03078698.2006. 9674340) 73. Guilford T, A˚kesson S, Gagliardo A, Holland RA, Mouritsen H, Muheim R, Wiltschko R, Wiltschko W, Bingman VP. 2011 Migratory navigation in birds: new opportunities in an era of fast-developing tracking technology. J. Exp. Biol. 214, 3705–3712. (doi:10.1242/jeb.051292) 58. Mouritsen H, Derbyshire R, Stalleicken J, Mouritsen O, Frost BJ, Norris RD. 2013 An experimental displacement and over 50 years of tag-recoveries show that monarch butterflies are not true 44. Milwright RDP. 2006 Redwing Turdus iliacus migration and wintering areas as shown by recoveries of birds ringed in the breeding season in on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from on February 2, 2017 http://rsif.royalsocietypublishing.org/ Downloaded from Navigation The process of reaching a remote goal from familiar or unfamiliar locations. Optimal orientation Time-minimizing orientation in any hori- zontal flow, resulting in fastest arrival at the goal. Orientation The determination and maintenance of heading relative to the ground. rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 Orientation strategy An orientation behaviour to negotiate flow, typically involving endogenous headings (see vector orientation) and/ or exogenous factors (in this study, flow conditions and navigational cues). Over- compensation Adjustment of heading resulting in lateral drift opposite to that of the ambient flow. Over-drift Adjustment of heading which increases lateral drift. Preferred direction The intended travel direction. In this study, the preferred direction is ultimately the goal direction but headings differ instan- taneously according to the orientation strategy and flow configuration. Self-speed An animal’s self-propelled speed relative to the moving flow (not adjusted to flow in this study). Typically referred to as the airspeed and swim speed in air and water, respectively. Shear flow A gradient in flow speed along any direction; here used to mean a gradient in lateral flow along the initial goal direction. Travel speed Speed of travel including non-movement periods. With non-stop movement, equivalent to mean ground speed. True navigation The ability to determine the direction to the goal from anywhere within the navigable range, even when drifted or displaced from intended paths. Vector orientation An orientation strategy, characterized by (a sequence of) constant headings. Typically proposed to be a juvenile migration strategy, we here also simu- late vector orientation by animals navigating in predictable flow by allowing headings to be adjusted on departure according to goal location and flow conditions en route. Glossary Glossary The process of reaching a remote goal from familiar or unfamiliar locations. Navigation Optimal orientation Orientation Orientation strategy Over- compensation Over-drift Preferred direction Self-speed Shear flow Travel speed True navigation Vector orientation The process of reaching a remote goal from familiar or unfamiliar locations. 11 rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 11 Advection Motion induced by the ambient flow. Balanced flow Flow for which full drift results in no cumulative lateral drift. Direction of travel The angle of an animal’s trajectory (clock- wise from geographic north). Compensation Adjustment of self-speed and/or heading to prevent or diminish lateral drift. Simulations in this study presume con- stant self-speeds. Drift Advection by lateral flow, i.e. perpen- dicular to the migrant’s preferred direction. The term passive drift, some- times used to describe movement in water, refers to drift without self-speed. Endogenous heading An inherited preferred migratory direc- tion or heading (in the latter case possibly adapted to prevalent flow). Flow support Travel speed minus self-speed, i.e. the differ- ence in speed from that in the absence of flow. Hence we quantify flow support over the entire trajectory as opposed to locally(cf.localmeasures of flowassistance in [4]). Flow strength Ratio of flow speed to self-speed. Full compensation Reaction to flow, restricted in this study through adjustment of heading, resulting in a constant direction of travel, i.e. pre- cluding drift. Only possible when the self-speed exceeds the lateral flow speed component relative to the heading. Full drift Non-adjustment to flow, i.e. continually heading in the initial goal direction. Goal orientation An orientation strategy whereby the animal repeatedly heads towards the goal. This does not preclude drift, i.e. goal orien- tation is distinct from full compensation. Ground speed An animal’s instantaneous (horizontal) speed relative to the ground. Heading The angle of an animal’s body axis rela- tive to the ground (clockwise from geographic north). Lateral flow The flow component perpendicular to a particular direction, here the preferred, i.e. goal direction. Time-minimizing orientation in any hori- zontal flow, resulting in fastest arrival at the goal. Advection Motion induced by the ambient flow. Balanced flow Flow for which full drift results in no cumulative lateral drift. Direction of travel The angle of an animal’s trajectory (clock- wise from geographic north). Compensation Adjustment of self-speed and/or heading to prevent or diminish lateral drift. Simulations in this study presume con- stant self-speeds. Drift Advection by lateral flow, i.e. perpen- dicular to the migrant’s preferred direction. The term passive drift, some- times used to describe movement in water, refers to drift without self-speed. Endogenous heading An inherited preferred migratory direc- tion or heading (in the latter case possibly adapted to prevalent flow). Flow support Travel speed minus self-speed, i.e. Vector orientation rsif.royalsocietypublishing.org J. R. Soc. Interface 11: 20140588 11 the differ- ence in speed from that in the absence of flow. Hence we quantify flow support over the entire trajectory as opposed to locally(cf.localmeasures of flowassistance in [4]). The determination and maintenance of heading relative to the ground. hing.org J. R. Soc. Interface 11: 20140588 The intended travel direction. In this study, the preferred direction is ultimately the goal direction but headings differ instan- taneously according to the orientation strategy and flow configuration. An animal’s self-propelled speed relative to the moving flow (not adjusted to flow in this study). Typically referred to as the airspeed and swim speed in air and water, respectively. Flow strength Ratio of flow speed to self-speed. Full compensation Reaction to flow, restricted in this study through adjustment of heading, resulting in a constant direction of travel, i.e. pre- cluding drift. Only possible when the self-speed exceeds the lateral flow speed component relative to the heading. Ratio of flow speed to self-speed. Reaction to flow, restricted in this study through adjustment of heading, resulting in a constant direction of travel, i.e. pre- cluding drift. Only possible when the self-speed exceeds the lateral flow speed component relative to the heading. A gradient in flow speed along any direction; here used to mean a gradient in lateral flow along the initial goal direction. Speed of travel including non-movement periods. With non-stop movement, equivalent to mean ground speed. True navigation The ability to determine the direction to the goal from anywhere within the navigable range, even when drifted or displaced from intended paths. An orientation strategy, characterized by (a sequence of) constant headings. Typically proposed to be a juvenile migration strategy, we here also simu- late vector orientation by animals navigating in predictable flow by allowing headings to be adjusted on departure according to goal location and flow conditions en route. An animal’s instantaneous (horizontal) speed relative to the ground. The angle of an animal’s body axis rela- tive to the ground (clockwise from geographic north). The flow component perpendicular to a particular direction, here the preferred, i.e. goal direction.
https://openalex.org/W2801523106	https://hess.copernicus.org/articles/22/2511/2018/hess-22-2511-2018.pdf	English	null	Managing uncertainty in flood protection planning with climate projections	Hydrology and earth system sciences	2,018	cc-by	13,839	Managing uncertainty in ﬂood protection planning with climate projections Received: 24 September 2017 – Discussion started: 1 November 2017 Revised: 19 January 2018 – Accepted: 30 March 2018 – Published: 24 April 2018 Abstract. Technical ﬂood protection is a necessary part of integrated strategies to protect riverine settlements from extreme ﬂoods. Many technical ﬂood protection measures, such as dikes and protection walls, are costly to adapt af- ter their initial construction. This poses a challenge to de- cision makers as there is large uncertainty in how the re- quired protection level will change during the measure life- time, which is typically many decades long. Flood protec- tion requirements should account for multiple future uncer- tain factors: socioeconomic, e.g., whether the population and with it the damage potential grows or falls; technological, e.g., possible advancements in ﬂood protection; and climatic, e.g., whether extreme discharge will become more frequent or not. This paper focuses on climatic uncertainty. Specif- ically, we devise methodology to account for uncertainty associated with the use of discharge projections, ultimately leading to planning implications. For planning purposes, we categorize uncertainties as either “visible”, if they can be quantiﬁed from available catchment data, or “hidden”, if they cannot be quantiﬁed from catchment data and must be es- timated, e.g., from the literature. It is vital to consider the “hidden uncertainty”, since in practical applications only a limited amount of information (e.g., a ﬁnite projection en- semble) is available. We use a Bayesian approach to quan- tify the “visible uncertainties” and combine them with an estimate of the hidden uncertainties to learn a joint prob- ability distribution of the parameters of extreme discharge. The methodology is integrated into an optimization frame- work and applied to a pre-alpine case study to give a quanti- tative, cost-optimal recommendation on the required amount of ﬂood protection. The results show that hidden uncertainty ought to be considered in planning, but the larger the uncer- tainty already present, the smaller the impact of adding more. The recommended planning is robust to moderate changes in uncertainty as well as in trend. In contrast, planning without consideration of bias and dependencies in and between un- certainty components leads to strongly suboptimal planning recommendations. 1 Introduction The frequency of large ﬂuvial ﬂood events is expected to increase in Europe due to climate change (Alﬁeri et al., 2015). Therefore, planning authorities increasingly incorpo- rate discharge projections into the assessment of future ﬂood protection needs, rather than considering past observations alone. However, projections differ widely in terms of the level and trend of extreme discharge that they forecast. Fu- ture discharge extremes therefore should be modeled prob- abilistically for ﬂood protection planning (Aghakouchak et al., 2013). This raises two main questions: (1) how does one quantify a relevant uncertainty spectrum and (2) how is this then further used to identify a protection strategy? Recent studies have aimed at quantifying individual un- certainties in (extreme) discharge (Bosshard et al., 2013; Hawkins and Sutton, 2011; Sunyer, 2014). Sunyer (2014) has pointed out the usefulness of ﬁnding a methodology to com- bine uncertainties for ﬂood protection planning. In the ﬁrst part of this paper we present such a methodology for deriving a probabilistic model of extreme discharge; it is a pragmatic approach to handling the limited available data in practical problems. We quantitatively incorporate climate uncertainty from multiple information sources as well as an estimate of the “hidden uncertainty” into learning the probability distri- bution of parameters of extreme discharge. The term hidden Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 https://doi.org/10.5194/hess-22-2511-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 https://doi.org/10.5194/hess-22-2511-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Published by Copernicus Publications on behalf of the European Geosciences Union. This is done such that bias is inte- grated out and projections later on the horizon are assigned diminishing weights, making use of the hidden uncertainty shares (see Sect. 3.2). (4) The likelihoods of individual pro- jections are combined using the method of effective projec- tions (Pennell and Reichler, 2011; Sunyer et al., 2013b) in or- der to account for dependencies among them (see Sect. 3.3). (5) The Bayesian decision framework of Dittes et al. (2018) is used to obtain (6) a protection recommendation based on the likelihood of extreme discharge. The qualitative basis of the framework is outlined in Sect. 3.4. Once established, the question is then how to deal with the uncertainty in ﬂood risk estimates when conducting ﬂood protection planning. Multiple approaches have been pro- posed (Hallegatte, 2009; Kwakkel et al., 2010), including the addition of a planning margin to the initial design. The plan- ning margin is the protection capacity implemented in excess of the capacity that would be selected without taking into ac- count the uncertainties. Such reserves are used in practice; for example, in Bavaria, a planning margin of 15 % is ap- plied to the design of new protection measures to account for climate change (Pohl, 2013; Wiedemann and Slowacek, 2013). Planning margins are typically implemented based on rule-of-thumb estimates rather than a rigorous quantitative analysis (KLIWA, 2005, 2006; De Kok et al., 2008). y We have previously proposed a fully quantitative Bayesian decision-making framework for ﬂood protection (Dittes et al., 2018). Bayesian techniques are a natural way to model discharge probabilistically (Coles et al., 2003; Tebaldi et al., 2004). They also make it easy to combine several sources of information (Viglione et al., 2013). Furthermore, Bayesian methods support updating the discharge distribution in the future, when new information becomes available (Graf et al., 2007). Our framework probabilistically updates the distribu- tion of extreme discharge with hypothetical observations of future discharge, which are modeled probabilistically. This is an instance of a sequential (or “preposterior”) decision analysis (Benjamin and Cornell, 1970; Davis et al., 1972; Kochendorfer, 2015; Raiffa and Schlaifer, 1961). This en- ables a sequential planning process, where it is taken into consideration that the measure design may be revised in the future. Furthermore, it naturally takes into account the uncer- tainty in the parameters of extreme discharge. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projection 2512 n Bayesian decision framework (incl. parameter uncertainty) o Protection recommendation k Hidden uncertainty l Account for uncertainty and bias within projections m Account for dependency among projections j Flood projections Figure 1. Process of ﬁnding the recommended planning margin from projections and hidden uncertainty estimate. j Flood projections k Hidden uncertainty m Account for dependency among projections o Protection recommendation Figure 1. Process of ﬁnding the recommended planning margin from projections and hidden uncertainty estimate. (such as the 100-year ﬂood). To protect for the 100-year ﬂood is common European practice (Central European Flood Risk Assessment and Management in CENTROPE, 2013) and is also the requirement in the case study. uncertainty refers to uncertainty components that cannot be quantiﬁed from the given projections and data. For example, if the same hydrological model has been used for all projec- tions, then the hydrological model uncertainty is “hidden”, since one effectively has only a single sample of hydrologi- cal model output. It is vital to consider the hidden uncertainty since in practical applications only a limited amount of infor- mation and models is available and hidden uncertainty will always be present. In this paper, we show how to incorporate into the ﬂood planning process the “visible uncertainty” from an ensem- ble of climate projections as well as hidden uncertainties that cannot be quantiﬁed from the ensemble itself but may be es- timated from the literature. In the process of combining these uncertainties, we account for uncertainty and bias in projec- tions as well as for dependencies among different projec- tions. We provide reasoned estimates of climatic uncertain- ties for a pre-alpine catchment, followed by an application of the previously proposed Bayesian decision framework, sensitivity and robustness analysis. The process is shown in Fig. 1: (1) projections of annual maximum discharges (see Sect. 2.2) and (2) an estimate of the shares of various uncer- tainties that are not covered by the projection ensemble (see Sect. 2.5) form the inputs to the analysis. (3) For each projec- tion individually, a likelihood function of annual maximum discharge is computed. www.hydrol-earth-syst-sci.net/22/2511/2018/ B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2513 under a wide range of scenarios. We consider our approach to be complementary: rather than prescribing a protection system for the study site, it gives a recommendation for the optimal protection capacity. Expert judgement remains cen- tral for identifying robust protection systems to provide the recommended protection, e.g., by implementing a protection system that consists of several different, possibly spatially distributed, measures. Such an approach leads to more robust protection in which ﬂoods in excess of the design ﬂood do not quickly lead to very high damages or even failure (Blöschl et al., 2013b; Custer and Nishijima, 2013). ! 1880 m a.s.l. 440 m a.s.l. Planned flood polder Mangfall Mangfall catchment Rosenheim Elevation Lake Tegernsee Germany 0 10 km 5 Figure 2. Digital elevation model of the pre-alpine Mangfall catch- ment with its river network. The catchment is characterized by its highly heterogeneous topography leading to different discharge be- havior between the northern and southern regions of the river basin (Geobasisdaten© Bayerische Vermessungsverwaltung). ! Mangfall Mangfall catchment Rosenheim Lake Tegernsee The paper is structured as follows: in Sect. 2, we introduce the pre-alpine case study catchment together with the avail- able data and relevant uncertainties, concluding in an esti- mate of the hidden uncertainties. In Sect. 3, we show how to combine the different sources of uncertainty to use in the de- cision framework of Dittes et al. (2018). The resulting recom- mendations are presented and discussed in Sect. 4, together with the sensitivity analysis. Finally, a discussion is given in Sect. 5 and conclusions in Sect. 6. Figure 2. Digital elevation model of the pre-alpine Mangfall catch- ment with its river network. The catchment is characterized by its highly heterogeneous topography leading to different discharge be- havior between the northern and southern regions of the river basin (Geobasisdaten© Bayerische Vermessungsverwaltung). both characteristics of mountainous and lowland areas (Kun- stmann and Stadler, 2005; RMD Consult, 2016; Magdali, 2015). 2 Uncertainty in extreme discharge in a pre-alpine case study catchment Precipitation in the catchment is strongly affected by the adjacent Alpine arch leading to annual mean amounts of 1800 mm in mountainous and 1000 mm in low-altitude zones. The watershed receives most precipitation in July, of- ten in form of convective, high-intensity precipitation (Mag- dali, 2015; Deutscher Wetterdienst, 2018). Therefore, this study focusses on the uncertainty analysis for summer dis- charge, since it poses the greatest threat to the city of Rosen- heim. Planning authorities give the 100-year design dis- charge at the Rosenheim gauge as 480 m3 s−1 (RMD Con- sult, 2016). Figure 2 shows the topography of the Mangfall catchment alongside its river network. The available historic record at the Mangfall gauge in Rosenheim is reproduced in Supplement S1. In this section, we introduce individual components of un- certainty in estimates of extreme future discharge. This is done on the example of a pre-alpine catchment with a short historic record and a limited set of available climate projec- tions, which do not exhaustively cover the spectrum of cli- mate uncertainties. The resulting problem of planning under uncertainty is typical in practice. We introduce the case study catchment in Sect. 2.1, followed by the available discharge projections in Sect. 2.2. We then move on to describe cli- matic uncertainties in Sect. 2.3 and 2.4 and give an estimate of their magnitude for our analysis in Sect. 2.5. We end by in- troducing the mathematical modeling of uncertainties and the respective uncertainty of model parametrization in Sect. 2.6. Published by Copernicus Publications on behalf of the European Geosciences Union. The output of the framework is a cost-optimal capacity recommendation of ﬂood protection measures, given a ﬁxed protection criterion It is stressed that this paper focusses on the engineering as- pect of planning ﬂood protection under climate change. We aim to demonstrate how different sources of uncertainty can be combined probabilistically to make decisions, taking into account future developments. This is to aid decision making under climate uncertainty, when there are limited data and models available. Some authors advocate not using a proba- bilistic approach when the uncertainty is very large. This is because of the potential of surprises under large uncertainty (Hall and Solomatine, 2008; Merz et al., 2015; Paté-Cornell, 2011). Instead, they recommend an approach focussed on ro- bustness: the ability of the protection system to work well www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2.2 Available ensemble of discharge projections Our case study site is the river Mangfall at gauge Rosen- heim, shortly before it ﬂows into the Inn river. Rosenheim is a city in Bavaria that has suffered severe ﬂood losses from Mangfall ﬂooding in the past (Wasserwirtschaftsamt Rosenheim, 2014). With an area of 1102 km2, the Mang- fall is a medium-sized catchment exhibiting a highly hetero- geneous topography. Elevations within the catchment range from 443 to 1988 m a.s.l. with a mean value of approxi- mately 1000 m a.s.l., indicating the pre-alpine nature of the river basin. Southern sub-catchments in the Mangfall moun- tains are steep and rocky, resulting in a rapid runoff response. On the contrary, northern regions in the Alpine foothills show a more moderate discharge behavior due to gentle slopes. Thus, the discharge pattern of the Mangfall combines Table 1 lists the projections available at the case study gauge. Several projections have identical modeling chains and differ only in the model run; 6 of the 10 regional cli- mate models (RCMs) are nested in the same global climate model (GCM), ECHAM5; and all GCM–RCMs are based on the same IPCC SRES (Special Report on Emissions Scenar- ios) emission scenario, A1B. Furthermore, all climate mod- els are coupled to the same hydrological model (WaSiM) and same downscaling technique (quantile mapping). The ensemble is limited in that it does not cover a wide range of modeling uncertainties, and it is imperfect in that the pro- jections of the ensemble are not independent. Such a set of available projections is quite typical of what is encountered www.hydrol-earth-syst-sci.net/22/2511/2018/ www.hydrol-earth-syst-sci.net/22/2511/2018/ www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 2514 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections Table 1. RCMs used in this study, driving GCMs, source of the RCMs, downscaling and hydrological model. R1–R3 denote distinct model runs. Name GCM RCM Source Downscaling Hydrological model CLM1 ECHAM5 R1 CLM consort. Consortium Quantile mapping CLM2 ECHAM5 R2 CLM consort. Consortium (German federal CCLM HadCM3Q0 CCLM ETH institute of hydrology BfG), SCALMET (Willems and Stricker, 2011) WaSiM REMO1 ECHAM5 R1 REMO MPI Quantile mapping v8.06.02, Inn, REMO2 ECHAM5 R2 REMO MPI (Bavarian daily, 1 km2 REMO3 ECHAM5 R3 REMO MPI environmental RACMO ECHAM5 R3 RACMO2 KNMI agency LfU), HadRM HadCM3Q3 HadRM3Q3 Hadley Center SCALMET HadGM HadCM3Q3 RCA3 SMHI (Schmid et al., BCM BCM RCA3 SMHI 2014) B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2514 in ﬂood protection planning. The projections are reproduced in Supplement S2. in ﬂood protection planning. The projections are reproduced in Supplement S2. a nonlinear fashion. Just as uncertainties can add up, it is conceivable that they may not be relevant for future steps in the modeling chain (Refsgaard et al., 2013). In the fol- lowing, we brieﬂy introduce the individual modeling steps required to obtain projections of (extreme) discharge. The uncertainty from the interaction of consecutive steps in the modeling chain is called “interaction uncertainty” (Bosshard et al., 2013). The uncertainties in the climate modeling chain are in principle epistemic, yet it is debatable whether they can and will be reduced in the foreseeable future (Hawkins and Sutton, 2009). 2.3 Internal variability The term “internal variability” describes the irreducible un- certainty component in extreme discharge: even with per- fect knowledge, it cannot be predicted deterministically what the annual maximum discharge of a year will be, and thus how the design ﬂood estimate will change. This is because discharge realizations occur spontaneously, due to interac- tions of components within the climate system (IPCC, 2013). In the available projections, the absolute amount of internal variability did not change in time signiﬁcantly and is thus modeled as stationary. In projections of future discharge, however, the relative importance of internal variability de- creases with time as climatic uncertainties increase with in- creasing projection horizon. In a small pre-alpine catchment, such as considered in our case studies, the internal variabil- ity is large and dominates the uncertainty spectrum, poten- tially masking existing trend signals in heavy precipitation (and thus extreme discharge) for the entire projection horizon up to the year 2100 (Maraun, 2013). Alternative terms for the internal variability are “inherent randomness” or “noise”. The forcing of the climate through greenhouse gas emis- sions (GHGs) is the ﬁrst element in the climate modeling chain. The future socioeconomic, political and technolog- ical development determines the amount of GHGs emit- ted. The SRES scenarios – ﬁrst introduced by the IPCC in 2000 (IPCC, 2000) – were recently substituted by rep- resentative concentration pathways (RCPs), which directly refer to the amount of GHGs emitted rather than complex scenarios (Moss et al., 2010). For our case study, only projec- tions based on SRES scenario A1B, a widely used scenario with moderate socioeconomic and technological changes, are available. Thus, we have to take into account the uncertainty of what the projection results might have been under other forcing scenarios. However, in Europe, forcing uncertainty only becomes relevant in the far future and is of particularly low signiﬁcance for local extreme precipitation (Hawkins and Sutton, 2011; Maraun, 2013; Tebaldi et al., 2015). 2.5 Estimate of climatic uncertainty shares in extreme discharge for case study The uncertainty contribution of the downscal- ing is likely to be large (Hundecha et al., 2016; Sunyer et al., 2015b). It would be beneﬁcial to use not one but several downscaling techniques, similarly to how one uses an ensem- ble of GCM–RCMs (Arnbjerg-Nielsen et al., 2013; Sunyer et al., 2015a), as well as several calibration datasets (Sunyer et al., 2013a). – the role of forcing uncertainty and hydrological model is minor; the former becomes relevant only very late in the projection horizon; – uncertainty from interaction of the individual compo- nents may be of some signiﬁcance. A methodology to quantify the size of the internal variability, model response and forcing uncertainty in mean precipita- tion and corresponding results for different regions and sea- sons has been presented in Hawkins and Sutton (2009, 2011). We base our estimate of these components on equivalent re- sults for summer precipitation in Europe obtained from Ed Hawkins (email communication, 17 February 2017). We con- sider precipitation results to be transferable to discharge in the given catchment since extreme summer precipitation has in the past been the dominant trigger of high discharge in the Mangfall. A comparison of uncertainty shares for mean ver- sus extreme discharge is available in Bosshard et al. (2013) and is used to adapt the results. Quantitative estimates of the shares of model response, downscaling, hydrological model and interactions for a different pre-alpine catchment are also provided in Bosshard et al. (2013). We combine the quanti- tative results with the catchment-speciﬁc qualitative knowl- edge to produce the estimate. The uncertainty spectrum is shifted towards the later projection horizon to account for the longer dominance of internal variability in a pre-alpine catchment with small-scale, extreme summer precipitation as the ﬂood-triggering process. This results in a near-term contribution of the internal variability of at least 80 % of to- tal uncertainty, as expected. The shift also reduces the un- certainty share attributed to model response and emission Up to and including statistical downscaling, the climate modeling chain produces not discharge but various other cli- matic variables that are translated to discharge in a speciﬁc catchment through a hydrological model. Catchment param- eters (such as surface roughness) are typically found in an elaborate calibration procedure (Labarthe et al., 2014; Li et al., 2012). The parameters are usually assumed to be sta- tionary, but they might in fact be non-stationary (Merz et al., 2011). 2.5 Estimate of climatic uncertainty shares in extreme discharge for case study (IPCC, 2013). Multi-model ensembles (MMEs) such as the one available for the case study reproduce part of this spread. They do not reproduce it completely because they consist of a ﬁnite number of possibly biased and dependent models that typically have to be chosen based on availability rather than on statistical considerations (Knutti et al., 2013; Tebaldi and Knutti, 2007). To mitigate this problem, some researchers as- sign weights to individual models, but there is an ongoing de- bate about this: some researchers are making a general case for the beneﬁts of weighting (Ylhäisi et al., 2015) or its draw- backs (Aghakouchak et al., 2013), some are detailing when it may make sense on the basis of model performance (Ref- sgaard et al., 2014; Rodwell and Palmer, 2007) or geneal- ogy (Masson and Knutti, 2011), but all approaches are dis- puted. The relative importance of model response increases with projection lead time and is particularly signiﬁcant for extreme summer precipitation (Bosshard et al., 2013). Since ﬂooding in the case study catchment is dominated by extreme summer precipitation, we expect model response to form the second most important uncertainty contribution (after inter- nal variability). In this section, we estimate the relative contribution of cli- matic uncertainties, using internal variability as a reference. Note that this is done as a rough estimate, since uncertainty quantiﬁcation is not the focus of this paper. As will be- come clear in Sects. 4.2 and 5; an exact quantiﬁcation is also not necessary for the proposed decision-making process. To summarize the previous two sections, the following qualita- tive statements can be made about the contribution of rel- evant sources of uncertainty in the considered midsize pre- alpine catchments with ﬂoods driven by summer precipita- tion: – internal variability is dominant throughout most of the coming century; – model response is the second largest source of uncer- tainty, growing with lead time; – the impact of downscaling is also considerable, again particularly later in the projection horizon; The available projections underwent statistical downscal- ing using quantile mapping, which is often recommended for extreme events (Bosshard et al., 2011; Dobler et al., 2012; Hall et al., 2014; Themeßl et al., 2010). Statistical downscal- ing is frequently used to align GCM–RCM outputs with his- toric records, but its use is still controversial (Chen et al., 2015; Ehret et al., 2012; Huang et al., 2014; Maurer and Pierce, 2014). 2.4 Uncertainties in the climate modeling chain Discharge projections are the result of a complex multi- step climate modeling process. In the literature, this is of- ten termed the climate modeling “chain”, which, as new un- certainties are introduced at each modeling step, leads to the “uncertainty cascade” (Mitchell and Hulme, 1999; Fo- ley, 2010). It is worth pointing out that the uncertainty cas- cade does not necessarily lead to an increase in uncertainty at each step, as the modeling steps depend on each other in For climate change impact studies, it is customary to use ensembles of multiple combinations of global and regional climate models (GCMs–RCMs) (Huang et al., 2014; Muerth et al., 2012; Rajczak et al., 2013). The differences in GCM– RCM output when driven by the same emission forcing are termed “model response uncertainty” or “model spread” www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 2515 2.5 Estimate of climatic uncertainty shares in extreme discharge for case study The shares are adjusted to better represent the particular modeling and topography: the share of model response is set to peak at around 40 %. For downscaling, shares of up to 25 % are expected. Uncer- tainties stemming from interactions are anticipated to lie on the order of 10 %. Contributions attributed to hydrological modeling are set to remain below 5 % over the whole time horizon. The results of the estimation are shown in Fig. 3. Figure 3a shows the resulting relative uncertainty shares and Fig. 3b the resulting absolute uncertainties for the projection CCLM. Forcing, downscaling, hydrological model and inter- action components are hidden uncertainties in the case study. As will be shown in Sect. 3, the sum of hidden uncertainties rather than individual components is used in the Bayesian estimation. Thus, it does not matter if the share of any one of these uncertainties has been slightly over- or underesti- mated. The question of sensitivity will be discussed further in Sect. 4. The estimated variance shares of the hidden un- certainty components and internal variability with respect to total uncertainty for Rosenheim are given in Supplement S3. 1 −FQ(t)\|θ q(T )\|θ = 1 T ↔q(T ) := F −1 Q(t)\|θ 1 −1 T \|θ , (1) where FQ(t)\|θ is the cumulative distribution function (CDF) and F −1 Q(t)\|θ is the inverse CDF of the annual maximum dis- charge Q(t). In a Bayesian setting, the posterior joint PDF of the parameters θ can be estimated from N years of annual maximum discharges q = [q1, ..., qN] (from historic record or projections) as follows: where FQ(t)\|θ is the cumulative distribution function (CDF) and F −1 Q(t)\|θ is the inverse CDF of the annual maximum dis- charge Q(t). In a Bayesian setting, the posterior joint PDF of the parameters θ can be estimated from N years of annual maximum discharges q = [q1, ..., qN] (from historic record or projections) as follows: fθ\|Q(t)(θ\|q) ∝L(θ\|q)fθ(θ), (2) (2) where fθ(θ) is the prior distribution of the parameters and L(θ\|q) is the likelihood describing the discharge data q. The discharge maxima can be assumed to be independent be- tween individual years (Coles, 2004). Neglecting measure- ment error, the likelihood function in Eq. (2) can hence be formulated as L(θ\|q) = N Y t=1 fQ(t)\|θ (qt\|θ). (3) (3) 2.5 Estimate of climatic uncertainty shares in extreme discharge for case study Furthermore, the calibration might mask model errors by tuning the catchment parameters to balance them. Thus, the parameter estimates strongly depend on the cali- bration period (Brigode et al., 2013). Several approaches ex- ist to quantify the uncertainty stemming from the hydrolog- ical model (Götzinger and Bárdossy, 2008; Velázquez et al., 2013). Overall, however, the error from the choice of hydro- logical model is small, in particular for high-ﬂow indicators (Velázquez et al., 2013). It is likely smaller than or compara- ble to forcing uncertainty (Wilby, 2005). www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2516 10 20 30 40 50 60 70 80 90 Projection horizon (years) 0 10 20 30 40 50 60 70 80 90 100 Share of total variance (%) Internal variability Model response Forcing Downscaling Hydrological model Interactions 10 20 30 40 50 60 70 80 90 Projection horizon (years) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Uncertainty variance (m s ) 3 104 Internal variability Model response Forcing Downscaling Hydrological model Interactions (a) (b) -1 Figure 3. (a) Share of different uncertainty components (variance) for extreme discharge in Rosenheim. (b) Resulting absolute uncertainties for CCLM. Uncertainties that are “visible” in our case study are shaded yellow–orange and “hidden” ones blue–green. 10 20 30 40 50 60 70 80 90 Projection horizon (years) 0 10 20 30 40 50 60 70 80 90 100 Share of total variance (%) Internal variability Model response Forcing Downscaling Hydrological model Interactions (a) 10 20 30 40 50 60 70 80 90 Projection horizon (years) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Uncertainty variance (m s ) 3 104 Internal variability Model response Forcing Downscaling Hydrological model Interactions (b) -1 Uncertainty variance (m s ) 3 -1 Figure 3. (a) Share of different uncertainty components (variance) for extreme discharge in Rosenheim. (b) Resulting absolute uncertainties for CCLM. Uncertainties that are “visible” in our case study are shaded yellow–orange and “hidden” ones blue–green. The discharge q(T ) of a design ﬂood associated with a re- turn period T is deﬁned as a function of θ as forcing, which, following Ed Hawkins (email communica- tion, 17 February 2017), explained over 90 % of total un- certainty by the end of the century. 3.2 Accounting for uncertainty and bias in projections In this section, we propose an approach for combining dif- ferent uncertainty components when using projections to estimate the parameters θ of the time-variant distribution fQ(t)\|2(q\|θ) of annual maximum discharge Q(t) in year t (see Sect. 2.6). This distribution is ﬁrst estimated for each projection of extreme discharges individually. For projection, we increase the distribution spread in a time-dependent man- ner using the estimate of hidden uncertainty from Sect. 2.5. Since the uncertainty increases with time, projections late on the horizon are naturally assigned less information value. We then combine the distributions from different projections. When using discharge projections, it is important to account for uncertainty and bias within them. As discussed in Sect. 2, climatic uncertainties increase with the projection horizon and thus the information value of a projection made late on the horizon is smaller than that of an earlier one. For exam- ple, a projection for the year 2100 is associated with higher uncertainty than one that is made for the coming year and should have less weight when estimating the parameters θ of the distribution of annual maximum discharge from climate projections. In the following, we develop a methodology that accounts for this. In Sect. 3.1, we categorize uncertainties in such a way that it is conducive for our application. We then combine these uncertainties within a Bayesian approach. In Sect. 3.2, we show how the likelihood L(θ\|q) for the joint parameter PDF is estimated for any individual projection, taking into account uncertainty estimates from the literature. In Sect. 3.3, we show how to combine the likelihoods of the projection en- semble. Finally, we give a summary of planning and decision making under uncertainty in Sect. 3.4. We introduce the standard deviation σ (u) i,t , in which the su- perscript (u) describes which type of uncertainty is consid- ered (internal or hidden), the subscript i denotes the projec- tion and the subscript t the time dependence. The internal variability in a projection, [σ (internal) i ]2, can be quantiﬁed fol- lowing Hawkins and Sutton (2009). Note that the subscript t is excluded here since internal variability is assumed to be in- dependent of time. Relative variance shares of the individual uncertainties, including hidden ones, can be estimated using literature (Bosshard et al., 2013; Hawkins and Sutton, 2011) and expert judgement, as was done in Sect. 2.5. 2.6 Parameter uncertainty With increasing number of records of annual maximum dis- charges qt, the uncertainty in the parameters θ is reduced. Statistical modeling of extreme discharge Q is commonly based on ﬁtting a suitable extreme value distribution to the available data, e.g., a Gumbel or a generalized extreme value (GEV) distribution. These are described by their prob- ability density function (PDF), fQ\|θ(q\|θ), in which θ is the set of parameters of the distribution function that are esti- mated from the data. Estimating θ from ﬁnite data will result in a probability distribution over θ, which describes parame- ter uncertainty (Kennedy and O’Hagan, 2001). The Bayesian method requires the selection of a prior dis- tribution fθ(θ) in Eq. (2). For the application to ﬂood pro- tection planning, one may wish to select a prior distribution that is only weakly informative in q(T ). We propose to use the following distribution for this purpose (dropping the time dependence t for readability): www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 www.hydrol-earth-syst-sci.net/22/2511/2018/ B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2517 fθ(θ) ∝ 1 fQ(T ) q(T ) = 1 fQ(T ) F −1 Q(T )\|θ 1 −1 T \|θ , (4) where fQ(T )(q(T )) is the PDF of q(T ) based on a prior dis- tribution that is uniform in θ and Eq. (1) has been applied in the equality. fθ(θ) ∝ 1 fQ(T ) q(T ) = 1 fQ(T ) F −1 Q(T )\|θ 1 −1 T \|θ , (4) – Hidden uncertainty, which is the remaining uncertainty and can, at best, be estimated. For example, in the pro- jection ensemble of the case study, forcing uncertainty is hidden since all projections are based on the same emission scenario. In real planning situations, hidden uncertainty is typically signiﬁcant because of limited and imperfect projections and data; it can therefore not be neglected. (4) where fQ(T )(q(T )) is the PDF of q(T ) based on a prior dis- tribution that is uniform in θ and Eq. (1) has been applied in the equality. In the following sections, a methodology will be presented to estimate the distribution of parameters of annual maximum discharge using these uncertainties. 3.2 Accounting for uncertainty and bias in projections The share of an individual uncertainty component in the total variance is here labeled η(u) t , with the indexing as for σ. The uncertainty shares are assumed to be general for a given location, in- dependent of the projection. Thus, the absolute value of the hidden uncertainty can be found from the absolute internal variability and the uncertainty variance shares of Sect. 2.5 (reproduced numerically in Supplement S3) as B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections these sets, the likelihood function can then be formulated as the product of the likelihoods L(j) i (θ\|pi) of the set members, since they are assumed to contain independent information: Li,t θ\|pi,t,1i,t = fQ(t)\|2 pi,t −1i,t\|θ , (6) (6) where fQ(t)\|2 is the PDF of the extreme value distribution describing Q(t). The likelihood Li,t(θ\|pi,t, 1i,t) determines the estimation of the PDF of parameters θ from projections, similar to Eq. (2). L(j)(θ\|p) = IY i=1 L(j) i θ\|pi . (9) (9) (9) The bias 1i,t is modeled as a normal random variable with zero mean and standard deviation σ (hidden) i t : Climatological rationale is applied to determine the division of the ensemble into sets: in line with the concept of effective projections, the projections in each set should be as distinct as possible, adding a maximum of additional information. 1i,t = zσ (hidden) i,t = zσ (internal) i v u u t η(hidden) t η(internal) t , (7) (7) Based on their genealogy, we partition the available pro- jections (Table 1) as follows. – When using two sets of ﬁve effective projections: with z being a standard normal random variable. By model- ing all 1i,t as a function of the same z, it is assumed that the 1i,t are fully dependent within one projection i. This treat- ment is conservative, since it minimizes the amount of learn- ing from projected discharges. Due to the large impact of the projection on the bias, it is a better depiction of reality than the assumption of independent 1i,t within one projection i. From this follows the likelihood for a complete projection time series pi as – Set 1: CLM1, CCLM, REMO2, HadGM, RACMO. – Set 2: CLM2, REMO1, REMO3, HadRM, BCM. – Set 1: CLM1, CCLM, REMO2, HadGM, RACMO. – Set 2: CLM2, REMO1, REMO3, HadRM, BCM. – When using three sets of three effective projections (dropping REMO3): – When using three sets of three effective projections (dropping REMO3): – Set 1: CLM1, REMO2, HadRM. – Set 2: CLM2, REMO1, HadGM. – Set 3: CCLM, RACMO, BCM. – Set 1: CLM1, REMO2, HadRM. – Set 2: CLM2, REMO1, HadGM. – Set 3: CCLM, RACMO, BCM. – Set 1: CLM1, REMO2, HadRM. Li θ\|pi = ∞ Z −∞ " N′ Y t=1 fQ(t)\|2 pi,t −zσ (hidden) i,t \|θ # ν(z)dz, (8) (8) The set likelihood L(j)(θ\|p) from Eq. 3.4 Planning under uncertainty Protection requirements (“criterions”) are based on the T - year discharge q(T ) (see Eq. 1), most commonly the 100 year discharge. Since the estimate of q(T ) – the peak of the PDF – changes as new data become available, the capacity of the ﬂood protection system will be re-evaluated in the fu- ture and possibly be adjusted. The probability that adjust- ment becomes necessary is determined by the level of un- certainty: the higher the uncertainty in the future extreme discharges, the more likely it is that an adjustment of the protection system will become necessary in the future. To understand why this is, consider Fig. 4: after initial plan- ning, new discharges are observed (lilac dots). If, as pictured here, the observed discharges are higher than expected, the design ﬂood estimate q(T ) will increase. (We show the esti- mate of q(1) to be able to display observations on the same scale. Note that the 99th percentile of the shown PDF does not correspond to q(100).) If the uncertainty is large at the time of initial planning – as is the case here, visualized with the blue, original PDF – then the additional information from Protection requirements (“criterions”) are based on the T - year discharge q(T ) (see Eq. 1), most commonly the 100 year discharge. Since the estimate of q(T ) – the peak of the PDF B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections (9) is used to compute the joint set posterior of parameters, f (j) 2\|Q(t)(θ\|p), similar to Eq. (2). The set posteriors are then averaged to result in an overall posterior f2\|Q(t)(θ\|p) of learning from projections under climate uncertainty. The averaging over posteriors ex- presses that we place equal trust in distributions estimated from the different sets. where ν is the standard normal PDF. Internal variability is included in Eq. (8) naturally via pi,t, as is parameter uncer- tainty, which is a function of the length of projections. The estimate of hidden uncertainty, as from Sect. 2.5, is included via σ (hidden) i,t . While we are focussing on climate uncertainty here, in principle, any kind of additional uncertainty can be incorporated via the hidden uncertainty parameter σ (hidden) i,t in Eq. (8). Model response uncertainty is included in the com- bination of the likelihoods Li(θ\|pi) from different projec- tions i, as described in the following section. 3.1 Uncertainty categorization Different categorizations of uncertainty have been proposed in the literature. In Sect. 2.3–2.6 for example, we have pre- sented the uncertainties in extreme discharge by source. An- other common way to categorize uncertainties is the distinc- tion between aleatory (irreducible) and epistemic (reducible) uncertainties (Der Kiureghian and Ditlevsen, 2009; Refs- gaard et al., 2013). This categorization is useful in that it underlines in which areas future research could lead to un- certainty reduction. Other authors focus their categorization on the different effects of uncertainties (Merz et al., 2015). σ (hidden) i,t = σ (internal) i v u u t η(hidden) t η(internal) t . (5) (5) (5) In the context of estimating ﬂood extremes under climate change with limited information, we distinguish between the following: For estimating the joint PDF of the parameters θ of the annual maximum discharge distribution, we treat the i = 1, ..., M discharge projections pi = [pi,t=1, . .., pi,t=N′] as samples of the true future discharge τt with a bias 1i,t: τt = pi,t −1i,t. We express the likelihood Li,t(θ\|pi,t, 1i,t) describing the annual maximum discharge of projection i in year t as For estimating the joint PDF of the parameters θ of the annual maximum discharge distribution, we treat the i = 1, ..., M discharge projections pi = [pi,t=1, . .., pi,t=N′] as samples of the true future discharge τt with a bias 1i,t: τt = pi,t −1i,t. We express the likelihood Li,t(θ\|pi,t, 1i,t) describing the annual maximum discharge of projection i in year t as – Visible uncertainty, which is known and can be quan- tiﬁed. For an ensemble of discharge projections, this would, for example, be the internal variability, the model response uncertainty and parameter uncertainty. Parameter uncertainty is also visible in that it is straight- forward to quantify, but it is not a climatic uncertainty. www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 2518 3.3 Accounting for dependency among projections Individual projections are not independent. Hence, one can- not combine Li(θ\|pi) into a joint likelihood L(θ\|p) via a simple product over projections pi. Dependence among mul- tiple projections is due to common model biases, be it be- cause they, for example, share code from the same institu- tion or because our understanding of climate processes is not perfect (Knutti et al., 2013; Tebaldi and Knutti, 2007). Con- sequently, conﬁdence in the prediction variance should not increase linearly with the number of projections in an ensem- ble. Instead, the ensemble should be seen as consisting of an effective number I of quasi-independent projections (adding independent pieces of knowledge) that is smaller than the en- semble size M (Pennell and Reichler, 2011; Sunyer et al., 2013b). We thus partition the ensemble into J sets of I pro- jections, where J is the integer quotient of M I . For each of www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2519 0 50 100 150 200 250 300 Annual max. discharge (m s ) 3 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 PDF Original PDF Updated PDF Protection level boundary New observations Adjustment needed -1 Figure 4. Original and updated PDF based on a period of high new observations of annual maximum discharge. Because the original PDF is so broad, the period of extreme observations results in a strongly shifted updated PDF, the peak of which (corresponding to the estimate of q(1)) crosses the protection boundary level. Thus, the protection system must be adjusted. ing that period or – more precisely – based on the q(100) as resulting from the updated distribution of annual maximum discharges. To probabilistically model this future updating (before these data are actually available), future realizations of annual maximum discharge q are sampled from the dis- charge distribution fQ(t)\|2(q\|θ) estimated initially. Optimal decisions are then identiﬁed via backwards induction opti- mization (Raiffa and Schlaifer, 1961), which works by ﬁrst determining the system that should be installed at the last adjustment, conditional on the existing protection and dis- charges observed by then. 3.3 Accounting for dependency among projections The obtained recommendation is then used to ﬁnd the system that should be installed at the second to last adjustment and so forth until arriving at a recommendation for the system that should be installed ini- tially. We employ this optimization framework in the follow- ing case study. 0 50 100 150 200 250 300 Annual max. discharge (m s ) 3 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 PDF Original PDF Updated PDF Protection level boundary New observations Adjustment needed -1 Note that, since there is often a discrepancy between the level of observed past discharge at a speciﬁc gauge and the corresponding regional climate projections, we take the com- monly used approach (Fatichi et al., 2013; Pöhler et al., 2012) of computing relative rather than absolute values from the climate projections. Here, this means that we ﬁnd a planning margin γ based on the projection ensemble and uncertainty estimates from the literature, which may then be applied to the absolute protection (100-year ﬂood) as estimated from historic records. Figure 4. Original and updated PDF based on a period of high new observations of annual maximum discharge. Because the original PDF is so broad, the period of extreme observations results in a strongly shifted updated PDF, the peak of which (corresponding to the estimate of q(1)) crosses the protection boundary level. Thus, the protection system must be adjusted. the new observation has a larger weight in predicting fu- ture extreme discharges. The change in q(T ) is larger than if the distribution of extreme discharges had been more in- formative (i.e., more “certain”, less “spread out”). In prac- tice, the protection will only be adjusted when a signiﬁcant change in q(T ) has occurred that cannot be compensated by the freeboard and planning margin present (represented by the protection level boundary from which onward adjustment is needed). To avoid the need for frequent adjustments and increase robustness, the optimization framework of Dittes et al. (2018) thus recommends a higher planning margin when the system is constructed under higher uncertainty initially, as will become apparent in the results. 4 Case study We present the integration of the uncertainty quantiﬁcation of extreme discharge in the pre-alpine Mangfall gauge at Rosenheim as shown in Sect. 2 with the uncertainty com- bination methodology of Sect. 3 and the decision framework of Dittes et al. (2018). Section 4.1 gives details of the imple- mentation, followed by the protection recommendation and sensitivity results in Sect. 4.2. Because the – as yet uncertain – future discharge real- izations determine future decisions, they have an impact on the optimality of the initial decision. Therefore, it is sensi- ble to model protection planning as sequential, with prob- abilistic future discharge observations, updating of the dis- charge PDF and corresponding decisions on adjustment in regular time intervals. A Bayesian network approach do- ing so for decisions on adapting infrastructure to a chang- ing climate has been presented by Nishijima (2015) and a POMDP (partially observable Markov decision process) ap- proach applied to ﬂood protection, using climate scenarios, has been described by Špaˇcková and Straub (2016). An al- ternative sampling-based approach, which takes the full joint parameter PDF into account, has been proposed by Dittes et al. (2018). The planning horizon is divided into a num- ber of time periods. After each period, the current protec- tion level is re-evaluated and possibly adjusted based on the annual maximum discharges that have been observed dur- www.hydrol-earth-syst-sci.net/22/2511/2018/ B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections The joint PDF of parameters of annual maximum dis- charge estimated from the climate projections is used as the basis for future updating with discharge realizations. To ob- tain this PDF, the climate projections are estimated on a prior distribution that is weakly informative in the 100-year design discharge of the ﬁrst time step (years 1–30) as by Eq. (4). Computationally, the prior distribution is constructed by uni- form sampling of parameters over a large space, computing the respective 100-year ﬂood estimate for the ﬁrst time step for each sampled parameter vector, and performing rejection sampling to obtain samples on the order of 6 × 105 following Eq. (4). The PDFs shown in Fig. 5 are used as input to the op- timization framework of Dittes et al. (2018) to obtain rec- ommendations for the planning margin. Section 3.4 gave an intuitive understanding of how these relate to the 100-year PDF. The planning margin that is recommended when es- timating based on the historic record only is 111.8 %, ver- sus 81.9, 16.5, 12.5 and 2.6 % for 1, 3, 5 and 10 effective projections, respectively. These results are summarized in Table 2. Using a similar ensemble of climate projections over Den- mark, Sunyer et al. (2013b) established that an ensemble of 10 projections corresponds to 5 effective projections for 20- year heavy summer precipitation. Despite some issues with transferability – as will be discussed in Sect. 5 – we thus use ﬁve effective projections and hence a planning reserve of 12.5 % as the recommended protection margin from the ex- treme summer precipitation ﬂoods observed at the Mangfall in Rosenheim. To ﬁnd the optimal ﬂood protection considering the full sequential decision process, it is necessary to simulate future discharge data, from which new ﬂood estimates will be esti- mated (see Sect. 3.4). For this purpose, we used 300 samples of annual maximum discharge in the period 1–30 years and 70 samples of annual maximum discharge in the period 31– 60 years. Using fewer discharge samples in later periods is computationally preferable and still comes with a high accu- racy, as the absolute number of samples in the second period overall is 300 × 70 = 21 000. This choice of number of sam- ples leads to a relative error of less than 4 % in the protection recommendation. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2520 y p p g p j 200 300 400 500 600 700 800 900 1000 1100 1200 100-year discharge (m s ) 3 0 2 4 6 8 10 12 14 16 18 PDF 10 effective projections 5 effective projections 3 effective projections 1 effective projection Historic record x10-3 -1 Figure 5. The 100-year discharge PDF from initial parameter distri- bution when estimated based on the historic record (dashed) versus different numbers of effective projections, for years 1–30. describes the cost of the construction/extension of the pro- tection system and a discounting rate of 2 % annually is em- ployed. In Dittes et al. (2018), we considered a measure of ﬂexibility which describes how costly it is to adapt measures later in their lifetime. In this contribution, we give results for the non-ﬂexible case only, which implies that future adjust- ments to the system are expensive. Introducing some ﬂexibil- ity into the protection system would lead to lower planning margin results than those obtained here. 200 300 400 500 600 700 800 900 1000 1100 1200 100-year discharge (m s ) 3 0 2 4 6 8 10 12 14 16 18 PDF 10 effective projections 5 effective projections 3 effective projections 1 effective projection Historic record x10-3 -1 Following model plausibility testing on the projections (MacKay, 1992), a GEV distribution is chosen to model the annual maximum discharges. It is described by shape pa- rameter k, scale parameter β > 0 and location parameter µ. We employ a linear trend in the scale and location parame- ters, which is common practice in the literature (Coles, 2004; Delgado et al., 2010; Hanel and Buishand, 2011; Maraun, 2013). The scale is expressed as β = β0 + β1t and the lo- cation as µ = µ0 + µ1t (Coles, 2004; Hanel and Buishand, 2011). Thus, θ = (kβ0µ0, β1µ1). 100-year discharge (m s ) 3 -1 Figure 5. The 100-year discharge PDF from initial parameter distri- bution when estimated based on the historic record (dashed) versus different numbers of effective projections, for years 1–30. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections To investigate the effect of hidden uncertainty on the pro- tection recommendation, we repeated the optimization, once using no hidden uncertainty and once doubling the hid- den uncertainty variance shares estimated in Sect. 2.5 (re- produced numerically in Supplement S3), with an effective model number of ﬁve. The recommended planning margins lay in the expected order, with the “no uncertainty” recom- mendation the smallest at 8.1 % and the “double uncertainty” recommendation the largest at 13.8 %. Finally, we studied the effect of changing the trend in the projections of annual max- imum discharge. Detrending the projected annual maxima lead to a recommendation of 12.2 %. We then used the pro- jected annual maxima with doubled trend: from the observed average of 0.25 m3 s−1 per year (corresponding to an 11 % rise in mean annual maximum discharge during the 90-year 4.1 Implementation We conduct our case study for the Mangfall river in Rosen- heim, which has been introduced in Sect. 2.1. We consider the designed ﬂood protection systems to have a lifetime of 90 years and to be designed in such a way as to protect from the 100-year ﬂood, with design discharge q(100). The deci- sion on the protection capacity will be revised every 30 years, taking into account the discharge records that will be avail- able at these points in time. When estimating climate param- eters – especially trends – from a time step, 30 years is an often used compromise between the desire to minimize sta- tistical uncertainty and that to capture recent climate devel- opments (IPCC, 2013; Kerkhoff et al., 2015; Laprise, 2014; Pöhler et al., 2012). The protection requirement corresponds to the maximal required protection during the time step in question. As in Dittes et al. (2018), a square root function www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 5 Discussion It is apparent from the results that the number of effective projections has a large impact on the recommended plan- ning margin. Hence, we recommend that planners make use of the concept of effective projections and partition ensem- bles accordingly, rather than just average over all members of a projection ensemble. Our assumption that ﬁve effec- tive projections are applicable for the 10-member ensemble at Rosenheim can be questioned. The transferability of the corresponding results of Sunyer et al. (2013b) might be hin- dered by the difference in considered location (a southern German catchment versus an averaging over Denmark), en- semble (some members differ) and extreme index (100-year event versus 20-year event). From other results presented in Sunyer et al. (2013b) using an alternative measure of pro- jection dependence as well as higher extreme indices, we believe that the 12.5 % recommendation given here is con- servative and a slightly lower recommendation for the plan- ning margin (based on a slightly higher number of effective projections) may be applicable. However, the transferability remains questionable for the location and ensemble and thus the study ideally ought to be repeated for the given catchment and ensemble, in particular with respect to the large impact of the number of effective projections on the protection rec- ommendation. We turn to the sensitivity analysis. First, the trend: the fact that signals that emerge late on the planning horizon are masked by noise and rendered less relevant by discount- ing explains why changing the trend signal leads to only in- signiﬁcant changes in recommended planning margin. This is compounded by the fact that the trend signal is weak, which is to be expected from the location of the case study catch- ment (Madsen et al., 2014; Maraun, 2013) and is potentially ampliﬁed by projections underestimating trends in extreme precipitation (Haren et al., 2013). It should be added that not all scientists are comfortable with linear trend projections in extreme precipitation and discharge and that there is also an argument to be made for cyclical components (Gregersen et al., 2014) or “ﬂood-rich” versus “ﬂood-poor” periods (Hall et al., 2014; Merz et al., 2014), though these may not be ap- plicable to ﬂoods of large return periods such as studied here (Merz et al., 2016). We assumed a linear trend in the case study for simplicity, but the proposed methodology is gen- eral. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2521 Table 2. Recommended planning margin when using the historic record versus differing numbers of effectiv initial parameter space. nded planning margin when using the historic record versus differing numbers of effective projections for esti ace. Effective number of projections (or historic) historic 1 3 5 10 Recommended planning margin (%) 111.8 81.9 16.5 12.5 2.6 Effective number of projections (or historic) historic 1 3 5 10 Recommended planning margin (%) 111.8 81.9 16.5 12.5 2.6 Table 3. Recommended planning margin (%) when using ﬁve ef- fective projections and varying hidden uncertainty and trend. Quantity\applied change None Reference Double Hidden uncertainty 8.1 12.5 13.8 Trend in annual max. discharge 12.2 12.7 lifetime) to 0.5 m3 s−1 per year. The recommended planning margin increased only very slightly, from 12.5 to 12.7 %. The results are summarized in Table 3. Table 3. Recommended planning margin (%) when using ﬁve ef- fective projections and varying hidden uncertainty and trend. Table 3. Recommended planning margin (%) when using ﬁve ef- fective projections and varying hidden uncertainty and trend. Table 3. Recommended planning margin (%) when using ﬁve ef- fective projections and varying hidden uncertainty and trend. Quantity\applied change None Reference Double Hidden uncertainty 8.1 12.5 13.8 Trend in annual max. discharge 12.2 12.7 4.2 Protection recommendation and sensitivity Figure 5 shows the 100-year discharge PDF (weighted mean) from the initial parameter distribution for the ﬁrst 30 years of planning when estimated from the 39-year-long historic record versus 10, 5, 3 and 1 effective projections of 90-year length. Ten effective projections correspond to multiplying all posteriors and one effective projection corresponds to av- eraging all posteriors. For ﬁve and three effective projections, we split the projections into sets as given in Sect. 3.3. Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 www.hydrol-earth-syst-sci.net/22/2511/2018/ B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections Author contributions. BD developed the concepts of this paper un- der the guidance of OŠ and DS. BD wrote the code and performed the simulations. The results presented in Sect. 2 were obtained by LS. BD prepared the manuscript with contributions from all co- authors. mendation to the size of the hidden uncertainty in the pre- sented case study can be explained by the considerable visi- ble uncertainty present: the capacity to project the future ex- treme discharge is already extremely limited and can barely be reduced by adding more uncertainty. While this may ap- pear disheartening, it can also be a wake-up call to stop wait- ing for (doubtful) uncertainty reductions in climate modeling and start making (robust) decisions (Arnbjerg-Nielsen et al., 2013; Curry and Webster, 2011; Hawkins and Sutton, 2011). mendation to the size of the hidden uncertainty in the pre- sented case study can be explained by the considerable visi- ble uncertainty present: the capacity to project the future ex- treme discharge is already extremely limited and can barely be reduced by adding more uncertainty. While this may ap- pear disheartening, it can also be a wake-up call to stop wait- ing for (doubtful) uncertainty reductions in climate modeling and start making (robust) decisions (Arnbjerg-Nielsen et al., 2013; Curry and Webster, 2011; Hawkins and Sutton, 2011). Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. We would like to thank Holger Komischke of Bayerisches Landesamt für Umwelt (LfU) for fruitful discussions. The LfU also provided the discharge records and projections used in the case study. The discharge projections were modeled within the cooperation KLIWA and the Interreg IVB project AdaptAlp. They were based either on ENSEMBLES data funded by the EU FP6 Integrated Project ENSEMBLES (contract number 505539), whose support is gratefully acknowledged, or additional available climate projections. These are REMO1 (“UBA”) and REMO2 (“BfG”) (Umweltbundesamt, 2018), as well as CLM1 and CLM2 (Hollweg et al., 2008). This work was supported by Deutsche Forschungsge- meinschaft (DFG) through the TUM International Graduate School of Science and Engineering (IGSSE). 6 Conclusions Estimates of future extreme discharge are fraught with sig- niﬁcant uncertainties that need to be accounted for in ﬂood protection planning. In particular, the following points must be considered when estimating the parameters of future ex- treme discharge distributions: 1. an estimate of the uncertainty that cannot be quantiﬁed from the available data (the hidden uncertainty) must be included, since projections and data at hand cover only a limited range of the uncertainty spectrum (the visible uncertainty); This work was supported by the German Research Foundation (DFG) and the Technische Universität München within the funding programme 2. the time development of the uncertainty, so as to give less weight to projections far on the projection horizon; Open Access Publishing. 3. dependency between projections, since projection en- sembles often include several projections sharing code or assumptions. Edited by: Uwe Ehret Reviewed by: two anonymous referees Edited by: Uwe Ehret Reviewed by: two anonymous referees Edited by: Uwe Ehret Reviewed by: two anonymous referees In the proposed methodology, we quantitatively include these aspects in estimating the probabilistic distribution of ﬂood discharge. Both “visible” and hidden uncertainty are in- cluded in a time-dependent Bayesian likelihood function. Dependence between projections is accounted for by using the concept of effective projection number. The uncertainty analysis proposed in this paper was used with the optimiza- tion framework of Dittes et al. (2018) to ﬁnd protection rec- ommendations for a pre-alpine case study catchment. The re- sults show that when there is sizable visible uncertainty, the protection recommendation is robust to further uncertainty and moderate changes in trend. However, hidden uncertainty should not be neglected in planning as this would lead to in- sufﬁcient protection recommendations. 5 Discussion To use a different trend representation, one just has to change the deﬁnition of θ (see Sect. 4.1) accordingly. Finally, we discuss the impact of varying size of uncer- tainty on planning. To investigate this, we evaluated the rec- ommended planning margin when not adding any hidden un- certainty, when using the estimated amount and when us- ing double the estimated amount of hidden uncertainty (see Sect. 4.2). The effect was small, in particular between adding the estimate versus double the estimate of hidden uncertainty. The share of hidden uncertainty is larger in the farther fu- ture, where its effect is limited because of discounting. We conclude that hidden uncertainty should be considered in de- cision making, yet the sensitivity to its exact amount is low and, when there is already a considerable level of uncertainty, including more has little effect. This is why we do not engage in detailed discussion on whether the size of the hidden un- certainty has been gauged correctly and whether additional uncertainty components should be included, despite this cer- tainly being debatable (Grundmann, 2010; Refsgaard et al., 2013; Seifert, 2012; Sunyer, 2014; Velázquez et al., 2013). We believe that the low sensitivity of the protection recom- It is striking that the recommended planning margin from the historic record alone is very large. A main reason is that we use a GEV distribution with two trend parame- ters (i.e., ﬁve parameters overall) to pick up climate sig- nals in the projections. We are using the same distribu- tion for the historic record for comparability. In reality, one should not attempt to estimate such a high number of pa- rameters from such a small set of data (38 annual maxima in the historic record); instead, one would assume stationar- ity or a ﬁxed trend. We repeated the analysis for a station- ary GEV (no trend parameters), resulting in a planning mar- gin recommendation of 75.1 %. This is still high, conﬁrming that it is not recommendable to plan based on a short his- toric record alone. Additional information should always be used, e.g., climate projections (as in this study), tools from runoff prediction in ungauged basins and climate analogues (Arnbjerg-Nielsen et al., 2015; Blöschl et al., 2013a). www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 2522 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2523 in an Alpine watershed, Hydrol. Earth Syst. Sci., 16, 4343–4360, https://doi.org/10.5194/hess-16-4343-2012, 2012. Processes, in: Places and Scales, Cambridge University Press, Cambridge, 2013a. Processes, in: Places and Scales, Cambridge University Press, Cambridge, 2013a. in an Alpine watershed, Hydrol. Earth Syst. Sci., 16, 4343–4360, https://doi.org/10.5194/hess-16-4343-2012, 2012. Blöschl, G., Nester, T., Komma, J., Parajka, J., and Perdigão, R. A. P.: The June 2013 ﬂood in the Upper Danube Basin, and compar- isons with the 2002, 1954 and 1899 ﬂoods, Hydrol. Earth Syst. Sci., 17, 5197–5212, https://doi.org/10.5194/hess-17-5197-2013, 2013b. Ehret, U., Zehe, E., Wulfmeyer, V., Warrach-Sagi, K., and Liebert, J.: Should we apply bias correction to global and regional cli- mate model data?, Hydrol. Earth Syst. Sci., 16, 3391–3404, https://doi.org/10.5194/hess-16-3391-2012, 2012. Fatichi, S., Rimkus, S., Burlando, P., Bordoy, R., and Molnar, P.: Elevational dependence of climate change impacts on wa- ter resources in an Alpine catchment, Hydrol. Earth Syst. Sci. Discuss., 10, 3743–3794, https://doi.org/10.5194/hessd-10- 3743-2013, 2013. Bosshard, T., Kotlarski, S., Ewen, T., and Schär, C.: Spec- tral representation of the annual cycle in the climate change signal, Hydrol. Earth Syst. Sci., 15, 2777–2788, https://doi.org/10.5194/hess-15-2777-2011, 2011. Bosshard, T., Carambia, M., Goergen, K., Kotlarski, S., Krahe, P., Zappa, M., and Schär, C.: Quantifying uncer- tainty sources in an ensemble of hydrological climate- impact projections, Water Resour. Res., 49, 1523–1536, https://doi.org/10.1029/2011WR011533, 2013. Foley, A. M.: Uncertainty in regional climate modelling: A review, Progress in Physical Geography, 34(5), 647–670, doi:10.1177/0309133310375654, 2010. Götzinger, J. and Bárdossy, A.: Generic error model for calibration and uncertainty estimation of hydro- logical models, Water Resour. Res., 44, W00B07, https://doi.org/10.1029/2007WR006691, 2008. Brigode, P., Oudin, L., and Perrin, C.: Hydrological model parame- ter instability: A source of additional uncertainty in estimating the hydrological impacts of climate change?, J. Hydrol., 476, 410–425, https://doi.org/10.1016/j.jhydrol.2012.11.012, 2013. Graf, M., Nishijima, K., and Faber, M.: Bayesian updating in nat- ural hazard risk assessment, Aust. J. Struct. Eng., 9, 35–44, https://doi.org/10.1080/13287982.2009.11465008, 2007. Central European Flood Risk Assessment and Management in CENTROPE: Current standards for ﬂood protection, available at: http://www.ﬂoodcba2.eu/site/wp-content/uploads/CEframe_ 363_Current_standards_for_ﬂood_protection.pdf (last access: 22 April 2018), 2013. Gregersen, I. B., Madsen, H., Rosbjerg, D., and Arnbjerg- Nielsen, K.: Long term variations of extreme rainfall in Den- mark and southern Sweden, Clim. Dynam., 44, 3155–3169, https://doi.org/10.1007/s00382-014-2276-4, 2014. Grundmann, J.: Analyse und Simulation von Unsicherheiten in der Flächendifferenzierten Niederschlags-Abﬂuss-Modellierung, in: Dresdner Schriften zur Hydrologie, PhD Thesis, 165 pp., https: //d-nb.info/1008804363/34 (last access: 22 April 2018), 2010. Chen, J., Brissette, F. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections P., and Lucas-picher, P.: Assessing the lim- its of bias-correcting climatemodel outputs for climate change impact studies, J. Geophys. Res.-Atmos., 120, 1123–1136, https://doi.org/10.1002/2014JD022635, 2015. Coles, S.: An Introduction to Statistical Modeling of Extreme Val- ues, Springer, London, 2004. Hall, J. and Solomatine, D.: A framework for uncertainty analysis in ﬂood risk management decisions, Int. J. River Basin Manage., 6, 85–98, https://doi.org/10.1080/15715124.2008.9635339, 2008. Coles, S., Pericchi, L. R., and Sisson, S.: A fully probabilistic ap- proach to extreme rainfall modelling, J. Hydrol., 273, 35–50, https://doi.org/10.1016/S0022-1694(02)00353-0, 2003. Hall, J., Arheimer, B., Borga, M., Brázdil, R., Claps, P., Kiss, A., Kjeldsen, T. R., Kriaucuniene, J., Kundzewicz, Z. W., Lang, M., Llasat, M. C., Macdonald, N., McIntyre, N., Mediero, L., Merz, B., Merz, R., Molnar, P., Montanari, A., Neuhold, C., Parajka, J., Perdigão, R. A. P., Plavcová, L., Rogger, M., Salinas, J. L., Sauquet, E., Schär, C., Szolgay, J., Viglione, A., and Blöschl, G.: Understanding ﬂood regime changes in Europe: A state- of-the-art assessment, Hydrol. Earth Syst. Sci., 18, 2735–2772, https://doi.org/10.5194/hess-18-2735-2014, 2014. Curry, J. A. and Webster, P. J.: Climate science and the un- certainty monster, B. Am. Meteorol. Soc., 92, 1667–1682, https://doi.org/10.1175/2011BAMS3139.1, 2011. Custer, R. and Nishijima, K.: Hierarchical decision making for ﬂood risk reduction, in: 11th International Conference on Structural Safety & Reliability, ICOSSAR, New York, 4865–4872, 2013. Davis, D. R., Kisiel, C. C., and Duckstein, L.: Bayesian decision theory applied to design in hydrology, Water Resour. Res., 8, 33– 41, 1972. Hallegatte, S.: Strategies to adapt to an uncertain cli- mate change, Global Environ. Change, 19, 240–247, https://doi.org/10.1016/j.gloenvcha.2008.12.003, 2009. De Kok, J. L., Hoekstra, A. Y., Defence, F., and Change, C.: Liv- ing with peak discharge uncertainty: The self-learning dike, in 4th Biennial Meeting of the International Congress on Environ- mental Modelling and Software, iEMSs, Barcelona, 1542–1549, 2008. Hanel, M. and Buishand, T. A.: Analysis of precipitation ex- tremes in an ensemble of transient regional climate model sim- ulations for the Rhine basin, Clim. Dynam., 36, 1135–1153, https://doi.org/10.1007/s00382-010-0822-2, 2011. Hawkins, E. and Sutton, R.: The potential to narrow uncertainty in regional climate predictions, B. Am. Meteorol. Soc., 90, 1095– 1107, https://doi.org/10.1175/2009BAMS2607.1, 2009. Delgado, J. M., Apel, H., and Merz, B.: Flood trends and variabil- ity in the Mekong river, Hydrol. Earth Syst. Sci., 14, 407–418, https://doi.org/10.5194/hess-14-407-2010, 2010. Hawkins, E. and Sutton, R.: The potential to narrow uncertainty in projections of regional precipitation change, Clim. Dynam., 37, 407–418, 2011. References Aghakouchak, A., Easterling, D., Hsu, K., Schubert, S., and Sorooshian, S. (Eds.): Extremes in a Changing Climate, Springer, Dordrecht, 2013. Alﬁeri, L., Burek, P., Feyen, L., and Forzieri, G.: Global warming increases the frequency of river ﬂoods in Europe, Hydrol. Earth Syst. Sci., 19, 2247–2260, https://doi.org/10.5194/hess-19-2247- 2015, 2015. Arnbjerg-Nielsen, K., Willems, P., Olsson, J., Beecham, S., Pathi- rana, A., Bülow Gregersen, I., Madsen, H., and Nguyen, V. T. V: Impacts of climate change on rainfall extremes and urban drainage systems: A review, Water Sci. Technol., 68, 16–28, https://doi.org/10.2166/wst.2013.251, 2013. Arnbjerg-Nielsen, K., Funder, S. G., and Madsen, H.: Iden- tifying climate analogues for precipitation extremes for Denmark based on RCM simulations from the EN- SEMBLES database, Water Sci. Technol., 71, 418–425, https://doi.org/10.2166/wst.2015.001, 2015. Code availability. The code is available upon request. Code availability. The code is available upon request. Data availability. Data are available in the Supplement. Data availability. Data are available in the Supplement. Data availability. Data are available in the Supplement. Benjamin, J. R. and Cornell, C. A.: Probability, Statistics and Deci- sions for Civil Engineers, Mc Graw – Hill Book Company, New York City, 1970. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/hess-22-2511-2018-supplement. Blöschl, G., Sivapalan, M., Wagener, T., Viglione, A., and Savenije, H.: Runoff Prediction in Ungauged Basins: Synthesis Across www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections Merz, B., Vorogushyn, S., Lall, U., Viglione, A, and Blöschl, G.: Charting unknown water – on the role of surprise in ﬂood risk as- sessment and management, Water Resour. Res., 51, 6399–6416, https://doi.org/10.1002/2014WR016259, 2015. KLIWA: Heft 9 – Regionale Klimaszenarien für Süddeutschland, Arbeitskreis KLIWA, Mannheim, 2006. Knutti, R., Masson, D., and Gettelman, A.: Climate model geneal- ogy: Generation CMIP5 and how we got there, Geophys. Res. Lett., 40, 1194–1199, https://doi.org/10.1002/grl.50256, 2013. Merz, B., Dung, N. V., and Vorogushyn, S.: Temporal clustering of ﬂoods in Germany: Do ﬂood-rich and ﬂood-poor periods exist?, J. Hydrol., 541, 824–838, https://doi.org/10.1016/j.jhydrol.2016.07.041, 2016. Kochendorfer, M. J.: Decision Making Under Uncertainty, The MIT Press, Cambridge, Massachusetts, 2015. Merz, R., Parajka, J., and Blöschl, G.: Time stability of catchment model parameters: Implications for cli- mate impact analyses, Water Resour. Res., 47, 6119, https://doi.org/10.1029/2010WR009505, 2011. Kunstmann, H. and Stadler, C.: High resolution dis- tributed atmospheric–hydrological modelling for Alpine catchments, J. Hydrol., 314, 105–124, https://doi.org/10.1016/j.jhydrol.2005.03.033, 2005. Mitchell, T. D. and Hulme, M.: Predicting regional climate change: living with uncertainty, Prog. Phys. Geogr., 23, 57–78, https://doi.org/10.1191/030913399672023346, 1999. Kwakkel, J., Walker, W., and Marchau, V.: Grappling with uncer- tainty in the long-term development of infrastructure systems, in: 3rd International Conference on Infrastructure Systems and Services: Next Generation Infrastructure Systems for Eco-Cities, INFRA, Shenzhen, 2010. Moss, R. H., Edmonds, J. a, Hibbard, K. a, Manning, M. R., Rose, S. K., van Vuuren, D. P., Carter, T. R., Emori, S., Kainuma, M., Kram, T., Meehl, G. A., Mitchell, J. F. B., Nakicenovic, N., Ri- ahi, K., Smith, S. J., Stouffer, R. J., Thomson, A. M., Weyant, J. P., and Wilbanks, T. J.: The next generation of scenarios for climate change research and assessment, Nature, 463, 747–756, https://doi.org/10.1038/nature08823, 2010. Labarthe, B., Abasq, L., de Fouquet, C., and Flipo, N.: Step- wise calibration procedure for regional coupled hydrological- hydrogeological models, in: EGU General Assembly Conference Abstracts (Vol. 16), May 2014, Vienna, Austria, 2014. Laprise, R.: Comment on “The added value to global model pro- jections of climate change by dynamical downscaling: A case study over the continental U.S. using the GISS-ModelE2 nad WRF models” by Racherla et al., J. Geophys. Res.-Atmos., 119, 3877–3881, https://doi.org/10.1002/2013JD019945, 2014. Muerth, M., St.-Denis, B. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2524 tions of extreme precipitation and ﬂoods in Europe, J. Hydrol., 519, 3634–3650, https://doi.org/10.1016/j.jhydrol.2014.11.003, 2014. Global Scenarios, CLM Technical Report, 20, 857–861, https://doi.org/10.1089/lap.2010.0351, 2008. Global Scenarios, CLM Technical Report, 20, 857–861, https://doi.org/10.1089/lap.2010.0351, 2008. Huang, S., Krysanova, V., and Hattermann, F.: Projections of cli- mate change impacts on ﬂoods and droughts in Germany using an ensemble of climate change scenarios, Reg. Environ. Change, Magdali, M.: Calibration of the hydrological model WaSiM for the Mangfall, MS Thesis, Technical University of Munich, Munich, 2015. 15, 461–473, https://doi.org/10.1007/s10113-014-0606-z, 2014. Maraun, D.: When will trends in European mean and heavy daily precipitation emerge?, Environ. Res. Lett., 8, 14004, https://doi.org/10.1088/1748-9326/8/1/014004, 2013. Hundecha, Y., Sunyer, M. A., Lawrence, D., Madsen, H., Willems, P., Martinkova, M., Vormoor, K., Bürger, G., Hanel, M., Kri- auˇciuniene, J., Loukas, A., Osuch, M., and Yücel, I.: Inter- comparison of statistical downscaling methods for projection of extreme ﬂow indices across Europe, Hydrol. Earth Syst. Sci., 19, 1827–1847, https://doi.org/10.5194/hess-19-1827-2015, 2016. Masson, D. and Knutti, R.: Climate model genealogy, Geophys. Res. Lett., 38, L08703, https://doi.org/10.1029/2011GL046864, 2011. Maurer, E. P. and Pierce, D. W.: Bias correction can modify cli- mate model simulated precipitation changes without adverse ef- fect on the ensemble mean, Hydrol. Earth Syst. Sci., 18, 915– 925, https://doi.org/10.5194/hess-18-915-2014, 2014. IPCC: IPCC Special Report – Emission Scenarios, edited by: Na- kicenovic, N. and Swart, R., Cambridge University Press, Cam- bridge, England, 2000. IPCC: Climate Change 2013: The Physical Science Basis, Cam- bridge University Press, Cambridge, 2013. Merz, B., Aerts, J., Arnbjerg-Nielsen, K., Baldi, M., Becker, A., Bichet, A., Blöschl, G., Bouwer, L. M., Brauer, A., Ciofﬁ, F., Delgado, J. M., Gocht, M., Guzzetti, F., Harrigan, S., Hirschboeck, K., Kilsby, C., Kron, W., Kwon, H. H., Lall, U., Merz, R., Nissen, K., Salvatti, P., Swierczynski, T., Ulbrich, U., Viglione, A., Ward, P. J., Weiler, M., Wilhelm, B., and Nied, M.: Floods and climate: Emerging perspectives for ﬂood risk assess- ment and management, Nat. Hazards Earth Syst. Sci., 14, 1921– 1942, https://doi.org/10.5194/nhess-14-1921-2014, 2014. Kennedy, M. C. and O’Hagan, A.: Bayesian calibration of computer models, J. Roy. Stat. Soc. B, 63, 425–464, https://doi.org/10.1111/1467-9868.00294, 2001. Kerkhoff, C., Künsch, H. R., and Schär, C.: A Bayesian hierarchi- cal model for heterogeneous RCM-GCM multimodel ensembles, J. Climate, 28, 6249–6266, https://doi.org/10.1175/JCLI-D-14- 00606.1, 2015. KLIWA: Der Klimawandel in Bayern für den Zeitraum 2021–2050, Arbeitskreis KLIWA, Mannheim, 2005. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections Deutscher Wetterdienst: Deutscher Klimaatlas, available at: https://www.dwd.de/DE/klimaumwelt/klimaatlas/klimaatlas_ node.html, last access: 22 April 2018. Hollweg, H.-D., Böhm, U., Fast, I., Hennemuth, B., Keuler, K., Keup-Thiel, E., Lautenschlager, M., Legutke, S., Radtke, Dittes, B., Špaˇcková, O., and Straub, D.: Managing uncertainty in design ﬂood magnitude: Flexible protection strategies vs. safety factors, J. Flood Risk Manage., accepted, 2018. Hollweg, H.-D., Böhm, U., Fast, I., Hennemuth, B., Keuler, K., Keup-Thiel, E., Lautenschlager, M., Legutke, S., Radtke, K., Rockel, B., Schubert, M., Will, A., and Michael Woldt, C. W.: Ensemble Simulations over Europe with the Re- gional Climate Model CLM forced with IPCC AR4 K., Rockel, B., Schubert, M., Will, A., and Michael Woldt, C. W.: Ensemble Simulations over Europe with the Re- gional Climate Model CLM forced with IPCC AR4 Dobler, C., Hagemann, S., Wilby, R. L., and Stätter, J.: Quantify- ing different sources of uncertainty in hydrological projections www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections 2525 Sunyer, M. A., Madsen, H., Rosbjerg, D., and Arnbjerg-Nielsen, K.: Regional interdependency of precipitation indices across Den- mark in two ensembles of high-resolution RCMs, J. Climate, 26, 7912–7928, https://doi.org/10.1175/JCLI-D-12-00707.1, 2013b. Pennell, C. and Reichler, T.: On the effective num- ber of climate models, J. Climate, 24, 2358–2367, https://doi.org/10.1175/2010JCLI3814.1, 2011. Pohl, R.: Freibordbemessung an Hochwasserschutzanlagen, 36 Dresdner Wasserbaukolloquium “Technischer und or- ganisatorischer Hochwasserschutz”, Technische Universität Dresden, Institut für Wasserbau und technische Hydromechanik, Dresden, 2013. Sunyer, M. A., Gregersen, I. B., Rosbjerg, D., Madsen, H., Luchner, J., and Arnbjerg-Nielsen, K.: Comparison of different statistical downscaling methods to estimate changes in hourly extreme pre- cipitation using RCM projections from ENSEMBLES, Int. J. Cli- matol., 35, 2528–2539, https://doi.org/10.1002/joc.4138, 2015a. Pöhler, H., Schultze, B., and Scherzer, J.: KLIWA: Vergleichende Analyse der neuen globalen Klimaprojektionen aus CMIP5 für Süddeutschland, Abschlussbericht, Arbeitskreis KLIWA, Mannheim, 2012. Sunyer, M. A., Hundecha, Y., Lawrence, D., Madsen, H., Willems, P., Martinkova, M., Vormoor, K., Bürger, G., Hanel, M., Kri- auˇci¯unien˙e, J., Loukas, A., Osuch, M., Yücel, I., Kriauˇciu- niene, J., Loukas, A., Osuch, M., Yücel, I., Kriauˇci¯unien˙e, J., Loukas, A., Osuch, M., and Yücel, I.: Inter-comparison of sta- tistical downscaling methods for projection of extreme precip- itation in Europe, Hydrol. Earth Syst. Sci., 19, 1827–1847, https://doi.org/10.5194/hess-19-1827-2015, 2015b. Raiffa, H. and Schlaifer, R.: Applied Statistical Decision Theory, 5th Edn., The Colonial Press, Boston, 1961. Rajczak, J., Pall, P., and Schär, C.: Projections of extreme precip- itation events in regional climate simulations for Europe and the Alpine Region, J. Geophys. Res.-Atmos., 118, 3610–3626, https://doi.org/10.1002/jgrd.50297, 2013. Tebaldi, C. and Knutti, R.: The use of the multi-model ensemble in probabilistic climate projections, Philos. T. Roy. Soc. A, 365, 2053–2075, https://doi.org/10.1098/rsta.2007.2076, 2007. Refsgaard, J. C., Arnbjerg-Nielsen, K., Drews, M., Halsnæs, K., Jeppesen, E., Madsen, H., Markandya, A., Olesen, J. E., Porter, J. R., and Christensen, J. H.: The role of uncertainty in cli- mate change adaptation strategies – A Danish water manage- ment example, Mitig. Adapt. Strat. Global Change, 18, 337–359, https://doi.org/10.1007/s11027-012-9366-6, 2013. Tebaldi, C., Smith, R., Nychka, D., and Mearns, L.: Quantifying uncertainty in projections of regional climate change: a Bayesian approach to the analysis of multimodel ensembles, J. Climate, 18, 1524–1540, 2004. Refsgaard, J. C., Madsen, H., Andréassian, V., Arnbjerg-Nielsen, K., Davidson, T. A., Drews, M., Hamilton, D. P., Jeppesen, E., Kjellström, E., Olesen, J. E., Sonnenborg, T. O., Trolle, D., Willems, P., and Christensen, J. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections H.: A framework for testing the ability of models to project climate change and its impacts, Climatic Change, 122, 271–282, https://doi.org/10.1007/s10584- 013-0990-2, 2014. Tebaldi, C., O’Neill, B., and Lamarque, J.-F.: Sensitivity of regional climate to global temperature and forcing, En- viron. Res. Lett., 10, 74001, https://doi.org/10.1088/1748- 9326/10/7/074001, 2015. Themeßl, J., Gobiet, A., and Leuprecht, A.: Empirical-statistical downscaling and error correction of daily precipitation from regional climate models, Int. J. Climatol., 31, 1530–1544, https://doi.org/10.1002/joc.2168, 2010. RMD Consult: Erläuterungsbericht – Hochwasserrückhaltebecken Feldolling, Munich, Germany, 2016. Umweltbundesamt: Einführung in Klimaprojektionen, available at: https://www.umweltbundesamt.de/themen/klima-energie/ klimafolgen-anpassung/folgen-des-klimawandels/ klimamodelle-szenarien/einfuehrung-in-klimaprojektionen# textpart-1, last access: 22 April 2018. Rodwell, M. J. and Palmer, T. N.: Using numerical weather predic- tion to assess climate models, Q. J. Roy. Meteorol. Soc., 133, 937–948, https://doi.org/10.1002/qj.23, 2007. Schmid, F. J., Willkofer, F., and Ludwig, R.: Endbericht Einﬂuss der Biaskorrektur dynamischer regionaler Klimamodelldaten auf die Wasserhaushaltsmodellierung und Klimafolgeabschätzung in Bayerischen Flussgebieten – Erstellung eines Klimamodell- Audits und ergänzende Untersuchungen (BI-KLIM-2014), Lud- wig Maximilian Universität, München, 2014. Van Haren, R., Van Oldenborgh, G. J., Lenderink, G., and Hazeleger, W.: Evaluation of modeled changes in extreme pre- cipitation in Europe and the Rhine basin, Environ. Res. Lett., 8, 14053, https://doi.org/10.1088/1748-9326/8/1/014053, 2013. Velázquez, J. A., Schmid, J., Ricard, S., Muerth, M. J., Gauvin St- Denis, B., Minville, M., Chaumont, D., Caya, D., Ludwig, R., and Turcotte, R.: An ensemble approach to assess hydrological models’ contribution to uncertainties in the analysis of climate change impact on water resources, Hydrol. Earth Syst. Sci., 17, 565–578, https://doi.org/10.5194/hess-17-565-2013, 2013. Seifert, P.: Mit Sicherheit wächst der Schaden. Überlegungen zum Umgang mit Hochwasser in der räumlichen Planung, Geschäftsstelle des Regionalen Planungsverbandes Oberes Elb- tal/Osterzgebirge, Meissen, 2012. Špaˇcková, O. and Straub, D.: Long-term adaption decisions via fully and partially observable Markov decision processes, Sus- tain. Resil. Infrastruct., 2, 37–58, 2017. Viglione, A., Merz, R., Salinas, J. L., and Blöschl, G.: Flood fre- quency hydrology: 3. A Bayesian analysis, Water Resour. Res., 49, 675–692, https://doi.org/10.1029/2011WR010782, 2013. Sunyer, M. A.: Uncertainties in extreme precipitation under climate change conditions, Technical University of Denmark, available at: http://www.orbit.dtu.dk Copenhagen, 2014. Wasserwirtschaftsamt Rosenheim: Das Hochwasser vom Juni 2013, Wasserwirtschaftsamt Rosenheim, Rosenheim, 2014. Sunyer, M. A., Sørup, H. J. D., Christensen, O. B., Mad- sen, H., Rosbjerg, D., Mikkelsen, P. S., and Arnbjerg- Nielsen, K.: On the importance of observational data proper- ties when assessing regional climate model performance of ex- treme precipitation, Hydrol. Earth Syst. Sci., 17, 4323–4337, https://doi.org/10.5194/hess-17-4323-2013, 2013a. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections G., Ludwig, R., and Caya, D.: Evaluation of different sources of uncertainty in climate change impact re- search using a hydro-climatic model ensemble, in: 6th Biennial Meeting of the International Congress on Environmental Mod- elling and Software, iEMSs, Leipzig, 2012. Li, M., Yang, D., Chen, J., and Hubbard, S. S.: Calibration of a distributed ﬂood forecasting model with input uncertainty us- ing a Bayesian framework, Water Resour. Res., 48, W08510, https://doi.org/10.1029/2010WR010062, 2012. Nishijima, K.: Concept of decision graphical framework for optimising adaptation of civil infrastructure to a changing climate, Struct. Infrastruct. Eng., 12, 477–483, https://doi.org/10.1080/15732479.2015.1020496, 2015. p g MacKay, D. J. C.: Bayesian interpolation, Neural Computation, 4, 415–447, https://doi.org/10.1162/neco.1992.4.3.415, 1992. Paté-Cornell, E.: On “Black swans” and “Perfect storms”: Risk analysis and management when statistics are not enough, Risk Analysis, 32, 1823–1833, 2011. Madsen, H., Lawrence, D., Lang, M., Martinkova, M., and Kjeld- sen, T. R.: Review of trend analysis and climate change projec- www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 Ylhäisi, J. S., Räisänen, J., Masson, D., Räty, O., and Järvinen, H.: How does model development affect climate projections?, At- mos. Sci. Lett., 16.3, 414–419, https://doi.org/10.1002/asl2.577, 2015. Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections Wiedemann, C. and Slowacek, W.: Hochwasserrückhalte- becken Feldolling: Zweck, Betrieb Bemessung und Funk- tionsweise, Wasserwirtschaftsamt Rosenheim, Rosenheim, available at: http://www.wwa-ro.bayern.de/hochwasser/ hochwasserschutzprojekte/mangfalltal/doc/hrb_feldolling/ funktion_hrb_feldolling.pdf (last access: 22 April 2018), 2013. www.hydrol-earth-syst-sci.net/22/2511/2018/ Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections water resource model parameters used ct assessment, Hydrol. Process., 19, K.: Klimawandel und Wasserhaushalt: g zum Einﬂuss des Klimawandels auf Ylhäisi, J. S., Räisänen, J., Masson, D., Räty, O., and Järvinen, H.: How does model development affect climate projections?, At- mos. Sci. Lett., 16.3, 414–419, https://doi.org/10.1002/asl2.577, 2015. B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections ater resource model parameters used assessment, Hydrol. Process., 19, Klimawandel und Wasserhaushalt: Ylhäisi, J. S., Räisänen, J., Masson, D., Räty, O., and Järvinen, H.: How does model development affect climate projections?, At- mos. Sci. Lett., 16.3, 414–419, https://doi.org/10.1002/asl2.577, 2015. 2526 B. Dittes et al.: Managing uncertainty in ﬂood protection planning with climate projections Wilby, R. L.: Uncertainty in water resource model parameters used for climate change impact assessment, Hydrol. Process., 19, 3201–3219, 2005. Willems, W. and Stricker, K.: Klimawandel und Wasserhaushalt: AdaptAlp – Untersuchung zum Einﬂuss des Klimawandels auf Wasserbilanzen und Abﬂüsse für das Inneinzugsgebiet mittels verschiedener Klimaszenarien. Endbericht, Bayerisches Lan- desamt für Umwelt, Hof, 2011. Hydrol. Earth Syst. Sci., 22, 2511–2526, 2018 www.hydrol-earth-syst-sci.net/22/2511/2018/
W3156050488.txt	https://vniigis.elpub.ru/jour/article/download/710/625	en		Early morphogenesis of metacercariae of trematodes Paratimonia sp. Prevot and Bartoli, 1967 in gastropods Hydrobia acuta (Draparnaud, 1805) in the Black Sea environment	Rossijskij parazitologičeskij žurnal	2,021	cc-by	2,380	ФАУНА, МОРФОЛОГИЯ И СИСТЕМАТИКА ПАРАЗИТОВ УДК 619:616.995.122 DOI: 10.31016/1998-8435-2021-15-1-11-15 Оригинальная статья Досрочный морфогенез метацеркарий трематод Paratimonia sp. Prevot, Bartoli, 1967 у брюхоногих моллюсков Hydrobia acuta (Draparnaud, 1805) в акватории Черного моря Юлия Витальевна Белоусова Федеральный исследовательский центр «Институт биологии южных морей имени А. О. Ковалевского РАН», 299011, Российская Федерация, г. Севастополь, пр. Нахимова, 2, e-mail: julls.belousova@gmail.com Поступила в редакцию: 15.07.2020; принята в печать: 12.01.2021 Аннотация Цель исследований: изучить морфологию монорхидных личинок трематод рода Paratimonia и установить возможные причины их ускоренного морфогенеза. Материалы и методы. Материалом для данного исследования служили собственные сборы личинок (метацеркарий) трематод от 283 экз. брюхоногих моллюсков Hydrobia acuta, сделанные в марте 2012 г. в эстуарии р. Черная (г. Севастополь, Черное море, 44°27′49″ с. ш. 33°51′37″ в. д.). Моллюсков исследовали компрессорным методом на наличие гельминтов. Часть особей найденных трематод исследовали живыми, других – фиксировали в 70%-ном этиловом спирте, затем окрашивали ацетокармином по стандартной методике. Морфологические особенности метацеркарий изучали на живых личинках. Результаты и обсуждение. При гельминтологическом исследовании гастропод рода Hydrobia зарегистрированы метацеркарии семейства Monorchiidae. Неинцистированные метацеркарии располагались свободно в полости тела хозяина. Ключевые слова: трематоды, Monorchiidae, метацеркарии, гастроподы, Hydrobia acuta, Черное море Прозрачность финансовой деятельности: автор не имеет финансовой заинтересованности в представленных материалах или методах Конфликт интересов отсутствует Для цитирования: Белоусова Ю. В. Досрочный морфогенез метацеркарий трематод Paratimonia sp. Prevot, Bartoli, 1967 у брюхоногих моллюсков Hydrobia acuta (Draparnaud, 1805) в акватории Черного моря // Российский паразитологический журнал. 2021. Т. 15. № 1. С. 11–15. https://doi.org/10.31016/1998-8435-2021-15-1-11-15 © Белоусова Ю. В., 2021 Контент доступен под лицензией Creative Commons Attribution 4.0 License. The content is available under Creative Commons Attribution 4.0 License. Russian Journal of Parasitology / Российский паразитологический журнал 2021;15(41):11-15 11 12 FAUNA, MORPHOLOGY AND SYSTEMATICS OF PARASITES Original article Early morphogenesis of metacercariae of trematodes Paratimonia sp. Prevot and Bartoli, 1967 in gastropods Hydrobia acuta (Draparnaud, 1805) in the Black Sea environment Yulia V. Belousova Federal State Budgetary Institution of Science Federal Research Center “Institute of Biology of the Southern Seas named after A. O. Kovalevsky RAS”, 2, Nakhimova avenue, Sevastopol, Russian Federation, 299011, e-mail: julls.belousova@gmail.com Received on: 15.07.2020; accepted for printing on: 12.01.2021 Abstract The purpose of the research is studying the morphology of larvae of Trematoda -Monorchiidae of the genus Paratimonia and establishing possible causes of their accelerated morphogenesis. Materials and methods. The material for this study was trematode larvae (metacercariae) from 283 specimens of gastropods Hydrobia acuta that we collected in March 2012 at the estuary of the River Chernaya (Sevastopol, the Black Sea, 44°27′49″ N 33°51′37″ E). The gastropods were studied for helminths using the compression method. Some of specimens of the found trematodes were studied alive and others were fixed in 70% ethyl alcohol and then stained with acetocarmine according to the standard method. The morphological features of metacercaria were studied on live larvae. Results and discussion. During helminthological research of gastropods Hydrobia, we recorded metacercariae of the family Monorchiidae. Non-encysted metacercariae were located freely in the host’s body cavity. Keywords: trematodes, Monorchiidae, metacercariae, gastropods, Hydrobia acuta, the Black Sea Financial Disclosure: The author has no financial or property interest in any material or method mentionеd There is no conflict of interests For citation: Belousova Yu. V. Early morphogenesis of metacercariae of trematodes Paratimonia sp. Prevot, Bartoli, 1967 in gastropods Hydrobia acuta (Draparnaud, 1805) in the Black Sea environment. Rossiyskiy parazitologicheskiy zhurnal = Russian Journal of Parasitology. 2021; 15 (1): 11–15. (In Russ.). https://doi.org/10.31016/1998-8435-2021-15-1-11-15 © Belousova Yu. V., 2021 Введение Представители трематод семейства Monorchiidae в большинстве случаев паразиты пресноводных, реже – морских рыб. Спороцисты монорхид обычно развиваются в двухстворчатых моллюсках [8]. На сегодняшний день описано 16 видов монорхидных церкарий из северного полушария, но только для 7 из них расшифрован жизненный цикл [6, 13–16]. При гельминтологическом исследовании черноморских моллюсков Hydrobia acuta мы обнаружили метацеркарии монорхидных трематод, определенные нами как представители 2021;15(1):11-15 рода Paratimonia. Жизненные циклы трематод этого рода в Черном море не расшифрованы, сведений о морфогенезе метацеркарий трематод рода Paratimonia у брюхоногих моллюсков H. acuta в акватории Черного моря нет, что и определило цель нашего исследования. Материалы и методы Материалом для нашего исследования служили собственные сборы личинок (метацеркарий) трематод от 283 экз. брюхоногих моллюсков H. acuta, сделанные в марте 2012 г. в эстуарии р. Черная (г. Севастополь, Черное море, 44°27′49″ с. ш. 33°51′37″ в. д.). Russian Journal of Parasitology / Российский паразитологический журнал ФАУНА, МОРФОЛОГИЯ И СИСТЕМАТИКА ПАРАЗИТОВ При гельминтологическом исследовании этих моллюсков зарегистрированы метацеркарии семейства Monorchiidae. Неинцистированные метацеркарии располагались свободно в полости тела хозяина. Моллюсков на наличие гельминтов исследовали компрессорным методом [1]. Часть особей найденных трематод исследовали живыми, других – фиксировали в 70%-ном этиловом спирте, затем окрашивали ацетокармином по стандартной методике [4]: окраска дифференцировалась «железной водой» (H2O + Fe2O3) и подкисленным спиртом (70% этанола + 3% HCl), после дегидратации в серии спиртов (70–100º) и просветления в гвоздичном масле гельминты заключались в канадский бальзам. Морфологические особенности метацеркарий изучали на живых личинках. Промеры метацеркарий выполняли на двух фиксированных личинках Paratimonia sp.; промеры приведены в микронах. Фотографии, рисунок и промеры сделаны с использованием микроскопа XY-B2 и цифровой фотокамеры Canon A650 с программным обеспечением Infinity Analyze. Рисунки выполнены в редакторе векторной графики Scalable Vector Graphics (SVG) в программе Inkscape 0.48.2.-1 [9]. Результаты и обсуждение Экстенсивность инвазии метацеркариями Paratimonia sp. составила 1,06%, интенсивность инвазии 1–2 (1,33 ± 0,33) экз./особь, индекс обилия 0,014 ± 0,009 экз./особь. Обнаруженные нами метацеркарии монорхид имеют овальную форму тела. Длина тела 868–882 µm, ширина – 364–560 µm. У живых особей просматриваются мелкие шипики на тегументе. Мощная брюшная присоска длиной 252–280 µm и шириной 182–266 µm, немного больше ротовой (длина – 177–197 µm, ширина – 154–182 µm). Префаринкса нет. Фаринкс крупный, круглой или овальной формы. Короткий пищевод разветвляется над брюшной присоской. Кишечные ветви достигают нижней границы верхнего семенника (рис. 1). Экскреторный пузырь вытянутой формы, в виде ампулы. Семенники размером 200– 230 µm лежат по диагонали друг над другом, между ветвями кишечника. Цельнокрайний яичник лежит над семенниками и сдвинут вправо. В задней части тела четко просма- тривается сформированная половая бурса. Семенной пузырек маленький. Половая пора видна. Яйца отсутствовали у всех обследованных особей трематод. Впервые спороцисты и церкарии рода Paratimonia были описаны от моллюсков Abra segmentum (Philippi, 1836) у побережья Франции под названием Paratimonia gobii (Mailard, 1975). Зрелые церкарии покидали двустворчатых моллюсков через выводной сифон, некоторое время плавали самостоятельно в водной среде и с потоком воды попадали через сифоны в тело моллюска этого же вида. Далее церкарии инцистировались в теле второго промежуточного хозяина, формируя метацеркарий. В качестве окончательного хозяина зарегистрированы рыбы Pomatoschistus microps (Krøyer, 1838) (Mailard, 1975). Краткое описание метацеркарий и половозрелых особей Paratimonia sp. в Черном море впервые приведено Найденовой в 1970 г. [2]. В Черном море зарегистрированы мариты одного вида этого рода – Paratimonia gobii. Последние сведения о морфологии марит рода Paratimonia приведены в работе Bray, Gibson [7]. Согласно литературным данным [2, 7], отсутствие префаринкса, наличие короткого пищевода и кишечных ветвей, достигающих нижней границы семенника, позволяют нам отнести обнаруженных метацеркарий к роду Paratimonia. Литературные данные свидетельствуют, что метацеркарии сем. Monorchiidae паразитируют Рис. 1. Метацеркария Paratimonia sp. Prevot et Bartoli, 1967 от брюхоногого моллюска H. acuta (Draparnaud, 1805) из акватории Черного моря (масштабная линейка – 500 µm) Russian Journal of Parasitology / Российский паразитологический журнал 2021;15(1):11-15 13 14 FAUNA, MORPHOLOGY AND SYSTEMATICS OF PARASITES у морских двустворчатых моллюсков, а взрослые особи – в кишечнике костистых рыб [8]. По всей вероятности, брюхоногие моллюски H. acuta, выступающие в нашем случае в качестве дополнительного хозяина, могли заразиться в результате контакта с личинками, покинувшими через выводной сифон промежуточных хозяев – двухстворчатого моллюска Abra segmentum, который в большинстве случаев разделяет биотоп с гастроподами H. acuta в исследуемой акватории [3]. Окончательными хозяевами трематоды Paratimonia sp. в Черном море являются бычки P. marmoratus (Kvach, 2010) Обнаруженные метацеркарии Paratimonia sp., достигнув половой зрелости в моллюске H. acuta, не производили жизнеспособные яйца. Таким образом, применять термин «прогенез» относительно обнаруженных нами метацеркарий некорректно. В нашем исследовании мы использовали формулировку «досрочный морфогенез», при котором метацеркария достигает зрелости репродуктивной системы без образования яиц. Судить о зрелости репродуктивной системы можно, опираясь на размеры семенников и яичника, а также на структуру тканей этих органов. Появление такого хода жизненного цикла может быть условной стратегией в ответ на различное влияние окружающей среды, указывающее на низкую вероятность передачи личинки трематоды следующему хозяину. Досрочный морфогенез чаще всего наблюдают в морях северного полушария [10]; в акватории Черного моря это явление отмечено нами впервые. Следует отметить, что моллюски H. acuta имеют годичный жизненный цикл [5]. Максимальное число зрелых гастропод и пик размножения гидробий приходится на апрель-май, к июлю число зрелых особей резко сокращается (40% особей составляют молодь и 1–2% – это зрелые гастроподы). Большинство обследованных нами моллюсков были зрелыми к началу весеннего сезона и зараженными метацеркариями семейства Monorchiidae. На смену зрелым моллюскам стали появляться очень молодые, свободные от гельминтов гидробии. Таким образом, на наш взгляд, короткий промежуток жизни однолетних гастропод Hydrobia может способствовать ускоренному морфогенезу личинок трематод. 2021;15(1):11-15 Исследование выполнено в рамках государственного задания ФГБУН ФИЦ ИнБЮМ им. А. О. Ковалевского РАН (тема № АААА-А18-118020890074-2). Благодарности Автор выражает благодарность сотруднику отдела экологии бентоса ФГБУН ФИЦ ИнБЮМ к.б.н. Макарову Михаилу Валерьевичу за помощь в отборе проб и определении видового состава брюхоногих моллюсков. Литература 1. Быховская-Павловская И. Е. Паразитологическое исследование рыб. Ленинград: Наука, 1969. 108 с. 2. Гаевская А. В., Гусев А. В., Делямуре С. Л. Определитель паразитов позвоночных Черного и Азовского морей. Паразиты беспозвоночные рыб, рыбоядных птиц и морских млекопитающих. Киев: Наукова думка, 1975. 551 с. 3. Полевой Д. М., Макаров М. В., Прищепа Р. Е. Влияние солености на распределение Mollusca в контактной зоне «Река Черная – Севастопольская бухта» (юго-западный Крым, Черное море) // Материалы докладов молодежной науч. конф «Морские исследования и рациональное природопользование». М.: МГУ, 2018. С. 252-255. 4. Роскин Г. И., Левинсон Л. Б. Микроскопическая техника: учебное пособие для вузов. М.: Советская Наука, 1957. 466 с. 5. Чухчин В. Д. Экология брюхоногих моллюсков Черного моря. Киев: Наукова думка, 1984. 176 с. 6. Bartoli P., Jousson O., Russel-Pinto F. The life cycle of Monorchis parvus (Digenea: Monorchiidae) demonstrated by developmental and molecular data. J. Parasitol., 2000; 86: 479-489. DOI: 10.1645/00223395(2000)086[0479:TLCOMP]2.0. CO;2 7. Bray R. A, Gibson D., Jones A. Keys to the Trematoda. Volume 3. CAB International and Natural History Museum. London, 2008. 824 p. 8. Cremonte F., Kroeck M. A., Martorelli S. R. A new monorchiid cercaria (Digenea) parasitising the purple clam Amiantis purpurata (Bivalvia: Veneridae) in the Southwest Atlantic Ocean, with notes on its gonadal effect. Folia Parasitologica, 2001; 48: 217223. DOI: 10.1645/0022-3395(2000)086[0479:TLC OMP]2.0.CO;2 9. Inkscape 0.48.2.-1 (2011). Scalable Vector Graphics (SVG) (WEB: http://www.inkscape.org) 10. Galaktionov K. V., Dobrovolskij A. A. The Biology and Evolution of Trematodes. An Essay on the Russian Journal of Parasitology / Российский паразитологический журнал ФАУНА, МОРФОЛОГИЯ И СИСТЕМАТИКА ПАРАЗИТОВ Biology, Morphology, Life Cycles, Transmission, and Evolution of Digenetic Trematodes. Kluwer Academic Publishers, 2003; 594 p. DOI: 10.1007/978-94-017-3247-5 11. Kvach Y. Helminth fauna of gobiid fishes (Gobiidae) of the Tyligul Estuary of the Black Sea. Vestnik zoologii, 2010; 44 (6): 509-518. 12. Lefebvre F., Poulin R. Progenesis in digenean trematodes: a taxonomic and synthetic overview of species reproducing in their second intermediate hosts. Parasitology, 2005; 130 (6): 587-605. DOI: 10.1017/S0031182004007103 13. Maillard C. Cycle évolutif de Paratimonia gobii Prévot et Bartoli, 1967 (Trematoda: Monorchiidae). Acta Tropica. 1975; 32: 327-333. 14. Martin W. E. Studies on trematodes of Woods Hole. III. The life cycle of Monorcheides cumingiae (Martin) with special reference to its effect on the invertebrate host. Biol. Bull. (Woods Hole), 1940; 9: 131-144. 15. Stunkard H. W. The life history, developmental stages, and taxonomic relations of the digenetic trematode Lasiotocus minutus (Manter, 1931) Thomas, 1959. Biol. Bull. (Woods Hole).1981; 160: 146-154. DOI: 10.2307/1540908 16. Young R. T. Postmonorchis donacis, a new species of monorchiid trematode from the Pacific coast, and its life history. J. Wash. Acad. Science, 1953; 43: 88-93. References 1. Bykhovskaya-Pavlovskaya I. E. Parasitological study of fish. Leningrad, Nauka, 1969; 108. (In Russ.) 2. Gaevskaya A. V., Gusev A. V., Delyamure S. L. Key to vertebrate parasites in the Black and the Azov seas. Parasitic invertebrates of fish, fish eating birds and sea mammals. Kiev, Naukova Dumka, 1975; 551. (In Russ.) 3. Polevoy D. M., Makarov M. V., Prishchepa R. E. Influence of salinity on the distribution of Mollusca in the contact zone "Black River – Sevastopol Bay" (southwestern Crimea, Black Sea). Materialy dokladov molodezhnoy nauch. konf «Morskiye issledovaniya i ratsional'noye prirodopol'zovaniye» = Materials of reports of the Youth Scientific Conf. "Marine Research and Environmental Management". Moscow, MGU, 2018; 252-255. (In Russ.) 4. Roskin G. I., Levinson L. B. Microscopic technique: textbook for universities. Moscow, Sovetskaya Nauka, 1957; 466. (In Russ.) 5. Chukhchin V. D. Ecology of the Black Sea gastropods. Kiev, Naukova Dumka, 1984; 176. (In Russ.) 6. Bartoli P., Jousson O., Russel-Pinto F. The life cycle of Monorchis parvus (Digenea: Monorchiidae) demonstrated by developmental and molecular data. J. Parasitol., 2000; 86: 479-489. DOI: 10.1645/00223395(2000)086[0479:TLCOMP]2.0. CO;2 7. Bray R. A, Gibson D., Jones A. Keys to the Trematoda. Volume 3. CAB International and Natural History Museum. London, 2008; 824. 8. Cremonte F., Kroeck M. A., Martorelli S. R. A new monorchiid cercaria (Digenea) parasitising the purple clam Amiantis purpurata (Bivalvia: Veneridae) in the Southwest Atlantic Ocean, with notes on its gonadal effect. Folia Parasitologica, 2001; 48: 217223. DOI: 10.1645/0022-3395(2000)086[0479:TLC OMP]2.0.CO;2 9. Inkscape 0.48.2.-1 (2011). Scalable Vector Graphics (SVG) (WEB: http://www.inkscape.org) 10. Galaktionov K. V., Dobrovolskij A. A. The Biology and Evolution of Trematodes. An Essay on the Biology, Morphology, Life Cycles, Transmission, and Evolution of Digenetic Trematodes. Kluwer Academic Publishers, 2003; 594. DOI: 10.1007/978-94-017-3247-5 11. Kvach Y. Helminth fauna of gobiid fishes (Gobiidae) of the Tyligul Estuary of the Black Sea. Vestnik zoologii, 2010; 44 (6): 509–518. 12. Lefebvre F., Poulin R. Progenesis in digenean trematodes: a taxonomic and synthetic overview of species reproducing in their second intermediate hosts. Parasitology, 2005; 130 (6): 587–605. DOI: 10.1017/S0031182004007103 13. Maillard C. Cycle évolutif de Paratimonia gobii Prévot et Bartoli, 1967 (Trematoda: Monorchiidae). Acta Tropica. 1975; 32: 327-333. 14. Martin W. E. Studies on trematodes of Woods Hole. III. The life cycle of Monorcheides cumingiae (Martin) with special reference to its effect on the invertebrate host. Biol. Bull. (Woods Hole), 1940; 9: 131-144. 15. Stunkard H. W. The life history, developmental stages, and taxonomic relations of the digenetic trematode Lasiotocus minutus (Manter, 1931) Thomas, 1959. Biol. Bull. (Woods Hole).1981; 160: 146-154. DOI: 10.2307/1540908 16. Young R. T. Postmonorchis donacis, a new species of monorchiid trematode from the Pacific coast, and its life history. J. Wash. Acad. Science, 1953; 43: 88-93. Russian Journal of Parasitology / Российский паразитологический журнал 2021;15(1):11-15 15
https://openalex.org/W4300918758	https://zenodo.org/records/6586490/files/579-583.pdf	English	null	THE STUDY OF IMPERFECT COMPLEXES. PART I. CALORIMETRIC STUDY OF COMPLEXES BY JOB'S METHOD OF CONTINUED VARIATION	Zenodo (CERN European Organization for Nuclear Research)	1,948	cc-by	2,653	[Jour. Indian Cllem. Sor., Vol. liB, No. ~~. I-}48 ] [Jour. Indian Cllem. Sor., Vol. liB, No. ~~. I-}48 ] THE STUDY OF IMPERFECT COMPLEXES. PART I. CALORIMETRIC STUDY OF COMPLEXES BY JOB'S METHOD OF CONTINUED VARIATION Bv SusHrt, KUMAR SrnoHANTA Job's method of eontinned variation has been applied by several workers for the determination of the <"cmposition and the dissociation constant of complexes of low stability from a study of properties like extinction roefficient. conductivity, refraetivity, etc In the present work, it bas been shown that the molar heat content can be atili~ed as a property, the variation of which may be employed for the same purpose. Thermometric titrations were first introduced by Dutoit (]. chim. phys., xg21, 19, 324) to determine the composition of new compound or compounds formed in solu- tion by mixing solutions of two compounds. In this method the temperature change observed on adding gradually the solution of one compound to a definite voluo•e of that of another was plotted against the volume of the first solution added, and the com- positions of the new compounds formed in solution were taken to be indicated by the inflexion points. Such a simple method, in the opinion of the present author, is apt to give in many cases inaccurate information as to the composition of the new substance or substances formed. A detailed discussion on this point will be given in Part III of this series. Tit rations, using certain molecular properties, such as conductivity, as a guide for the determination of reacting proporiions are also in use for a long time. Such a method also, it may be remarked, gives a sharp inflexion point only when a perfect complex is formed and is apt to give inaccumte information about imperfect complexes having large dissociation constant (vide Part Ill of the series). Job's method of continued variation for the study of imperfect complexes (Job, Compt. '?'e-nd., 1925, 180, 928; An-n. Chim., 1928, x, 9, 113) using an additive mole- cular property as guide is, however, more reliable in cases where only one complex i:; formed by mixing two solutions. The method gives not only the composition of the complex formed, but its approximate dissociation constant in addition. In the following reaction between two molecules A and 11, where K is the dissociation constant of the complex. as c,p, m and n are finite constants, Hence, from a knowledge of the value of x where c. is maximum, the formula of the complex can be determined from the ratio m {11 usiug equation (2), taking simplest integral values of 111 and n provided that the two primary solutions used are of equimole- cular concentration. After dete11nining thus the values of m and 11, the value of.K can be determined by equation (r) using the value of x for maximum c. in the case of two primary solutions which are not equimolecular (i.e., where P ± r). The problem theu obviously reduces to the determination of the value of x for which the concentration C, of the complex A.,B,. is maximum. using both equimole- cular and non-equimolecular primary solutions of A and B. This offers no special diffi- culty when A,..B,. has a property not possessed by A and B; then the value of x, where this property is maximum, gives the maximum value of the concentration C, of the complex AmB.,. Usually, however, such a property is difficult to be found. In such cases, Job proposed to study as a function of x any molecular property which obeys the mixture law in the following way: Pmi:riure = ct.PA + f3Pn + 'Y Po+ Pmi:riure = ct.PA + f3Pn + 'Y Po+ where ex, /3, y... are the number of moles of A, B, C, ... present in the soh1tion and PA, PB, Pc ... are the respective molar values of the property P for A, B, C .... where ex, /3, y... are the number of moles of A, B, C, ... present in the soh1tion and PA, PB, Pc ... are the respective molar values of the property P for A, B, C .... Job has mathematically showed that the difference, Y, of the observed value of the property P for any mixture of x c.c. of a solution of B and (t-x) c.c. of a solution of A from that calculated for the mixture by the additivity rule with the assumption that no reaction takes place when plotted against the different corresponding values of x, gives a curve, the maximum (or minimum) point of which represents the value of x where C, is maximum. Job (lor. mA + nB= A,.B,. if (r- x) c.c. of a solution of A of molar concentration c per c.c. be added to x c.c. of a solution of B of molar concentration c' per c.c. (c1=Pc), and if C,, C2, c. be the respec- tive molar concentrations per c.c. of A, B, aJd A.,B., after the equilibrium has been attained, tht: following equations apply for any sut:h mixture. , g q pp y y C,'".C,"=K.C. (A) where K is the dissociation constant of the complex. C 1 +mC,=c(I-x) (B) C2 + nC. = pcx (Cl C,'".C,"=K.C. C,'".C,"=K.C. where K is the dissociation constant of the complex. where K is the dissociation constant of the complex. (B) (Cl C 1 +mC,=c(I-x) C2 + nC. = pcx 580 S. K. SIDDHANTA Starting from relations (A), (B), (C) Job showed that equation (t) is true only for tLat value of x for which c. is maximum. Starting from relations (A), (B), (C) Job showed that equation (t) is true only for tLat value of x for which c. is maximum. c"'+"-'. pn-1 {(Pm+n)x-n}"'+" =K. {n-(m+n)x}(p-r)m+<>-1 (I) tn"-z.nm J =K. {n-(m+n)x}(p-r)m+<>-1 (I) (I) wl1en P = r (i.e., for eqnimolecular parent solutions), the right hand ·side of equation ( r) is zero and therefore wl1en P = r (i.e., for eqnimolecular parent solutions), the right hand ·side of equation ( r) is zero and therefore (2) (2) m/n=J-x/x m/n=J-x/x as c,p, m and n are finite constants, as c,p, m and n are finite constants, as c,p, m and n are finite constants, cit.) chose molar extinction coefficient as the property to be studied and he determined it spectropbotometrically for various solutions of pure A and B and their mixtures in order to find out the value of Y for different values of x. The same property has been used by Vorsburgh and co·wmkers (]. Amer. Chem. Soc., 1941, 63, 437; 1942, 64i, r6.3o) to determine the composition of a number of imperfect complexes; these workers also modified Job's method to the study of composition in a few cases, where more than one complexes are formed in solution. Spacu and Popper (Bull. Soc. Stiinte, Cluj, 1934, 7, 400; 1934, 8, 5) have used molar refractivity as an indicative property to be studied by Job's method. Molar conductivity has also been used by a number of workers as a guide for the study of ,:omplex COlllJ?OUnds in solution by Job's method (Dutt, paper communicated to Indian STUDY OF IMPERFIWT COMPLEXES 581 Chemical Society; Purkayastha and Sen-Sarma, ]. India.n. Chem. Soc., 1946, 23, 31; Purkayastba, ibid., 1947,24, 257; 1948, 25,81; Biswas, ibid., 1947, 24, 345). Diswas (loc. cit.) has also studied as a function of the composition of the mixture, the differ- ence of the observed n+ ion concentration of the mixture from that calculated by ad- ditivity rule with the assumption of no reaction taking place in order to dett:rmine the composition of the complex formed in solution between molybdic and tartaric acids. In the present paper, it is proposed that molar heat content is a t>uitable property to be studied in Job's method of continued variation, so that the deviation Y from the additivity rule is given by the heat of reaction (-!!'..H). But since the reaction is to. be studied in solution, the heats of solution of the reactants and of the resultant should be considered. Thus, the heat content of (r- x) c.c. of a solution of A at concentration c =(I -x) 1C· HA + cqA. + II8 Hn,o ~ and the heat content of x c.c. of a solution of Bat concentration pc and the heat content of x c.c. of a solution of Bat concentration pc = x { pc . Ha+po qn+ 1~ Hn.o} where HA, HR and Ha. o are the respective molar heat contents of A, D and HaO and .q,. and p,qe are the respective heats of solution of c rnols. as c,p, m and n are finite constants, of A and :Pc mots. of B, both in r c.c. of water, supposing however, that no change of volume takes place on dissolution where HA, HR and Ha. o are the respective molar heat contents of A, D and HaO and .q,. and p,qe are the respective heats of solution of c rnols. of A and :Pc mots. of B, both in r c.c. of water, supposing however, that no change of volume takes place on dissolution of solutes and that r c.c. of any solution contains 1~ mols. of H,O. The sum of the heat contents of the two parent solutions before mixing is therefore given by Hreaol~nt• ""'(r-x) {c.HA+cq.o.}+ x{pc. Ha+pcqa}+ 1 1 8 HHzO· By mixing the two parent solutions, we get I c.c. of the mixture which after attainment of equilibrium contains (. 1 rnols. of A, c~ rnols. of Band Cs mols. of A.,B,. Proceed· ing as above with the use of analogous notation, and supposing that there is no volume change on mixing the two solutions and that I c.c. of any solution contains :s mols. of H 20, we can calculate Hrnsulfanl of I c,c. of this mixture. H 20, we can calculate Hrnsulfanl of I c,c. of this mixture. , H + r H H~resultont=C1 .HA+c1 qA.+C2.Ha+aoqn+Ca. Ame,.+asq•m•" Is flsO• The heat of reaction is therefore given by (-~H) = H t t H"'""'""'' 2 , H + r H H~resultont=C1 .HA+c1 qA.+C2.Ha+aoqn+Ca. Ame,.+asq•m•" Is flsO• The heat of reaction is therefore given by , H + r H H~resultont=C1 .HA+c1 qA.+C2.Ha+aoqn+Ca. Ame,.+asq•m•" Is flsO• , H + r H H~resultont=C1 .HA+c1 qA.+C2.Ha+aoqn+Ca. Ame,.+asq•m•" Is flsO• The heat of reaction is therefore given by The heat of reaction is therefore given by (-~H) = Hroact.nt. - H"'""'""'' = (r-x){c. HA+oqA.} +x{pc. Hf.l+pcqa}-{C1. H.&.+clqA+C.Ha+oaqD +C •• HA..a"+a.qA.~••} 582 s. K. SIDDHANTA Now, by the law of partial molal quantities, q .q. =heat of solution of c mols. of A in I c.c. of water where .q. is the differential heat of solution A at concentration c, i.e., heat cbange due to dissolution of I mol. of A in a la1'ge volume of a solution of A at concentration c and ,q (B:Q)• is the differential heat of dilution fm· a solution of A at concentration c i.e., heat d1ange due to the addition of I mol. CHEMICA!. r_ADORATORY, PHJ>SIDiiNCY COI..I.IiGI!, C.U.CUHA. as c,p, m and n are finite constants, of H 20 in a large volume of a solution of A at concentration c. If the solutions used be moderately dilute so that the heat change due to addition of further amount of water may be neglected, we may neglect the quantity c~H,O)A in equation (4). Also ,q, is practically equal to .i,, the heat of solution of r moL of A in a large volume of pure water (instead of in a solution of A at concentration c). Hence equation (4) can be written as Hence equation (4) can be written as cq,. =c. oq,... cq,. =c. oq,... cq,. =c. oq,... Proceeding similarly and using analogous notations, we may have (43) Proceeding similarly and using analogous notations, we may have pcqu = Pc · oqr; C4b) c1qA =C,. oqA (4c) c~qB = C,. Oql! (4d) (4d) c~qB = C,. Oql! Putting (4a, b, c, d, e) in (3) we have (-LlH)=(I-x) c {HA + oq ... }+ xpc{Ho + oqn}-[ c,{HA + OqA} + c.{Ho + oiio} + c.{ HAmil,.+ 0 qA,Iln }] (3 d, e) in (3) we have {HA + oq ... }+ xpc{Ho + oqn}-[ c,{HA + OqA} + c.{Ho + oiio} + c.{ HAmil,.+ 0 qA,Iln }] (3 (3a) (3a) Writing C 1 and C2 in equation ( ~a) in terms of Co using equations (BJ and (CJ, we have ( -LlH) =mc.{H ... + q,.} + nC. {HB + Oqll }- c.{HAmBn + oiiAmll•} {3b) Since HA, H11, HAmlin, 0q,., 0qB, 0qil. are constants we have by differentiating {Jb) with mBn respect to x, Since HA, H11, HAmlin, 0q,., 0qB, 0qil. are constants we have by differentiating {Jb) with mBn t t respect to x, d(-LlH) dx From equation (5) we have d(-L:l.H) dx <s> dC3 0 , when dx =o p , d(-LlH) dx From equation (5) we have <s> <s> From equation (5) we have 583 STUDY OF iMPERFECT COMPLEXES i. e., ( -AH) must be at a maximum or a minimum for that value of x for which c. iii! maximum. i. e., ( -AH) must be at a maximum or a minimum for that value of x for which c. iii! maximum. The heat of reaction ( -AH) due to mixing x c. c. of a solution of ammonia with (roo- x) c.c. Re•eivell july g, 1948. as c,p, m and n are finite constants, of a solution of copper sulphate at equimolecular and non-equimolecular con· centrations has been studied by Siddhanta and Guha (details will appear in Part III of this series). It bas been shown that for equimolecular solutions the value of x, where (-AH) is maximum, Hes at So c.c. So that m/n=1:4 by equation (2) i.e., complex ion formed is [Cu (NH.l.]++ ; also using non-equimolecular solutions the values of x, where (- AH) is maximum, have been utilised to calculate K, the dissociation constant of this complex, by applying equation (r). The value of K obtained in this way accords fairly well with that obtained by Job from the study of molar extinction coefficients by the method of continued variation. The special advantage of this process discussed above is that it directly gives the difference Y (i. e., -AH) from the additivity rule for any mixture by one reading only. But if any other property be chosen to be studi- ed, three readings of the property will be necessary to find out Y for any mixture--two n:adings for the two parent solutions and the third for the mixture. The process, intro- duced here, is tberefor·e more direct and takes much shorter time in compat:ison with any other prevalent process of study by Job's method of continued variation. p p y y Another remarkable feature of the process is that it gives the value of both K and (- AH) and hence the approximate value of the free energy change (- AF) and the entropy cl1ange (-AS) of the complex formation can be calculated by the well known relations (- .t.F) =RTln K (-AH)=(-AF)+T (-.t.S). (-AH)=(-AF)+T (-.t.S). There is, however, one serious limitation to the process that should be mentioned here. If the heat of dilution of the parent solutions u5ed be not negligible in compari- son with the heat of formation of the complex, the process will fail to yield good results. In the autlwr's estimate, the process is workable if tbe heats of dilution of the parent solutions used do not exceed 10% of the value of the heat of formaHon of the complex. The author's grateful thanks are due to Prof. P.Ray. Palit Professor of Chemistry, Calcutta University for his kind guidance and interest. Thanks are also due to Prof. N. K. Sen, Head of the Department of Chemistry, Presidency College, Calcutta, for taking interest and offering facilities. as c,p, m and n are finite constants, Re•eivell july g, 1948. Re•eivell july g, 1948.
https://openalex.org/W2168260838	https://europepmc.org/articles/pmc2783106?pdf=render	English	null	The role of diclofenack on inducing of aplasia cutis congenita: a case report	Cases journal	2,009	cc-by	1,987	Open Acc Case Report The role of diclofenack on inducing of aplasia cutis congenita: a case report Laura Pajaziti, Syzana Rexhepi, Ylfete Shatri-Muça and Mybera Ferizi Open Access Corresponding author Published: 12 October 2009 Cases Journal 2009, 2:150 doi:10.1186/1757-1626-2-150 Received: 16 September 2009 Accepted: 12 October 2009 This article is available from: http://www.casesjournal.com/content/2/1/150 © 2009 Pajaziti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 16 September 2009 Accepted: 12 October 2009 Received: 16 September 2009 Accepted: 12 October 2009 © 2009 Pajaziti 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. BioMed Central BioMed Central Abstract Background: Aplasia cutis congenita is a disorder where e newborn child is missing skin from certain areas. It is a rare condition with no particular race or sex more at risk. May occur by itself or be associated with other physical syndromes or disorders. A classification system exists for aplasia cutis congenital consisting of 9 groups, based on the number and location of the skin defects and the presence or absence of other malformations. Causes of aplasia congenital could be heredity, teratogenic substances, placental infarcts, intrauterine infections, ectodermal dysplasias etc. Diagnosis is made based on the clinical findings. Prognosis depends of the other organs malfunction level and lesions size. Case report: Our case was an 22 months old Albanian girl, who was recommended to dermatology for a consultation by a pediatric surgeon because of the changes she had on her parietal part of the scalp with missing hair areas. The child has stenosis congenita ani and to her was installed stoma. In order to investigate other accompanied anomalies of the disease, there are made specific consults by neurologist, orthopedist, cardiologist, nephrologists and citogenetics. Conclusion: It was found out a minor visual discoordination, Sy Floppy, Digiti V superductus pedis bill. Laxitas articularum generalisata. It was a great challenge for us to find out that during the first trimester of the pregnancy (unplanned pregnancy), her mother used Diclofenac. Since there is limited information regarding to teratogenic effects of diclofenac, we considered it interesting to present this case. Except sporadic cases, there are familiar cases, which are explained by autosomal -dominant heredity [4]. Causes of aplasia could be teratogenic substances, placental infarcts, intrauterine infections, ectodermal displasias etc [5,6]. Introduction Aplasia cutis congenita is one of the disorders of the skin embrional development. It is characterized by a limited defect of the skin where the skin layers and its adnexes are not created. This feature is rare, about 1 in 3000 alive born children [1-3]. The disorders during the skin development can include one or all skin layers. Sometimes the other deep structures Page 1 of 4 (page number not for citation purposes) Cases Journal 2009, 2:150 Cases Journal 2009, 2:150 http://www.casesjournal.com/content/2/1/150 The parietal part of the scalp appear two oval shaped areas without hair, confluated, with light depth and shinny bases Figure 1 The parietal part of the scalp appear two oval shaped areas without hair, confluated, with light depth and shinny bases. like fascia, ossa and dura are missing (which can be fol- lowed by different cosmetic defects of the skin until the deformities incompatible with life). Aplasia cutis congen- ita is manifested by the defects of the skin mostly oval, 1- 3 cm in diameter, with localization on the parietal part of scalp (60%) and rarely on the face and extremities. It belongs to the group of congenital cicatricial alopecia. Aplasia cutis congenita can be associated by a lot of anom- alies as: labium leporinum, polycystic kidneys, psycho- motor retardation, developing disorders of the eyes, spinal cord, ears, chromosomal aberationes, cutis mar- morata, congenital organoid nevus on the scalp and face, anomalies on extremities, intestines and genital organs. Based on the presence of these anomalies and their asso- ciation with the other diseases as bullous epidermolysis, growth and development stagnation, etc, congenital apla- sias of the skin are classified in 9 groups. The parietal part of the scalp appear two oval shaped areas without hair, confluated, with light depth and shinny bases Figure 1 The parietal part of the scalp appear two oval shaped areas without hair, confluated, with light depth and shinny bases. The parietal part of the scalp appear two oval shaped areas without hair, confluated, with light depth and shinny bases Figure 1 The parietal part of the scalp appear two oval shaped areas without hair, confluated, with light depth and shinny bases. Diagnosis is made based on the clinical findings, and the histopathology is not necessary. Prognosis depends of the other organs malfunction level and lesions size. 46, XX with no changes on the chromosome number and structure. Introduction The lesions can resolve spontaneously, but sometimes is needed surgery operation (punch-graft-transplant). The routine laboratory examinations resulted with com- mon values except the blood SE rate, which was increased (60/1 h). After the antibiotic ordination (Cephalexin sir. A 125 mg), the same fall down to 15/1 h. The patient was discharged home with recommendation to follow up the pediatric surgeon visits. Case presentation Patient aged 22 months, Albanian female, hospitalized in Clinic, by pediatric surgeon recommendation, because of the changes on the scalp with missing hair areas. This alteration persists from the birth without tendency for shape changing. Authors' contributions LP, SR, YSHM and MF analyzed and interpreted the patients' data. LP was a major contributor in writing the manuscript. All authors read and approved the final man- uscript. Conclusion Aplasia cutis congenita is very rare anomaly (1:3000). Causes of congenital aplasia could be teratogenic sub- On the parietal part of the scalp appear two oval shaped areas without hair, confluated, with light depth and shinny bases (Figure 1). Installed stoma (anus praeternaturalis) Figure 2 Installed stoma (anus praeternaturalis). Her mother mentioned that during the first trimester of the pregnancy (she did not know that she was pregnant) has taken Diclofenac amp. (five days) because she had abdominal pains. During pregnancy she did not have any infectious disease. Family history is negative. In the surgery clinic the child was operated and treated under the diagnoses: Stenosis congenita ani and was installed stoma (Figure. 2). For associated defects in other organs, there are made specific consults. Consultant orthopedist concluded the digiti V superduc- tus pedis bilateralis, laxitas articularum generalisata and Sy Floppy (Figure 3a and Figure 3b).Consultant neurolo- gist indicated minor visualmotor discoordination. Cardi- ologists and nephrolog consulting resulted on the common stage. Citogenetic report has shown Cariotype Installed stoma (anus praeternaturalis) Figure 2 Installed stoma (anus praeternaturalis). Page 2 of 4 (page number not for citation purposes) http://www.casesjournal.com/content/2/1/150 Cases Journal 2009, 2:150 a and b: Digiti V superductus pedis bill Figure 3 a and b: Digiti V superductus pedis bill. Laxitas articularum generalisata Sy Floppy. A B A A B a and b: Digiti V superductus pedis bill Figure 3 a and b: Digiti V superductus pedis bill. Laxitas articularum generalisata Sy Floppy. g p p g a and b: Digiti V superductus pedis bill. Laxitas articularum generalisata Sy Floppy. Diclofenac used in early trimester of pregnancy might induce those abnormalities and considered reasonable to present this case. Diclofenac used in early trimester of pregnancy might induce those abnormalities and considered reasonable to present this case. stances [7]. Diclofenac is a non-steroidal anti-inflamma- tory drug (NSAID), commonly used by reproductive age women for the treatment of variety of conditions. NSAID inhibit the biosynthesis of prostanoids. Women may inci- dentally become pregnant while receiving NSAID therapy (unplanned pregnancy) [8]. The diclofenac crosses the placenta readily in the first trimester of human pregnancy resulting in a fetal diclofenac concentration. which is the same as the maternal serum concentration [9]. The drug may also accumulate in fetal tissue with time [9]. Conclusion Usually diclofenac is given in multiple doses, so the possibility to reach teratogenic levels of fetal tissue concentrations is real in patients who are taking diclofenac. In our case the pregnant women received diclofenac 75 mg per day, five days. Diclofenac has also been shown to inhibit implan- tation and embryogenic development in rats when given on gestation day 5 [10]. A positive association between use of NSAID during pregnancy and miscarriages was reported by a study [11]. However, information regarding teratogenecity of NSAID during the critical period of org- anogenesis is lacking. Because aspirin and other NSAID share a similar mechanism of action (inhibition of pros- taglandin synthesis) it was postulated that NSAID might induce congenital abnormalities when given during the critical period of organogenesis [12]. Abbreviations NSAID: non-steroidal anti-inflammatory drug; Sy: Syn- drome. Competing interests The authors declare that they have no competing interests. Consent Written informed consent was obtained from the patient's mother for publication of this case report and accompany- ing images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Acknowledgements The authors would like to acknowledge Consultant Orthopedist, Cardiol- ogists and Nephrology consulting, Neurologist, and Cytogenetist who con- tributed on provided information about the patient. We present patient with aplasia cutis congenita accompa- nied by stenosis congenita ani, digiti V superductus pedis bill., laxitas articularum generalisata-sy Floppy and minor visualmotor discoordination. Based on mentioned stud- ies and real clinical situation we concluded that References 1. Braun-Falco O, Plewig G, Wolf HH, et al.: Dermatology. 2nd edi- tion. Springer; 1999. 2. Karadagliæ D, et al.: Dermatologija. Beograd 2000. 3. Mark A, Crowe MD: Aplasia cutis congenita. 2006. References References 1. Braun-Falco O, Plewig G, Wolf HH, et al.: Dermatology. 2nd edi- tion. Springer; 1999. 2. Karadagliæ D, et al.: Dermatologija. Beograd 2000. 3. Mark A, Crowe MD: Aplasia cutis congenita. 2006. 1. Braun-Falco O, Plewig G, Wolf HH, et al.: Dermatology. 2nd edi- tion. Springer; 1999. Page 3 of 4 (page number not for citation purposes) Page 3 of 4 (page number not for citation purposes) Cases Journal 2009, 2:150 http://www.casesjournal.com/content/2/1/150 http://www.casesjournal.com/content/2/1/150 4. Chitnis MR, Carachi R, Galea P: Familial aplasia cutis congenita. Eur J Pediatr Sturg 1996, 6:100-1. J g 5. White G Levene's color atlas of Dermatology. 2nd edition. Mosby-Wolfe; 1997. 6. Lipozencic J, et al.: Dermatovenerologija. Medicinska naklada Zagreb 1999. g 7. Dobriæ I, et al.: Dermatovenerologija. Zagreb 2005. 8. Chan LY, Chiu PY, Siu SSN, Lau TK: A study of diclofenac - induced teratogenicity during organogenesis sing a whole rat embryo culture model. Human Reproduction 2001, 16(11):2390-2393. 9. Siu SSN, Yeung JHK, Lau TK: A study on placental transfer of diclofenac in first trimester of human pregnancy. Human Reproduction 2000, 15(11):2423-2425. p ( ) 10. Carp HJA, Fein A, Nebel L: Effect of diclofenac on implantation and embryonic development in the rat. Eur J Obstet Gynecol Reprod Biol 1988, 28:273-277. 11. Nielsen GL, Sorensen HT, Larsen H, et al.: Risk of adevrse birth outcome and miscarriage in pregnant users of non-steroidal anti inflammatory drugs: population based observational study and case control study. Br Med J 2001, 322:266-270. y y J 12. Klein KL, Scott WJ, Clark KE: A possible mechanism of aspirin teratogenesis. Teratology 1980, 21:50. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 4 of 4 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge
https://openalex.org/W2242570199	https://dora.dmu.ac.uk/bitstream/2086/13005/1/Lambourn%20-%20Towards%20a%20Connected%20History%20of%20Equine%20Cultures%20TMG%202016.pdf	English	null	Towards a Connected History of Equine Cultures in South Asia: Bahrī (Sea) Horses and “Horsemania” in Thirteenth-Century South India	The Medieval globe	2,016	cc-by	23,848	© 2016, Arc Medieval Press, Kalamazoo and Bradford This work is licensed under a Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International Licence. The authors assert their moral right to be identified as the authors of their part of this work. Permission to use brief excerpts from this work in scholarly and educational works is hereby granted provided that the source is acknowledged. Any use of material in this work that is an exception or limitation covered by Article 5 of the European Union’s Copyright Directive (2001/29/EC) or would be determined to be “fair use” under Section 107 of the U.S. Copyright Act September 2010 Page 2 or that satisfies the conditions specified in Section 108 of the U.S. Copy- right Act (17 USC §108, as revised by P.L. 94-553) does not require the Publisher’s permission. ISSN 2377-3561 (print) ISSN 2377-3553 (online) ISSN 2377-3561 (print) ISSN 2377-3553 (online) www.arc-humanities.org THIS DOCUMENT MAY BE PRINTED FOR PRIVATE USE ONLY AND MAY NOT BE DISTRIBUTED WITHOUT PERMISSION FROM THE PUBLISHER. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress AY BE PRINTED FOR PRIVATE AY BE PRINTED FOR PRIVATE Copyeditor Shannon Cunningham Editorial Assistant Kelli McQueen Page design and typesetting Martine Maguire-Weltecke TOWARDS A CONNECTED HISTORY OF EQUINE CULTURES IN SOUTH ASIA: BAHRĪ (SEA) HORSES AND “HORSEMANIA” IN THIRTEENTH-CENTURY SOUTH INDIA In India, R. Chandrasekhara Reddy generously shared his own photographs of the Tripurantakam hero stones and gave permission for one to be reproduced here. At the American Institute of Indian Studies, Vandana Sinha, Director (Academic), and Sushil Sharma were as ever helpful in supplying the images required and giving permission for the reproduction of the image included here. This contribution began life as part of a panel on Regions of Global Exchange convened by Carol Symes at the 2013 Medieval Academy of America Annual Meeting in Knoxville, Ten nes see. Although the topic will now be unrecognizable to my co-panelists, Christian Raffensperger and Carla Nappi, and to anyone who was in the audience that day, I would like to thank all involved for their good company and interest in the global medieval, wherever it is to be found. 1 Landry, Noble Brutes, 14. The Medieval Globe 2.1 (2016) 10.17302/TMG.2-1.5 pp. 57–100 TOWARDS A CONNECTED HISTORY OF EQUINE CULTURES IN SOUTH ASIA: BAHRĪ (SEA) HORSES AND “HORSEMANIA” IN THIRTEENTH-CENTURY SOUTH INDIA ELIZABETH LAMBOURN ThiS aRTiCle eXPloReS ways that the concept of equine cultures, developed thus far principally in European and/or early modern and colonial contexts, might translate to premodern South Asia. As a first contribution to a history of equine mat- ters in this region, it focuses on the maritime circulation of horses from the Middle East to Peninsular India in the thirteenth century, examining how this phenomenon is recorded in textual and material sources and assessing their potential for writing a new, more connected history of South Asia and the Indian Ocean world. In Noble Brutes: How Eastern Horses Transformed English Culture, Donna Landry remarks upon “the imbalance between the equestrian saturation of early modern culture and today’s marginalization of matters equine.” 1 To many in the “First World,” the horse’s overwhelming association with elite sport and leisure, from polo to pony clubs, has obscured its profound importance in the history of daily life, both into the very recent past and in many parts of the world today. For its meat, its milk, its use as a draft animal, as a mount, and as a luxury object, the horse has been essential to many cultures and subcultures, and without these diverse uses the horse would not have become a global animal. Landry’s book has joined a small but growing num- I would like to thank the Leverhulme Trust for funding my Major Research Fellowship during 2011–13; the present article is a direct outcome of research conducted during that period as part of the project West Asia in the Indian Ocean 500–1500 CE. Special thanks go to Sharon Kinoshita for checking over my translations of the Franco-Italian text of Marco Polo’s Milione. In India, R. Chandrasekhara Reddy generously shared his own photographs of the Tripurantakam hero stones and gave permission for one to be reproduced here. At the American Institute of Indian Studies, Vandana Sinha, Director (Academic), and Sushil Sharma were as ever helpful in supplying the images required and giving permission for the reproduction of the image included here. I would like to thank the Leverhulme Trust for funding my Major Research Fellowship during 2011–13; the present article is a direct outcome of research conducted during that period as part of the project West Asia in the Indian Ocean 500–1500 CE. Special thanks go to Sharon Kinoshita for checking over my translations of the Franco-Italian text of Marco Polo’s Milione. 2 Landry, Noble Brutes, 14. See, e.g., Raber and Tucker, Culture of the Horse; Edwards, Enenkel, and Graham, Horse as Cultural Icon. Among the pioneering studies is Jardine and Brotton, Global Interests. eliZabeTh laMboURN Notwithstand- ing the importance of “Eastern”—that is, Arabian, Persian, and North African— horses to the development of European thoroughbreds, the wider equine cultures of Eurasia and Africa remain sparsely represented in the new research, and there is little dialogue with existing, though often more disciplinarily bounded, literatures on horses in Eurasia and Africa.4 In the gap between these two scholarly tracks lie 4 For a broad selection of essays on the horse in Asia, including during the medieval period, see Fragner et al., Pferde in Asien; on the Chinese horse trade specifically, see Ptak, “Pferde auf See.” For those engaged in a deeper dialogue with the new European equine 4 For a broad selection of essays on the horse in Asia, including during the medieval period, see Fragner et al., Pferde in Asien; on the Chinese horse trade specifically, see Ptak, “Pferde auf See.” For those engaged in a deeper dialogue with the new European equine eliZabeTh laMboURN 58 ber of studies redressing this imbalance through an imaginative combination of tex- tual, visual, and material sources that convey the breadth and depth of this forgot- ten “equine saturation,” as it is termed by Landry.2 Equine cultures as they emerge through this scholarship are an entangled complex of animate and inanimate things decidedly unbounded by subject-object oppositions: from grooms, horse merchants, veterinarians, and those who rode or worked horses, to horse furnishings and sta- bles, writings about horses (such as veterinary and military treatises), visual and sculptural representations, and of course horses themselves. Where earlier scholarship approached horses as commodities in a larger eco- nomic history, as military technologies in a broader history of warfare, or again as veterinary subjects in a wider history of premodern medicine, the new focus on equine cultures places horses at the center and brings these different disci- plines together to write deeper cultural and intellectual histories. As Landry’s title suggests, “Eastern” horses were an important locus of encounter between the English and the Orient: that encounter produced the English thoroughbred and substantially changed English equestrian practices in the eighteenth century, but also impacted national identity. Not only were new riding styles seen to differen- tiate the English from their Continental European neighbors, the “free forward movement” experienced by the gentry during cross-country riding became associ- ated with ideas of liberty, control, and adventure that were profoundly entangled with contemporary political thought and the British imperial enterprise.3 Horses, it transpires, are “good to think with.” This approach is, of course, part also of a larger movement toward writing non-anthropocentric histories, histories that see things other than humans as equally valid subjects of inquiry. Yet paradoxically, given the largely non-European origins of the modern horse (equus caballus), the new interdisciplinary approach to matters equine continues to marginalize the non-European and, in particular, the premodern. 3 Landry, Noble Brutes, 2–4. For the horse in pre-colonial Africa, see also Law, Horse in West African History; and for a useful overview of research, see Blench, “Prehistory.” Allsen’s Royal Hunt is far broader than the “royal hunt” of its title and offers an important panoramic view of the place of animals, including horses, within court cultures from antiquity to the modern period. Japanese scholars have been among those writing more connected histories of premodern equine cultures in Eurasia, although these publications have rarely been translated or referenced. See, in particular, Yokkaichi’s “Horses,” which is clearly indebted to Hikoichi Yajima’s pioneering work. 5 Jardine and Brotton, Global Interests. 6 The literature on horses in South Asia is mostly focused on the early modern and modern periods: for example, Gommans, “Horse Trade,” and Rise of the Indo-Afghan Empire. Scholar- ship on earlier periods is mainly scattered across other histories, or approaches horses from more bounded disciplinary angles. An essential study, undertaken as its title suggests from the direction of economic and military history, is Digby’s War-Horse and Elephant; for the history of military technologies, see Deloche’s various studies based on iconographic evidence, e.g., Le Cheval and “Techniques militaires.” For the horse in popular culture, see Doniger, “Deconstruction of Vedic Horselore,” and also “Symbol in Search of an Object.” 7 The similarity in terminology is not necessarily intended to signal deeper parallels although, as Anne Goldgar’s study, Tulipmania has revealed, the phenomenon was often treated as an example of speculative folly. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 59 important questions about the premodern and non-European equine cultures that contributed to the “equestrian saturation” of the early modern period and that made horses a truly “global interest” even up until the mid-twentieth century.5 In premodern South Asia, the study of matters equine is still a marginal enter- prise. Aside from a small body of specialist literature scattered across economic, military, religious, and art history,6 horses most often figure as “extras” in the background of other narratives. As a first contribution to a new history of equine matters in South Asia, this essay focuses on the importation of horses by sea from the Middle East to Peninsular India—the area comprising the modern-day states of Karnataka, Kerala, Andhra Pradesh, Telangana, and Tamil Nadu—in the thir- teenth century. In Arabic and Persian sources, horses imported by sea, bahr in Arabic, were often referred to as bahrī, “from or of the sea,” and this is the term I adopt here. Thirteenth-century sources describe a near-bankrupting consump- tion of expensive imported horses that I have chosen to call “horsemania,” in ref- erence to “tulipmania”: the consumer passion for the newly-introduced Ottoman tulip that consumed seventeenth century Holland.7 If horses had been transported to South Asia by sea since at least the early centuries of the Common Era, the thir- teenth century saw a boom in the volume of this trade and established patterns of circulation and consumption that continued into the early modern period and the Portuguese entry into this same trade. scholarship, and covering both Eurasia and Africa, see Bankoff and Swart, Breeds of Empire, and Mitchell, Horse Nations. For the horse in pre-colonial Africa, see also Law, Horse in West African History; and for a useful overview of research, see Blench, “Prehistory.” Allsen’s Royal Hunt is far broader than the “royal hunt” of its title and offers an important panoramic view of the place of animals, including horses, within court cultures from antiquity to the modern period. Japanese scholars have been among those writing more connected histories of premodern equine cultures in Eurasia, although these publications have rarely been translated or referenced. See, in particular, Yokkaichi’s “Horses,” which is clearly indebted to Hikoichi Yajima’s pioneering work. scholarship, and covering both Eurasia and Africa, see Bankoff and Swart, Breeds of Empire, and Mitchell, Horse Nations. 60 eliZabeTh laMboURN 60 Thus far, the boom in the maritime horse trade to South India in the thirteenth century has been written primarily as a commodity history or as part of Mongol history, since a significant part of this trade took place within larger networks of commercial and diplomatic exchange between Il Khanid Iran, the polities of South India, and Yuan China.8 This scholarship has largely been built around three con- temporary, non-Indic sources: passages from the two Iranian geographical histo- ries of the Il Khanid technocrat Hazrat-i Vassaf9 and the Il Khanid vizier Rashid al-Din,10 and the travel account of the Venetian merchant Marco Polo, itself heavily dependent on Il Khanid data.11 There is no denying the critical importance of these 8 See Digby’s discussion of the Il Khanid sources in War-Horse and Elephant, 29–33; Kauz, “Horse Exports,” 133–34, and “Maritime Trade”; and Yokkaichi, “Horses,” 90–91. 9 His personal name was Shihab al-Din ‘Abdallah Sharaf Shirazi, although he is commonly known simply as Vassaf. His history of the Il Khanids, the Tajziyat al-amsar va tazjiyat al- as‘ar, commonly known as the Ta’rikh-i vassaf, was begun in 699/1300, and volume four was completed in 712/1312; the fifth volume took another fifteen years and was completed in 727 or 728/1327 or 1328: see Jackson, “Wassaf.” Vassaf’s history is vital for Iran and the Gulf at this period but has never benefitted from a critical edition of the Persian text; the Bombay lithographed edition of 1853 (reprinted in Teheran, 1959–60) remains the fundamental source. Thus far, the highly florid Persian text has resisted any full English translation, although summaries or paraphrases of the sections on South India are given by numerous authors, including Digby, War-Horse and Elephant, 30–31; see also Aubin, “Les Princes d’Ormuz,” and Aigle, Le Fārs sous la domination mongole, 143–44, both of which are clearly derived in large part from Vassaf’s text. While aware of its shortcomings, I rely here on the English translation: Tazjiyatu-l amsar, translated by Elliot and Dowson in History of India. For the broader history of Vassaf’s text and its reception, see Pfeiffer, “Turgid History.” 10 Rashid al-Din’s Ta’rikh al-hind forms part of volume two of his larger Jawami‘ al-tawarikh and was completed in 1314. 60 eliZabeTh laMboURN In this essay, I have relied on the facsimiles of the Persian and Arabic manuscripts of the Ta’rikh al-hind edited by Jahn as Rashid al-Din’s History, as well as on rough English translations of key passages in Yule’s “Endeavour.” Rashid al-Din’s and Vassaf’s texts are clearly highly entangled in places, repeating key names and figures to the extent that the two sources are often quoted interchangeably, as if they were identical. The overlap is not surprising, not least because Rashid al-Din patronized Vassaf at the Il Khanid court; however, this information is presented within very different genres and discourses, complicating rather than corroborating our understanding of their relationship. Moreover, the genealogy of the borrowings is far from clear. In theory, Vassaf’s account of South India precedes that of Rashid al-Din by about two years, being completed in 1312; however, this section of Vassaf’s work is not stylistically coherent, and the passage including the hard data on the horse trade is in fact closer to Rashid al-Din’s prose style. The problem will only be resolved when a critical edition of Vassaf’s text is finally undertaken. 11 In this essay, I have used Gabriella Ronchi’s bilingual edition of Polo’s account, Milione, which gives both the Tuscan and Franco-Italian texts. I have followed the latter, based on Paris, Bibliothèque nationale de France, fr. 1116, which is judged to be one of the earliest 8 See Digby’s discussion of the Il Khanid sources in War-Horse and Elephant, 29–33; Kauz, “Horse Exports,” 133–34, and “Maritime Trade”; and Yokkaichi, “Horses,” 90–91. 9 His personal name was Shihab al-Din ‘Abdallah Sharaf Shirazi, although he is commonly known simply as Vassaf. His history of the Il Khanids, the Tajziyat al-amsar va tazjiyat al- as‘ar, commonly known as the Ta’rikh-i vassaf, was begun in 699/1300, and volume four was completed in 712/1312; the fifth volume took another fifteen years and was completed in 727 or 728/1327 or 1328: see Jackson, “Wassaf.” Vassaf’s history is vital for Iran and the Gulf at this period but has never benefitted from a critical edition of the Persian text; the Bombay lithographed edition of 1853 (reprinted in Teheran, 1959–60) remains the fundamental source. Thus far, the highly florid Persian text has resisted any full English translation, although summaries or paraphrases of the sections on South India are given by numerous authors, including Digby, War-Horse and Elephant, 30–31; see also Aubin, “Les Princes d’Ormuz,” and Aigle, Le Fārs sous la domination mongole, 143–44, both of which are clearly derived in large part from Vassaf’s text. While aware of its shortcomings, I rely here on the English translation: Tazjiyatu-l amsar, translated by Elliot and Dowson in History of India. For the broader history of Vassaf’s text and its reception, see Pfeiffer, “Turgid History.” ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 61 three sources, but this essay proposes that they have perpetuated an Irano-centric history, overly focused on one South Indian dynasty, that of the Pandyas, which had particularly close political and economic ties with Il Khanid Iran; this narra- tive is thus little interested in the subsequent lives of bahrī horses in India and the Indic equine cultures they joined, or in the wider importation of horses to the region. This essay accordingly argues for a more complex, multi-directional, and interconnected history of the horse in thirteenth-century South India, built on a broader reading of existing Il Khanid sources and the introduction of new textual and visual material from both indigenous and external sources. The sources pre- sented here have all been edited and translated and have already contributed to other histories—of commodities and tax regimes, military technologies, chivalric cultures, or animal husbandry—but they have not previously been combined in a history of equine cultures. This essay proceeds in two steps. It begins by broadening and complicating the history of the horse as a maritime commodity through the integration into the existing narrative of data from Yemeni and Indic textual sources, as well as Indic visual sources, to give a more rounded picture of the volume, patterns, and politics of the horse trade to Peninsular India in the thirteenth century. Prominent among these sources is a collection of late thirteenth-century documents from the cus- toms house of Aden, documents which were only published in 2003, and visual representations of horses and their equipment on Indian hero stones and in tem- ple sculpture. The second part of this essay moves beyond this revised commodity history and into the realm of equine histories proper, by arguing that the facts and figures pertaining to the horse trade as given in the influential accounts of Vas- saf, Rashid al-Din, and Marco Polo were never primarily “about” trade but were instead part of larger discourses surrounding civilizational differences and values, the encounter between the Middle East and South Asia, or (in Polo’s case) Europe and non-European Others. The study of “equine cultures” rather than “horses” therefore underlines the extent to which, much as in eighteenth-century England, horses mediated cross-cultural encounters and were vehicles for larger discourses. Recognizing animals as a fundamental component of the material and intellec- tual exchanges that took place around the Indian Ocean opens new interpretive possibilities beyond their history as trade commodities. The second section also broaches the challenge of following bahrī horses into their new “Indian lives” and the problems that South Asian sources and South Asian historiography pose to this task. I explore the potential of inscriptional and iconographic evidence from the and most complete manuscripts. All translations are my own, but I am grateful to Sharon Kinoshita for suggesting corrections where necessary. See also her essay in this issue. The Horse in South Asia Earlier bones from Harappan sites have now been identified as those of Asiatic wild asses (equus hemionus) rather than modern horses (equus caballus). The Horse in South Asia The ubiquity of the horse today tends to obscure its limited original natural habitat and the very active human interventions that made it a global animal. Domesticated and wild horses are now found on four of five continents, excluding only Antarctica, but at around 6000 BCE the wild horse populations that seeded the first domesticated horses were found only on the steppes of eastern Europe and central Asia. It is from this relatively circumscribed area that the modern horse has spread globally in just eight millennia,12 a remarkably rapid and pow- erful expansion in which premodern non-European actors played a central role. Horses are indigenous, and best adapt to semi-arid zones or steppe climates, dry but not desertic conditions that tend to support a short or scrubby vegetation of grasses or shrubs.13 Only a limited number of regions across the globe are able to sustain wild horse populations, and the horse’s wider global spread would have been impossible without human initiatives to export it beyond, and sustain it out- side, its original natural habitat—in effect, to domesticate it. Archaeological evidence points to the introduction of the modern horse to the north of the Indian subcontinent from at least the first millennium BCE,14 yet the 12 The first horses evolved some fifty-five million years ago and multiple wild horse species coexisted on the North American plains and in Europe and Asia. While these North American species became extinct around ten thousand years ago, and species dwindled in western Europe, it was in central Asia that the modern horse equus caballus was first domesticated, probably round 4000 BCE. The diversity of the modern horse genome suggests that different wild horse populations were domesticated in different places. Although first raised for their meat and milk, evidence from eastern Russia and Kazakhstan shows horses being used to pull chariots by around 2000 BCE. By ca. 2000–1500 BCE, riding horses had become common in Iran and Afghanistan. See Levine, “Domestication.” 13 For the full definition of semi-arid climates, see Köppen, “Das geographisca System.” For an updated world distribution map, see Peel, Finlayson, and McMahon, “Updated World Map.” 14 This is based on confirmed finds of horse bones at the site of Pirak in Pakistan, where terracotta horse figurines have also been found; see Van der Geer, Animals in Stone, 233. eliZabeTh laMboURN 62 Hoysala and the Kakatiya polities for future research into the place of horses within military networks and their translation into distinctive Indic chivalric cultures (see Map 3). I conclude by tracing the ways that these newly forged Indic equine cultures made reverse journeys back across the western Indian Ocean, entering Middle Eastern hippiatric texts as a distinct body of Indian equine knowledge. 12 The first horses evolved some fifty-five million years ago and multiple wild horse species coexisted on the North American plains and in Europe and Asia. While these North American species became extinct around ten thousand years ago, and species dwindled in western Europe, it was in central Asia that the modern horse equus caballus was first domesticated, probably round 4000 BCE. The diversity of the modern horse genome suggests that different wild horse populations were domesticated in different places. Although first raised for their meat and milk, evidence from eastern Russia and Kazakhstan shows horses being used to pull chariots by around 2000 BCE. By ca. 2000–1500 BCE, riding horses had become common in Iran and Afghanistan. See Levine, “Domestication.” THIS DOCUMENT MAY BE PRINTED FOR PRIVATE USE ONLY AND MAY NOT BE DISTRIBUTED WITHOUT PERMISSION FROM THE PUBLISHER. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 63 Map 3. South India Show ing Select Locations and Features. Map created by the author. story of the horse in South Asia is far from being a neat narrative of gradual north- south diffusion. Finds of horse-bits in South Indian Megalithic (Iron Age) burials, broadly datable to around 800 to 300 BCE,15 as well as representations of horses and riders in Megalithic rock art, offer clear indications of the early presence and high status of these animals in the Peninsular south.16 Detailed understanding of the horse’s diffusion across South Asia and its reception by, and translation into, the subcontinent’s hugely varied cultures and environments remains a task hin- 15 The so-called Megalithic period remains inconsistently dated and is often extended into the first centuries of the Common Era. For a reference to the “clear attestation of horses” in Mega lithic South India between 800–300 BCE, see Asouti and Fuller, Trees and Woodlands, 41. 16 To the best of my knowledge, the only survey of the archaeological material from South India is Leshnik’s “Some Early Indian Horse-Bits” (now dated). For a recent overview of Megalithic rock art, see Blinkhorn, Taçon, and Petraglia, “Rock Art Research,” 184 and 188. 15 The so-called Megalithic period remains inconsistently dated and is often extended into the first centuries of the Common Era. For a reference to the “clear attestation of horses” in Mega lithic South India between 800–300 BCE, see Asouti and Fuller, Trees and Woodlands, 41. 16 To the best of my knowledge, the only survey of the archaeological material from South India is Leshnik’s “Some Early Indian Horse-Bits” (now dated). For a recent overview of Megalithic rock art, see Blinkhorn, Taçon, and Petraglia, “Rock Art Research,” 184 and 188. 15 The so-called Megalithic period remains inconsistently dated and is often extended into the first centuries of the Common Era. For a reference to the “clear attestation of horses” in Mega lithic South India between 800–300 BCE, see Asouti and Fuller, Trees and Woodlands, 41. 15 The so-called Megalithic period remains inconsistently dated and is often extended into the first centuries of the Common Era. For a reference to the “clear attestation of horses” in Mega lithic South India between 800–300 BCE, see Asouti and Fuller, Trees and Woodlands, 41. 16 To the best of my knowledge, the only survey of the archaeological material from South India is Leshnik’s “Some Early Indian Horse-Bits” (now dated). For a recent overview of Megalithic rock art, see Blinkhorn, Taçon, and Petraglia, “Rock Art Research,” 184 and 188. eliZabeTh laMboURN Horses driven overland from their native habitats in central Asia or the eastern Himalayas were referred to in medieval Arabic and Persian sources as barrī or “horses from the land,” while (as noted above) those shipped by sea were bahrī (Arabic) or daryā’ī (Persian): literally “from the sea.” It is not uncommon in medieval Indian texts to find horses discussed alongside pearls as one of the bounties of the ocean.19 Within this broader scenario, Peninsular India appears to have suffered a double disadvantage, in that its climate is especially 19 A Silahara copper plate grant from Kohlapur, dated 1114 CE (Śaka 1037), describes the ocean giving pearls, fine cloth, and horses to the king; cited in Chauhan, “Arab Horses,” 392. 17 Barani, a fourteenth-century chronicler, discusses Sind and towns in the Punjab in a speech attributed to Sultan Balban which (Digby suggests) reflects specifically contemporary conditions. See Digby, War-Horse and Elephant, 21–22; the speech is also cited in Chakravarti, “Equestrian Demand and Dealers,” 149. The history of Marwari and Kathiawari horses is far from clear, although both are believed to be partly interbred with Arabian horses. 18 See Peel, Finlayson, and McMahon, “Updated World Map,” fig. 5. 19 A Silahara copper plate grant from Kohlapur, dated 1114 CE (Śaka 1037), describes the ocean giving pearls, fine cloth, and horses to the king; cited in Chauhan, “Arab Horses,” 392. 19 A Silahara copper plate grant from Kohlapur, dated 1114 CE (Śaka 1037), describes the ocean giving pearls, fine cloth, and horses to the king; cited in Chauhan, “Arab Horses,” 392. 17 Barani, a fourteenth-century chronicler, discusses Sind and towns in the Punjab in a speech attributed to Sultan Balban which (Digby suggests) reflects specifically contemporary conditions. See Digby, War-Horse and Elephant, 21–22; the speech is also cited in Chakravarti, “Equestrian Demand and Dealers,” 149. The history of Marwari and Kathiawari horses is far from clear, although both are believed to be partly interbred with Arabian horses. 18 See Peel, Finlayson, and McMahon, “Updated World Map,” fig. 5. eliZabeTh laMboURN 64 dered by a neglected base of textual sources, scattered iconographic evidence, and all too little secure archaeological data. This neglect is particularly puzzling given the seminal position of the horse within Hinduism and Indic kingship; the horse sacrifice or aśwamedha was one of the most important religious rites described in the Vedas, the foundational texts of Hinduism, and was performed by kings into the medieval period to celebrate their paramountcy. Nevertheless, even at this early stage it is clear that the story of the horse in South Asia is multi-directional and often paradoxical, profoundly intertwined with that of the elephant, India’s royal mount par excellence, and particularly complex in Peninsular India, where maritime supply routes from the Middle East and Bengal made the horse an Indian Ocean commodity from an early period. South Asia largely lacks the extensive grasslands needed to sustain stable populations of strong and healthy horses. A variety of sources—from the Kautilya Arthaśastra (a treatise on statecraft now agreed to date from the fourth century CE) to a fourteenth-century Sultanate history—affirm that the Punjab, Sind, and Saurashtra were the principal regions of the subcontinent with environments that could sustain stable horse populations and large-scale breeding.17 Not sur- prisingly, these are the very areas designated as semi-arid (BSk and BSh) in the Köppen-Geiger climate classification system.18 The climate beyond these areas was not suited to horse-keeping, fostering environments with little natural grass or fodder for equine diets while monsoonal humidity adversely affected horses’ health, resulting in both a shortened lifespan and small, sickly offspring. Through- out the history of South Asia, any “indigenous” horse populations which became established were repeatedly supplemented by imported animals driven overland or shipped by sea. 22 Pre-tenth-century Indian epigraphic materials and material culture certainly provide evidence for an earlier trade in horses from the west, but the scale of this is difficult to determine; see Chauhan, “Arab Horses.” 22 Pre-tenth-century Indian epigraphic materials and material culture certainly provide evidence for an earlier trade in horses from the west, but the scale of this is difficult to determine; see Chauhan, “Arab Horses.” 23 To the best of my knowledge, the history of this title has not been traced but it was adopted, for example, by the Sena rulers of Bihar and Bengal (r. 1095–1204); see discussion in Majumdar, Pusalker, and Majumdar, The Struggle for Empire, 118–31, line 41. 25 See Mazars, “Traditional Veterinary Medicine,” 444. Jayadatta’s Aśvavaidyaka is prob ably datable to the thirteenth century. 26 Doniger, “Deconstruction of Vedic Horselore,” 76–77. 20 See Cosmas Indicopleustes, Christian Topography, 372. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 65 challenging to horses, and it was often cut off from terrestrial supplies of barrī horses by polities in central India. South Indian polities therefore found them- selves much more dependent on animals imported by sea. We have records of Per- sian horses reaching Sri Lanka by sea as early as the sixth century CE,20 but some of the earliest evidence suggests that horses were also being shipped from the head of the Bay of Bengal to the Tamil south in the third century CE.21 Neverthe- less, epigraphic and textual references to Arab or Persian horses, many of which must have arrived by sea, only begin to appear regularly from the tenth and elev- enth centuries onwards.22 Horses never entirely displaced the indigenous elephant as the choice mount of Indian rulers, and elephants remained crucial in South Asian warfare; but in spite of the cost and difficulty of maintaining horses in much of the subcontinent, it is generally agreed that cavalries had become a staple of Indian warfare by the early medieval period. South Asian rulers took titles that expressed their com- mand of diverse armies: the king was Aśvapati or Advapati (Lord of Horses), Gaj- apati (Lord of Elephants), and Narapati (Lord of Men).23 The Kautilya Arthaśastra (fourth century CE) already includes advice on the administration of royal stables and the care of horses,24 and the first specifically hippiatric texts—SƵalihotra’s Aśvayurvedasiddhanta (Complete Ayurvedic System for Horses) and Nakula’s Aśvacikitsita (Therapeutics of Horses)—are generally held to have been composed in the ninth and early eleventh centuries, respectively.25 Yet in the popular recep- tion of horses in Indian folklore, as Wendy Doniger has remarked, the horse was always perceived as non-Indian and ephemeral, the mount of conquerors from the Kushanas to the British, an animal that entered but could never truly establish itself in India.26 It was always a little otherworldly, a semi-divine creature. 20 See Cosmas Indicopleustes, Christian Topography, 372. 21 Chakravarti, “Early Medieval Bengal,” 205–07; Chakravarti marshals evidence from a Chinese source and an Indian seal both from the third century CE, together with passages from roughly contemporary Tamil sangam literature. 21 Chakravarti, “Early Medieval Bengal,” 205–07; Chakravarti marshals evidence from a Chinese source and an Indian seal both from the third century CE, together with passages from roughly contemporary Tamil sangam literature. 20 See Cosmas Indicopleustes, Christian Topography, 372. 21 Chakravarti, “Early Medieval Bengal,” 205–07; Chakravarti marshals evidence from a Chinese source and an Indian seal both from the third century CE, together with passages from roughly contemporary Tamil sangam literature. 22 Pre-tenth-century Indian epigraphic materials and material culture certainly provide evidence for an earlier trade in horses from the west, but the scale of this is difficult to determine; see Chauhan, “Arab Horses.” 23 To the best of my knowledge, the history of this title has not been traced but it was adopted, for example, by the Sena rulers of Bihar and Bengal (r. 1095–1204); see discussion in Majumdar, Pusalker, and Majumdar, The Struggle for Empire, 118–31, line 41. 24 Kautilya, Arthaśāstra, 146–50. 25 See Mazars, “Traditional Veterinary Medicine,” 444. Jayadatta’s Aśvavaidyaka is prob ably datable to the thirteenth century. 26 Doniger, “Deconstruction of Vedic Horselore,” 76–77. Horse-Producing Regions and Supply Networks The price per head of horse was 220 dinars of red gold.28 In his contemporary Ta’rikh al-hind (History of India), the Il Khanid vizier Rashid al- Din counts a total of ten thousands head of horse exported annually to Ma‘bar and purchased at a cost of 2,200,000 dinars of red gold.29 Marco Polo, whose journey to China via India is one of the best known accounts of the European mercantile encounter with Eurasia at this time, gives similar quantities, reporting that two thousand or more horses were purchased by each of the five Pandya kings each year, for a total of ten thousand horses.30 Elsewhere in his account, Polo specifi- cally mentions Qalhat in northern Oman as an export depot for good destriers (warhorses): “because know that from this country […] large quantities of beauti- ful horses are taken to India, so many that you could hardly count them.”31 The 27 Earlier references to the maritime circulation of horses can be counted on the fingers of two hands; see scattered references in Chakravarti, “Horse Trade and Piracy,” “Early Medi- eval Bengal,” and “Equestrian Demand and Dealers.” 30 The Pandya polity operated via a system of sub-regents, which often caused outside observers to describe it as governed by multiple kings. 31 Polo, Milione, 609: “de cest contree […] se portent grant quantités de biaus chevaus en Y<n>de, si grant que a poine le poroit l’en contere.” eliZabeTh laMboURN 66 27 Earlier references to the maritime circulation of horses can be counted on the fingers of two hands; see scattered references in Chakravarti, “Horse Trade and Piracy,” “Early Medi- eval Bengal,” and “Equestrian Demand and Dealers.” Horse-Producing Regions and Supply Networks The thirteenth century saw a major rise in the volume of the horse trade between the Arabian Peninsula, the Gulf, and South India. The fact that horses were cir- culated into and around India by sea has long fascinated Western observers, not least Marco Polo; however, the shipping of large animals is not surprising in itself. Horses and animals far larger and more dangerous, such as elephants, were regu- larly transported around and across the Indian Ocean, and had been since at least the early first millennium CE. What is surprising are the multiple, corroborating references to this trade in sources from around the Indian Ocean rim, from Syria and the Yemen to China.27 For the first time, horses emerge as a major commodity of maritime trade, circulating in apparently very large numbers. In the 1290s, the Il Khanid historian and erstwhile fiscal administrator Vassaf al-Hazrat reports that 1,400 horses were due to be exported annually from the Gulf island of Kish (Qa’is) to Ma‘bar, the Tamil region of South India then under the Pandya dynasty, together with as many as could be sourced from other Il Khanid tributaries elsewhere in the Gulf: areas such as Qatif, Lahsa (al-Ahsa), Bahrayn, Hormuz, and Qalhat. eliZabeTh laMboURN 28 Vassaf, Tazjiyatu-l amsar, 33. 29 Rashid al-Din, Ta’rikh al-hind, 65. 27 Earlier references to the maritime circulation of horses can be counted on the fingers of two hands; see scattered references in Chakravarti, “Horse Trade and Piracy,” “Early Medi- eval Bengal,” and “Equestrian Demand and Dealers.” 28 Vassaf, Tazjiyatu-l amsar, 33. 29 Rashid al-Din, Ta’rikh al-hind, 65. 30 The Pandya polity operated via a system of sub-regents, which often caused outside observers to describe it as governed by multiple kings. 31 Polo, Milione, 609: “de cest contree […] se portent grant quantités de biaus chevaus en Y<n>de, si grant que a poine le poroit l’en contere.” ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia Another rare reference to equestrian training comes from a Hoysala inscription dated 1140 CE, which mentions that the wife of the Hoysala ruler Vishnuvardhana, Bammala-devi, managed a “crown riding school” along with Ananthapala Sahani, the Master of the Royal Stables; see Coelho, Hoysala Vamsa, 254 (citing Epigraphia Carnatica, vol. 5, inscription no. Ak 58). 37 See Jackson, “Wassaf,” 67–68; at one point Vassaf cites Jamal al-Din, suggesting a per- sonal acquaintance. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 67 consistency of the figures across these two last sources is remarkable and sug- gests that Polo had access to informants with reliable knowledge of the Il Khanid economy. A surprising aspect of the Il Khanid trade in horses with South India, given the hugely variable traits of individual animals, was the operation of a fixed price per head of horse. For the Il Khanid trade, both Vassaf and Rashid al-Din give the rate at 220 dinars of red gold per horse,32 Polo gives 500 saje d’or, which he states are worth more than 100 silver marks (mars).33 According to both Il Khanid sources, the price was set on the condition that every horse was paid for, whatever its condition upon arrival, whether or not it survived the journey. In Vassaf’s text and Polo’s, the Pandya kingdom (in what is now southern Tamil Nadu) emerges as a key market. As Polo observed during the course of his return west via the Pandya ports in the early 1290s, “in this kingdom no horses are born, and therefore all the treasure from the income (rende) that they have each year, or the greater part, is consumed in buying horses.”34 The final destination of the majority of these horses was the Pandya cavalry; as Vassaf observed, these horses were ridden by the troops of the Indians (junūd-i hindū) and used for war and equestrian exercises (li-l-harb wa furusiyya al-maydān).35 Of course, these figures might be disputed as conventional medieval hyperinflation, and Simon Digby has wisely cited the advice of an early thirteenth-century Sultanate author Fakhr-i Mudabbir, to the effect that “the king or the commander of an army may know the numbers of horse and foot, he must say two or three times this number” when boasting about his troops.36 Nevertheless, Rashid al-Din, as vizier, was well positioned to obtain fiscal information; while Vassaf, as a fiscal administrator in Fars for the Il Khanids, could have accessed, and perhaps even compiled, such fig- ures. Vassaf was, furthermore, personally acquainted with members of the Tibi family who dominated this Il Khanid trade from their island emporium of Kish.37 Moreover, these figures are altogether modest when compared to the hundreds 35 Vassaf, Tazjiyatu-l amsar, 34. 37 See Jackson, “Wassaf,” 67–68; at one point Vassaf cites Jamal al-Din, suggesting a per- sonal acquaintance. 36 Cited in Digby, War-Horse and Elephant, 23. 35 Vassaf, Tazjiyatu-l amsar, 34. Another rare reference to equestrian training comes from a Hoysala inscription dated 1140 CE, which mentions that the wife of the Hoysala ruler Vishnuvardhana, Bammala-devi, managed a “crown riding school” along with Ananthapala Sahani, the Master of the Royal Stables; see Coelho, Hoysala Vamsa, 254 (citing Epigraphia Carnatica, vol. 5, inscription no. Ak 58). 33 Polo, Milione, 556. 32 Vassaf, Tazjiyatu-l amsar, 33; and Rashid al-Din, Ta’rikh al-hind, 65. 33 Polo, Milione, 556. 34 Ibid. 35 Vassaf, Tazjiyatu-l amsar, 34. Another rare reference to equestrian training comes from a Hoysala inscription dated 1140 CE, which mentions that the wife of the Hoysala ruler Vishnuvardhana, Bammala-devi, managed a “crown riding school” along with Ananthapala Sahani, the Master of the Royal Stables; see Coelho, Hoysala Vamsa, 254 (citing Epigraphia Carnatica, vol. 5, inscription no. Ak 58). 36 Cited in Digby, War-Horse and Elephant, 23. 37 See Jackson, “Wassaf,” 67–68; at one point Vassaf cites Jamal al-Din, suggesting a per- sonal acquaintance. 32 Vassaf, Tazjiyatu-l amsar, 33; and Rashid al-Din, Ta’rikh al-hind, 65. eliZabeTh laMboURN , , 42 Jazim, Nur al-ma‘arif, 1:189: (al-sūliyān wa hum ahl al-raghba fī shirā’ al-khayl) and 1:190 (wa sā’ir al-hind qalīl ’an yarghubū fī shirā’ al-khayl ka-raghba al-sūliyan). These documents have recently been edited under the title Nūr al-ma‘ārif or Light of Knowledge, and they considerably deepen our understanding of the economic history of the period. Eric Vallet, in L’Arabie marchande, has worked most closely on those related to the horse trade, although they were also consulted by Hokoichi Yajima. 39 Aubin, “Les Princes d’Ormuz,” 92. 38 Figures from various sources are cautiously cited in Digby, War-Horse and Elephant, 23–25 and 49. 38 Figures from various sources are cautiously cited in Digby, War-Horse and Elephant, 23–25 and 49. 39 Aubin, “Les Princes d’Ormuz,” 92. 40 Cited in Raychaudhuri, Habib, and Kumar, Cambridge Economic History, 118. Another remarkable continuity of practice is that the Vijayanagara ruler also paid for the horses whether or not they arrived alive or even healthy. 41 Polo, Milione, 577: “charchiés de mercandies e de chevaus” and “de toute l’Arabe.” 42 Jazim, Nur al-ma‘arif, 1:189: (al-sūliyān wa hum ahl al-raghba fī shirā’ al-khayl) and 1:190 (wa sā’ir al-hind qalīl ’an yarghubū fī shirā’ al-khayl ka-raghba al-sūliyan). These documents have recently been edited under the title Nūr al-ma‘ārif or Light of Knowledge, and they considerably deepen our understanding of the economic history of the period. Eric Vallet, in L’Arabie marchande, has worked most closely on those related to the horse trade, although they were also consulted by Hokoichi Yajima. eliZabeTh laMboURN 68 of thousands of horses reportedly mobilized by Delhi Sultanate armies,38 and not unreasonable when we consider, for example, that Jamal al-Din Tibi was able to present 1,000 geldings to the Mongol Il Khan Gazan in 1296, along with Chinese and Indian gifts.39 The figures may even turn out to be remarkably consistent with subsequent trends, since the Portuguese horse trader Nuniz reported, in the first half of the sixteenth century, that the ruler of the large South Indian polity of Vijay- anagara imported thirteen thousand head of horse per annum via the port of Hor- muz.40 Environments can only sustain animal populations of a certain size, and it may be that the surplus available for export was only ever in the region of ten to thirteen thousand head of horse per annum, after internal Iranian demand for horses was met. Nevertheless, these figures represent only a portion of the total number of horse exports to South India in this period: horses were bred elsewhere than in Iran and the Gulf, and were also exported to other South Indian polities besides the Pandya. Departing from the heavily Irano-centric foci of Vassaf and Rashid al- Din, Marco Polo reports that the Pandya port of Qa’il (modern Kayalpattanam, on the Coromandel coast) received ships “laden with merchandise and horses” com- ing from Hormuz and Kish but also Aden, then under the Rasulid dynasty (r. 1229– 1454), and “all of Arabia.”41 A remarkable corpus of Rasulid customs documents from the port of Aden and dating to the 1290s confirm this, with one document explicitly underlining the scale of Pandyan purchases. Referring to the inhabit- ants as the Sūliyān—literally “Cholas,” since the Chola polity (r. ca. 850–1279) had until recently been the dominant power across the region—this document repeatedly notes this people’s desire for horses: “the Sūliyān, they are the people who wish to buy horses,” and again, “in all of India few desire to buy horses as much as the Sūliyān desire it.”42 The triliteral root used in both cases, RGHB, can be 38 Figures from various sources are cautiously cited in Digby, War-Horse and Elephant, 23–25 and 49. 40 Cited in Raychaudhuri, Habib, and Kumar, Cambridge Economic History, 118. Another remarkable continuity of practice is that the Vijayanagara ruler also paid for the horses whether or not they arrived alive or even healthy. 40 Cited in Raychaudhuri, Habib, and Kumar, Cambridge Economic History, 118. Another remarkable continuity of practice is that the Vijayanagara ruler also paid for the horses whether or not they arrived alive or even healthy. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 69 translated neutrally as “to wish” or “to desire” but might also be translated more forcefully as “to crave” or “to covet.” Whichever the translation, it is the particular purchasing enthusiasm of the Sūliyān that is underlined. One ultimate destination of these horses was undoubtedly the Pandya polity, as confirmed by Marco Polo. “All of Arabia” may also have included Syria, since a geographical treatise finished in 721/1321 by the Ayyubid prince and governor of Hamah Abu al-Fida’ includes the fact that horses from various places were brought to the capital city of Ma‘bar,43 while an account of the plunder of the Pandya royal stables in 1311 (by Delhi Sul- tanate forces) reports that the five thousand horses found there included bahrī, that is, Yemeni and Shami or Syrian animals.44 Importantly, the Rasulid customs documents also point to other horse-pur- chasing polities in South India. One document lists the prices that horses fetched at the northern Malabari ports of Mangalore and Barkur (Faknur in the Arabic), the principal ports of the small coastal polity of the Alupas in what is now south Karnataka. At Mangalore, a horse fetched 400 mīnī (equivalent to 200 Egyptian gold mithqāls) and, at Faknur, a healthy horse without blemishes was worth 420 mīnī.45 As Eric Vallet has noted, these two ports were most likely the transit points for horses destined for the cavalries of the Hoysala kingdom based on the Dec- can plateau to the east, with its capital at Dwarasamudra (located on the western edge of the Deccan plateau, near modern-day Halebid). The northern Malabari ports offered the Hoysalas the most direct access to goods coming from the Indian Ocean system. The same pattern of horse supply, sometimes via the same ports and passes through the Western Ghats, operated under the Hoysala’s Vijayanagara successors and was eventually taken over by the Portuguese.46 It is unclear what pricing system operated in the horse trade with Rasulid Yemen. The sources from Yemen are contradictory: some customs house docu- ments describe an annual auction of horses at Aden and thus suggest that prices were set by market demand,47 and the northern Malabari evidence suggests differ- 47 Jazim, Nur al-ma‘arif, 1: 505; discussed in Vallet, L’Arabie marchande, 223–25. 43 Discussed in his Taqwim al-buldan, 402–03. 44 Figures derived from Amir Khusraw Dihlavi’s Khaza’in al-futuh; cited by Digby, War- Horse and Elephant, 48. 45 See Jazim, Nur al-ma‘arif, 1:265; also discussed in Vallet, L’Arabie marchande, 570–71. One mīnī was worth half an Egyptian gold mithqāl, 570. 46 For the Portuguese trade, see Loureiro, “Portuguese Involvement.” There is a general overview in Raychaudhuri, Habib, and Kumar, Cambridge Economic History, 117–19, which also includes the account of the trade with Viyayanagara by the Portuguese horse merchant Fernão Nunes, also known as Fernao Nuniz. 47 Jazim, Nur al-ma‘arif, 1: 505; discussed in Vallet, L’Arabie marchande, 223–25. 44 Figures derived from Amir Khusraw Dihlavi’s Khaza’in al-futuh; cited by Digby, War- Horse and Elephant, 48. 43 Discussed in his Taqwim al-buldan, 402–03. 45 See Jazim, Nur al-ma‘arif, 1:265; also discussed in Vallet, L’Arabie marchande, 570–71. One mīnī was worth half an Egyptian gold mithqāl, 570. 47 Jazim, Nur al-ma‘arif, 1: 505; discussed in Vallet, L’Arabie marchande, 223–25. 46 For the Portuguese trade, see Loureiro, “Portuguese Involvement.” There is a general overview in Raychaudhuri, Habib, and Kumar, Cambridge Economic History, 117–19, which also includes the account of the trade with Viyayanagara by the Portuguese horse merchant Fernão Nunes, also known as Fernao Nuniz. eliZabeTh laMboURN Compare this to the earlier horse trade in Oman as described in Zhao Rugua’s Description of All Barbarians (ca. 1225 CE), where horses, pearls, and dates are noted as being bartered for cloves, cardamom seeds, and camphor; cited in Kauz, “Horse Exports,” 131. For more on networks of horse supply to Aden, see Vallet, L’Arabie marchande, 373–78. 48 Jazim, Nar al-ma‘arif, 1:189–90; discussed in Vallet, L’Arabie marchande, 226. 49 Vallet, L’Arabie marchande, 223–24. 50 Ibid., 226. 51 See Vallet, L’Arabie marchande, 225–27, building on the work of Yokkaichi, “Horses,” 91–93, and earlier work by Hikoichi Yajima. Compare this to the earlier horse trade in Oman as described in Zhao Rugua’s Description of All Barbarians (ca. 1225 CE), where horses, pearls, and dates are noted as being bartered for cloves, cardamom seeds, and camphor; cited in Kauz, “Horse Exports,” 131. 52 For more on networks of horse supply to Aden, see Vallet, L’Arabie marchande, 373–78. 53 Peel, Finlayson, and McMahon,“Updated World Map,” fig. 8. 51 See Vallet, L’Arabie marchande, 225–27, building on the work of Yokkaichi, “Horses,” 91–93, and earlier work by Hikoichi Yajima. Compare this to the earlier horse trade in Oman as described in Zhao Rugua’s Description of All Barbarians (ca. 1225 CE), where horses, pearls, and dates are noted as being bartered for cloves, cardamom seeds, and camphor; cited in Kauz, “Horse Exports,” 131. 53 Peel, Finlayson, and McMahon,“Updated World Map,” fig. 8. 52 For more on networks of horse supply to Aden, see Vallet, L’Arabie marchande, 373–78. eliZabeTh laMboURN 70 ent prices at different ports, while another document fixes the price of a horse at 800 dinars, stating that “horses worth 2,000 dinars, 1,000 and 500, 600, 400, 300, 200, all are sold for 800 [dinars], which has become a customary rule.”48 Neverthe- less, the precise mechanics of exchange for the Yemen are well understood thanks to Eric Vallet’s work on the Rasulid customs documents. These indicate that the dynasty operated a highly centralized system, at least in theory: the kingdom’s only sanctioned horsefair took place once a year in Aden during the month of August, a period known as mawsim al-khayl, the “horse season.”49 Those purchas- ing horses on behalf of Indian polities are described simply as nakhuda al-hind, the ship-owners and great merchants of India, and they settled their purchases and the taxes and dues, half in coin (using silver dirhams) and half in unsewn silk tex- tiles (fuwwat harīr).50 Vallet has also demonstrated how problematic the payment of such large sums of silver was for Indian buyers and has mapped the hugely complex chain of interregional exchanges, from China through to Egypt, which underlay these purchases.51 A comparable network analysis of the horses-for-gold system operating between Ilkhanid Iran and Pandya south India is long overdue. The Rasulid documents do not record the numbers of horses exported annually to India, or the annual revenue from this trade, but they do specify where some of the animals shipped from Aden were raised. Notwithstanding the fame of the “Ara- bian” horse, only select regions in the Arabian Peninsula are suitable for horse- breeding, and one document suggests that Yemeni horses were mainly bred on the great plains around San‘a and Damar to the north of Aden, and on the high coastal plateau of Hasi to its east.52 These regions are comparatively small compared to the semiarid regions found across Iran and Central Asia; so small, in fact, that they are not represented on current Köppen-Geiger maps of the Arabian Peninsula, and they cannot have sustained horse populations on the same scale.53 Although the modern notion of “breed” does not translate back to these prescientific, premod- ern milieus, it is clear that these areas produced a variety of horses. Polo gives by 51 See Vallet, L’Arabie marchande, 225–27, building on the work of Yokkaichi, “Horses,” 91–93, and earlier work by Hikoichi Yajima. 48 Jazim, Nar al-ma‘arif, 1:189–90; discussed in Vallet, L’Arabie marchande, 226. 49 Vallet, L’Arabie marchande, 223–24. 48 Jazim, Nar al-ma‘arif, 1:189–90; discussed in Vallet, L’Arabie marchande, 226. Sainte-Palaye et al., Dictionnaire historique, 9:378, “cheval etre deux selles”: a horse that is neither a courtault (work horse) nor a destrier. (The Tuscan version of Polo’s text is much shorter here and does not include the same amount of detail as included in the Franco-Italian.) 56 Polo, Milione, 608: “il hi portent maint buen destrer arabien [e] d’autres contree.” 57 This is in sharp contrast to northern India, where Delhi Sultanate sources provide the bulk of material for Digby’s seminal study War-Horse and Elephant . 57 This is in sharp contrast to northern India, where Delhi Sultanate sources provide the bulk of material for Digby’s seminal study War-Horse and Elephant . 58 See the Prabandhacintamani of Merutunga, cited in Chakravarti, “Equestrian Demand and Dealers,” 159. 59 See Chau Ju-Kua, Chu-Fan-Chi, 73. Some Middle Eastern horses even traveled the entire length of the sea route to eastern China. See, for example, Yokkaichi, “Horses,” 89–90, citing white horses brought back to China from Hormuz by the Chinese merchant emissary Yang Shu and a “western horse” presented by the Muslim merchant of Quanzhou Muhammad Andi to the Yuan court; the merchant’s residence at the eastern port city suggests that the horse was transported by sea rather than driven overland. 59 See Chau Ju-Kua, Chu-Fan-Chi, 73. Some Middle Eastern horses even traveled the entire length of the sea route to eastern China. See, for example, Yokkaichi, “Horses,” 89–90, citing white horses brought back to China from Hormuz by the Chinese merchant emissary Yang Shu and a “western horse” presented by the Muslim merchant of Quanzhou Muhammad Andi to the Yuan court; the merchant’s residence at the eastern port city suggests that the horse was transported by sea rather than driven overland. 55 Polo, Milione, 606: “portent les mercant mant buens detrier et manti buens chavalz de II selles en Endie, que molt sunt chier e de grant vailance;” cf. Sainte-Palaye et al., Dictionnaire historique, 9:378, “cheval etre deux selles”: a horse that is neither a courtault (work horse) nor a destrier. (The Tuscan version of Polo’s text is much shorter here and does not include the same amount of detail as included in the Franco-Italian.) 56 Polo, Milione, 608: “il hi portent maint buen destrer arabien [e] d’autres contree.” 54 Polo, Milione, 605: “maint biaus destrer arabien de grant vailance, dont les mercaant en font grant profit.” 57 This is in sharp contrast to northern India, where Delhi Sultanate sources provide the bulk of material for Digby’s seminal study War-Horse and Elephant . 58 See the Prabandhacintamani of Merutunga, cited in Chakravarti, “Equestrian Demand and Dealers,” 159. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 71 far the most detailed account of horse exports from the Arabian Peninsula, not- ing that merchants from Aden carry to India “many beautiful Arab destriers [war- horses] of great value, from which the merchants turn a great profit.” 54 From Shihr “merchants carry many good destriers and many good mixed-breeds [chevalz de II selles] to India, which are very expensive and very valuable.”55 Further north, at Dhofar, “they bring many good Arabian destriers from other countries,” which profit merchants greatly.56 It is unclear at present where the horses exported from Shihr and Dhofar were raised, but it seems clear that Rasulid Yemen would have been unable to supply Peninsular India on the same scale as Iran. Unfortunately, Indic sources for Peninsular India are almost totally silent about the horse economy and seldom specify where horses originated, let alone in what numbers.57 We are left with a string of general references to a contemporary trade in horses all along the western seaboard of the subcontinent, from Solanki Gujarat (where a Jain text describes the merchant minister Vastupala supervising the unloading of horses from ships at the port of Cambay)58 to Sri Lanka (where a mid-thirteenth-century Chinese source, the Description of the Barbarous Peoples, mentions horses traded at the island’s ports).59 However, we should not forget to look east for evidence. Polities such as the Pandyas and their northerly neighbors the Kakatiyas controlled ports on the Coromandel coast that were more immedi- ately tied into Bay of Bengal maritime networks. While horses from the Arabian Peninsula and Iran certainly reached Pandya ports via the maritime route, we 54 Polo, Milione, 605: “maint biaus destrer arabien de grant vailance, dont les mercaant en font grant profit.” 55 Polo, Milione, 606: “portent les mercant mant buens detrier et manti buens chavalz de II selles en Endie, que molt sunt chier e de grant vailance;” cf. Sainte-Palaye et al., Dictionnaire historique, 9:378, “cheval etre deux selles”: a horse that is neither a courtault (work horse) nor a destrier. (The Tuscan version of Polo’s text is much shorter here and does not include the same amount of detail as included in the Franco-Italian.) 55 Polo, Milione, 606: “portent les mercant mant buens detrier et manti buens chavalz de II selles en Endie, que molt sunt chier e de grant vailance;” cf. eliZabeTh laMboURN 61 Digby is careful to underline that these mountain ponies were different from so-called Tangana (Tanghan) horses, with which they are often confused; War-Horse and Elephant, 47. 62 Chakravarti, “Early Medieval Bengal,” 201–02. Important visual evidence not cited by Chakravarti are representations of Southeast Asian, perhaps Thai, horse traders depicted together with horses and boats in late sixteenth-century murals from the temple at Tirup- putaimarutur in the Tirunelveli District of Tamil Nadu; reproduced in Deloche, “Etudes.” 62 Chakravarti, “Early Medieval Bengal,” 201–02. Important visual evidence not cited by Chakravarti are representations of Southeast Asian, perhaps Thai, horse traders depicted together with horses and boats in late sixteenth-century murals from the temple at Tirup- putaimarutur in the Tirunelveli District of Tamil Nadu; reproduced in Deloche, “Etudes.” 63 Cited in Yang, “Horses, Silver, and Cowries,” 300; from another edition of Polo’s travels, see Polo, Travels, 10. 63 Cited in Yang, “Horses, Silver, and Cowries,” 300; from another edition of Polo’s travels, see Polo, Travels, 10. 64 Amir Khusraw Dihlavi, cited in Digby, War-Horse and Elephant, 48. 60 See Chakravarti, “Early Medieval Bengal,” and Yang, “Horses, Silver, and Cowries,” 294–300; the latter offers a comprehensive discussion of this trade incorporating Chakra- varti’s evidence for Bengal. 64 Amir Khusraw Dihlavi, cited in Digby, War-Horse and Elephant, 48. 61 Digby is careful to underline that these mountain ponies were different from so-called Tangana (Tanghan) horses, with which they are often confused; War-Horse and Elephant, 47. eliZabeTh laMboURN 72 should not discount the possibility that these polities also turned to other, closer horse-breeding areas. Important research by Ranabir Chakravarti and Bin Yang has suggested that horses from the eastern Himalayas and southwestern China entered the Bay of Bengal trading system.60 The region of Karambattan in the eastern Himalayas (likely corresponding to modern-day Bhutan or the fringes of Tibet) raised small hardy horses, what we would now term “ponies,” known sim- ply as kohī (mountain) horses.61 The unique climate of Yunnan in southwestern China had allowed horse husbandry to develop there from as early as the sixth century BCE. Horses from both regions were led into northern India via Bengal and, according to Chakravarti, were often re-exported to the eastern Deccan and even onwards to eastern Chinese ports.62 While the strongest evidence for this trade begins in the fifteenth century, there is scattered earlier evidence for similar patterns, notably for the thirteenth century, when Marco Polo again provides important testimony by observing that Amu (Yunnan) produced horses and oxen that were traded into India via Pagan and Bengal.63 Although it is likely that the larger horses from the Middle East were considered better suited to warfare than Himalayan and Yunnanese “ponies,” we cannot exclude the Bay of Bengal system as a source of horses for the Kakatiyas and even for the Pandyas to their south. When the armies of the Delhi Sultanate conquered the Kakatiya capital of Warangal in 1309, the Kakatiya ruler Rudradeva reportedly surrendered twenty thousand horses, a mix of kohī ponies from the Himalayas and bahrī horses, in this case almost certainly Arabian and Persian horses that had reached South Asia by sea.64 A proportion of these animals would certainly have been acquired as booty in engagements with neighboring poli- ties—this was always the fastest and cheapest way to obtain new horses, as the very surrender of these animals to the conquering forces of the Delhi Sultanate illustrates—but we should not dismiss the possibility that a proportion of these 60 See Chakravarti, “Early Medieval Bengal,” and Yang, “Horses, Silver, and Cowries,” 294–300; the latter offers a comprehensive discussion of this trade incorporating Chakra- varti’s evidence for Bengal. 61 Digby is careful to underline that these mountain ponies were different from so-called Tangana (Tanghan) horses, with which they are often confused; War-Horse and Elephant, 47. 60 See Chakravarti, “Early Medieval Bengal,” and Yang, “Horses, Silver, and Cowries,” 294–300; the latter offers a comprehensive discussion of this trade incorporating Chakra- varti’s evidence for Bengal. 61 Digby is careful to underline that these mountain ponies were different from so-called Tangana (Tanghan) horses, with which they are often confused; War-Horse and Elephant, 47. 62 Chakravarti, “Early Medieval Bengal,” 201–02. Important visual evidence not cited by Chakravarti are representations of Southeast Asian, perhaps Thai, horse traders depicted together with horses and boats in late sixteenth-century murals from the temple at Tirup- putaimarutur in the Tirunelveli District of Tamil Nadu; reproduced in Deloche, “Etudes.” 63 Cited in Yang, “Horses, Silver, and Cowries,” 300; from another edition of Polo’s travels, see Polo, Travels, 10. 64 Amir Khusraw Dihlavi, cited in Digby, War-Horse and Elephant, 48. The Birth of the Bahrī Trade Multiple sources, from Syria and the Yemen to China, point to the emergence of the horse as a major international commodity in Indian Ocean trade to Peninsular India by the later thirteenth century, an expenditure so important, according to some accounts, that it consumed the greater part of state revenues. While this trade certainly did not start ex nihilo, fixing a more precise “when” and “why” to this horsemania is by no means easy. Premodern sources for the Middle East and South Asia usually yield quantitative data only reluctantly—hence the attraction of the much repeated Il Khanid figures—but long-term fluctuations in the trade of a particular commodity are even more difficult to capture. Of all the sources noted above, only Vassaf gives some historical perspective on the horse trade from Fars and the Gulf, based on his knowledge of trade under the Salghurid Atabek of Fars, Abu Bakr (r. 1226–60), who was, for the latter part of his reign, a Mongol tributary.66 Vassaf notes that according to “authentic writers,” ten thousand horses a year were already being exported to Ma‘bar as well as to Kambayat (Cambay, the principal port of the Solanki kingdom) and other areas of India at this time for the same price of 220 dinars and under the same terms. 67 This is the clearest sugges- tion that the Tibi merchant princes of Kish may have taken over an existing trade agreement. If Vassaf is correct in this, this large-scale trade was already several generations old by the time he described it in the early fourteenth century. The 67 Vassaf, Tajziyat al-amsar, 33. Vassaf gives no indication of the pattern of horse trade between Abu Bakr’s death in 1260 and the Tibis’ takover in 1292; however, Aigle usefully sketches the outlines of a period of regional instability even if the consequences of this for the horse trade are unknown (Le Fārs sous la domination mongole, 113–36). ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 73 animals had also reached the Kakatiya polity as direct imports via the western and the eastern Indian Ocean. As all these examples underline, the large number of horses needed to supply Peninsular India had to be sourced from a wide area and through networks run- ning deep into the hinterlands of the actual shipping points. When Vassaf mentions horses from Qa’is and “the islands of Fars, Qatif, Lahsa [al-Ahsa], Bahrayn, Hormuz, Qalhat,” these were simply assembly points, perhaps formal horse fairs, for ani- mals that had been raised across the grasslands of Arabia and Iran. Indeed, “sea horses” first enter Polo’s narrative in his description of the kingdom of Persia and the importance of Kish and Hormuz in the export of good Persian horses to India.65 65 Polo, Milione, 342. 66 See discussion of the conflicting evidence in Aigle, Le Fārs sous la domination mongole, 101n241. 67 Vassaf, Tajziyat al-amsar, 33. Vassaf gives no indication of the pattern of horse trade between Abu Bakr’s death in 1260 and the Tibis’ takover in 1292; however, Aigle usefully sketches the outlines of a period of regional instability even if the consequences of this for the horse trade are unknown (Le Fārs sous la domination mongole, 113–36). 67 Vassaf, Tajziyat al-amsar, 33. Vassaf gives no indication of the pattern of horse trade between Abu Bakr’s death in 1260 and the Tibis’ takover in 1292; however, Aigle usefully sketches the outlines of a period of regional instability even if the consequences of this for the horse trade are unknown (Le Fārs sous la domination mongole, 113–36). 66 See discussion of the conflicting evidence in Aigle, Le Fārs sous la domination mongole, 101n241. 65 Polo, Milione, 342. eliZabeTh laMboURN 74 fact that the various polities of Fars were never conquered by the Mongols but entered tributary relationships with them would certainly have encouraged conti- nuity in local economic patterns. If a large-scale maritime trade in horses does go back to the mid- or even early thirteenth century, what had perhaps changed by the end of that century, at least with regards to the Gulf, was the distribution network. The mid-thirteenth-century Gulf trade, as described by Vassaf, supplied ten thousand horses per annum to a variety of locations in India, including Gujarat; however, by the early fourteenth century Vassaf and Rashid al-Din appear to describe a trade of around the same volume but this time apparently focused exclusively on supplying the Pandya pol- ity. If such a southwards shift is confirmed, it may reflect the growth of the mari- time trade route from the 1270s onwards due to Chagadai disruptions in central Asia, increased contact between Yuan China and Il Khanid Iran via this maritime route, and South India’s growing importance as a participant in these networks.68 Although the Mongol conquest of Iran eventually encouraged and facilitated the export of horses to South India by sea, that does not explain in itself the rise in demand. A number of historians, from Simon Digby to Ralph Kauz, have suggested a clear correlation between the general growth of the bahrī horse trade in the thir- teenth century and the challenge posed by the large cavalries of the Delhi Sultan- ate, which led to a “general structural militarization of the Indian subcontinent.”69 Bahrī horses it seems were part of a wider South Asian “arms race” precipitated If a large-scale maritime trade in horses does go back to the mid- or even early thirteenth century, what had perhaps changed by the end of that century, at least with regards to the Gulf, was the distribution network. The mid-thirteenth-century Gulf trade, as described by Vassaf, supplied ten thousand horses per annum to a variety of locations in India, including Gujarat; however, by the early fourteenth century Vassaf and Rashid al-Din appear to describe a trade of around the same volume but this time apparently focused exclusively on supplying the Pandya pol- ity. eliZabeTh laMboURN Digby carried out probably the most thorough assessment of military technologies and supplies for late twelfth-century India and concludes that there existed a general parity in military technologies between the Delhi Sultanate forces and their Indian oppo- nents; for Digby, the sole factor that explains the success of the Delhi Sultanate’s forces is the larger numbers of mounted troops, particularly archers, they could muster.70 Digby’s thesis is strongly supported by evidence from a variety of South Asian sources and would explain why horses became the single most important military import for other South Asian polities, notably those of the Peninsular 68 This Mongol period maritime axis is explored especially thoroughly by Kauz, “Horse Exports,” and by Yokkaichi, “Horses.” South India’s new centrality in these exchanges is certainly seen in the Tibis’ strategic positioning within the Pandya polity, but also in the over thirty diplomatic missions exchanged between Yuan China and South Indian polities between 1272 and 1296; see Sen, “Yuan Khanate and India,” 302. 69 Kauz, “Horse Exports,” 131–32: horse export became “a major component of the Indian Ocean trade from the thirteenth century onwards.” 69 Kauz, “Horse Exports,” 131–32: horse export became “a major component of the Indian Ocean trade from the thirteenth century onwards.” 70 Dibgy, War-Horse and Elephant, 13–22; see also the discussion of warfare at this period in the introduction to Gommans and Kolff, Warfare and Weaponry, 1–42. eliZabeTh laMboURN If such a southwards shift is confirmed, it may reflect the growth of the mari- time trade route from the 1270s onwards due to Chagadai disruptions in central Asia, increased contact between Yuan China and Il Khanid Iran via this maritime route, and South India’s growing importance as a participant in these networks.68 Although the Mongol conquest of Iran eventually encouraged and facilitated the export of horses to South India by sea, that does not explain in itself the rise in demand. A number of historians, from Simon Digby to Ralph Kauz, have suggested a clear correlation between the general growth of the bahrī horse trade in the thir- teenth century and the challenge posed by the large cavalries of the Delhi Sultan- ate, which led to a “general structural militarization of the Indian subcontinent.”69 Bahrī horses, it seems, were part of a wider South Asian “arms race” precipitated by the Ghurid conquest of northern India in the late twelfth century. Digby carried out probably the most thorough assessment of military technologies and supplies for late twelfth-century India and concludes that there existed a general parity in military technologies between the Delhi Sultanate forces and their Indian oppo- nents; for Digby, the sole factor that explains the success of the Delhi Sultanate’s forces is the larger numbers of mounted troops, particularly archers, they could muster.70 Digby’s thesis is strongly supported by evidence from a variety of South Asian sources and would explain why horses became the single most important military import for other South Asian polities, notably those of the Peninsular Bahrī horses, it seems, were part of a wider South Asian “arms race” precipitated by the Ghurid conquest of northern India in the late twelfth century. 68 This Mongol period maritime axis is explored especially thoroughly by Kauz, “Horse Exports,” and by Yokkaichi, “Horses.” South India’s new centrality in these exchanges is certainly seen in the Tibis’ strategic positioning within the Pandya polity, but also in the over thirty diplomatic missions exchanged between Yuan China and South Indian polities between 1272 and 1296; see Sen, “Yuan Khanate and India,” 302. 69 Kauz, “Horse Exports,” 131–32: horse export became “a major component of the Indian Ocean trade from the thirteenth century onwards.” 68 This Mongol period maritime axis is explored especially thoroughly by Kauz, “Horse Exports,” and by Yokkaichi, “Horses.” South India’s new centrality in these exchanges is certainly seen in the Tibis’ strategic positioning within the Pandya polity, but also in the over thirty diplomatic missions exchanged between Yuan China and South Indian polities between 1272 and 1296; see Sen, “Yuan Khanate and India,” 302. 69 Kauz, “Horse Exports,” 131–32: horse export became “a major component of the Indian Ocean trade from the thirteenth century onwards.” 70 Dibgy, War-Horse and Elephant, 13–22; see also the discussion of warfare at this period in the introduction to Gommans and Kolff, Warfare and Weaponry, 1–42. 70 Dibgy, War-Horse and Elephant, 13–22; see also the discussion of warfare at this period in the introduction to Gommans and Kolff, Warfare and Weaponry, 1–42. THIS DOCUMENT MAY BE PRINTED FOR PRIVATE USE ONLY AND MAY NOT BE DISTRIBUTED WITHOUT PERMISSION FROM THE PUBLISHER. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 75 ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 75 75 Figure 5. View of the Lower Level of the Kesava Temple at Somanathapur in Karnataka, Hoysala, ca. 1268 CE. In a distinctive Hoysala iconographic inno vation, a frieze of mounted horsemen has been added to the more traditional reper tory of elephant, goose, and makara friezes. Photograph reproduced courtesy of the American Institute of Indian Studies, Accession No 15366. Figure 5. View of the Lower Level of the Kesava Temple at Somanathapur in Karnataka, Hoysala, ca. 1268 CE. In a distinctive Hoysala iconographic inno vation, a frieze of mounted horsemen has been added to the more traditional reper tory of elephant, goose, and makara friezes. Photograph reproduced courtesy of the American Institute of Indian Studies, Accession No 15366. south, which were disadvantaged by both climate and distance from horse pro- ducing areas. Nevertheless, horses alone do not win battles: they are surrounded by larger assemblages of people and things, and it is this complex of factors that makes win- ning cavalry units. There is much we still do not know about the chronology of militarization in the Peninsular south and the place of horses within this. Indic sources will have an important role to play in clarifying this even if, at present, their use poses many challenges. Epigraphy, the mainstay of so much premod- ern South Asian history, has not yet been combed systematically for references to horses in the Peninsular south, while visual evidence is unevenly documented: some sites have been meticulously photographed and recorded, others are known eliZabeTh laMboURN 76 only through brief and often passing textual references, and nearly all are poorly studied. In the future, systematic surveys of both types of evidence may be able to uncover patterns that substantially clarify when and where in Peninsular India bahrī horses first became a major imported commodity, and when new equine technologies and perhaps skilled humans accompanied them. For the moment, the evidence we have is suggestive rather than conclusive. Perhaps some of the strongest evidence for understanding changes to the flow of horses in the thirteenth century is iconographic: Hoysala temple sculpture offers rich material. Friezes of prancing horses or mounted combat appear sud- denly on the basement walls of Hoysala temples from the early twelfth century onwards, where they are integrated with a more traditional repertory of elephant, lion, or makara friezes (Figure 5). Horse friezes were an iconographic innovation unique to Hoysala sculptors, a pointer to the high status of the horse and cavalry within that society. However, the first half of the thirteenth century stands out as a particularly intense period of horse representation; of the twelve temples with equine friezes studied by the military historian Jean Deloche, ten date between 1219 and 1268 CE,71 suggesting that horses had become, or were becoming, far more prominent and valued in Hoysala society at this period. Within this time- frame, the middle of the century stands out as particularly significant. As Deloche demonstrates, Hoysala sculpture began to represent a number of distinctively Turkic-Islamic equine technologies, such as stirrups, horseshoes, horse armor, and (most importantly) a new type of saddle with pommel and cantle that held the rider more securely than previous designs. The historian Philip Wagoner has suggested that these representations show “the first step in the adaptation of Tur- kic-Islamicate military technologies by local Indic military elites,” resulting from the Turkic conquest of northern India.72 Yet the Hoysala had not yet engaged the Delhi Sultanate in battle directly at this period, suggesting different mechanisms of exchange. In many scholarly discussions, direct military engagement is under- stood to be a necessary prequel to the transfer of military technologies, via cap- tured horses, horse equipment, or fighting men; and yet in the Peninsular south these changes appear to have occurred prior to direct military engagement. 71 Among the earliest examples is the Kesava temple at Beluru (Hasan district in the modern Karnataka state), dated to 1117 CE. See the (by no means comprehensive) table in Deloche, “Techniques militaires,” 149. A proper evaluation would require a complete inventory of Hoysala temples and their decoration, and an investigation perhaps of any relationships between the patron and the chosen iconography. 72 Wagoner, “Harihara, Bukka, and the Sultan,” 317. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 77 ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 77 Figure 6. Hero Stone Showing a Single Mounted Horseman in Battle, from Tripuran- takam, Kurnool District, Andhra Pradesh (Kakatiya Period, Thirteenth Century CE). This imagery is more commonly seen in western India and indicates the distinctive regional equine culture of the Andhra region at this period. Photograph reproduced with kind per mis- sion of Professor R. Chandrasekhara Reddy. Figure 6. Hero Stone Showing a Single Mounted Horseman in Battle, from Tripuran- takam, Kurnool District, Andhra Pradesh (Kakatiya Period, Thirteenth Century CE). This imagery is more commonly seen in western India and indicates the distinctive regional equine culture of the Andhra region at this period. Photograph reproduced with kind per mis- sion of Professor R. Chandrasekhara Reddy. 78 eliZabeTh laMboURN 78 It is important to bear in mind that incidental evidence for economic and tech- nological phenomena such as this can never be entirely reliable. Visual representa- tions follow their own conventions, fashions, and timescales: they do not represent the “real” world, let alone aim to supply economic historians with usable quan- titative data, and it is impossible to gauge at present the speed at which Hoysala sculptors reacted to social and technological changes.73 The introduction of a new equine iconography and its increased use arguably tell us more about the changing status of the horse in Hoysala society or its changed visibility than about the num- bers of animals imported or about the timing of technological transfers. Deloche also did not systematically examine evidence from Hoysala hero stones, which are clearly iconographically related to temple friezes, and such a study might help refine or revise his thesis (for a beautifully preserved Kakativa example, see Fig- ure 6).74 Nevertheless, if this interpretation of the Hoysala evidence is correct, it suggests a rise in the presence and status of the horse during the first half of the thirteenth century with increased adoption of certain Turkic-Islamic military tech- nologies from the middle of the century. This timeframe correlates broadly with Vassaf’s information about a large-scale horse trade from the Gulf to South Asia under Atabek Abu Bakr. The combined evidence further suggests that knowledge of military technolo- gies circulated within South Asia and between South Asia and surrounding regions before any military confrontation, indicating a phenomenon of preemptive rather than reactive technological change. Wagoner’s work on the fifteenth-century Dec- can has certainly underlined the mobility of military elites, notably Turkic Mus- lim nobles, across political and religious frontiers, and it is perhaps thanks to similar mobilities that Turkic-Islamic equine technologies traveled south through the Peninsula.75 We should also not forget that a wider Turkic-Islamic equine cul- ture had established itself across much of the central and eastern Islamic world since the first large-scale use of mounted Turkish slave soldiers by the Abbasid Caliphs in the ninth century. Subsequent waves of Turkic invasion consolidated the Turkic contribution to Islamic culture. In the thirteenth century, the “new” horse furnishings represented in Hoysala sculpture were as common in Iran and 73 Digby maintains that stirrups had been introduced across India much earlier, at least by the mid-tenth century; War-Horse and Elephant, 13–14. This raises the question of why stirrups appear so much later on Hoysala sculptures. 73 Digby maintains that stirrups had been introduced across India much earlier, at least by the mid-tenth century; War-Horse and Elephant, 13–14. This raises the question of why stirrups appear so much later on Hoysala sculptures. 74 The potential here is huge. In Memorial Stones, Settar and Sontheimer document 609 hero-stones from Karnataka, of which 363 are of the Hoysala period. 75 See Wagoner, “Fortuitous Convergences”; for the visual evidence, see Michell, “Migra- tions and Cultural Transmissions.” 74 The potential here is huge. In Memorial Stones, Settar and Sontheimer document 609 hero-stones from Karnataka, of which 363 are of the Hoysala period. stirrups appear so much later on Hoysala sculptures. 74 The potential here is huge. In Memorial Stones, Settar and Sontheimer document 609 hero-stones from Karnataka, of which 363 are of the Hoysala period. 75 See Wagoner, “Fortuitous Convergences”; for the visual evidence, see Michell, “Migra- tions and Cultural Transmissions.” 75 See Wagoner, “Fortuitous Convergences”; for the visual evidence, see Michell, “Migra- tions and Cultural Transmissions.” ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 79 the Arabian Peninsula as in the Delhi Sultanate. It is entirely possible, therefore, that these technologies were also transmitted to the Hoysala through the trade in bahrī horses from the Gulf or Yemen. Indian nākhūdas at Aden’s annual horse fair certainly came into direct contact with Turkic-Islamic equine cultures. The diplomatic exchanges that accompanied the wider Indian Ocean trade may also have assisted in the circulation of equine technologies, as it was common to send fully caparisoned horses as diplomatic gifts. Although we have as yet no con- crete examples of gifted horses from thirteenth-century Middle Eastern or Indic sources, the diplomatic activity of the period is well attested, and such a scenario is entirely possible. This is fragile data, but it points a way forward. Nevertheless, it remains impor- tant to calibrate both epigraphic and visual evidence carefully, and to be aware of their fundamentally recalcitrant natures. One of the most paradoxical aspects of horsemania in thirteenth-century South India remains the fact that, in spite of the Pandyas’ clear preeminence as importers of bahrī horses according to Yemeni, Iranian, and European sources, neither epigraphic nor visual sources hint at that dominance. References to horses as commodities and taxable items appear to be almost nonexistent in Pandya inscriptions: the historian Nilakantha Sastri is able to signal only one reference to a horse trader from southern Kerala in his history of the polity.76 Nor did horses enter Pandya visual culture. They are not represented in temple sculpture or on hero stones, the memorials carved and often inscribed to commemorate dead warriors. Without the Middle Eastern sources and Marco Polo, there would be little trace of the Pandya consumption of bahrī horses. Equine Knowledges and Civilizational Encounters The facts and figures on the bahrī horse trade provided by Vassaf, Polo, and others are rare for the thirteenth century, and it is perhaps no surprise that historians have clung to them. In so doing, however, they have largely ignored the discur- sive contexts in which these facts are situated. Restoring this data to its original context offers a remarkably effective means of using these sources as part of a broader history of equine cultures and illustrates the extent to which, well before the eighteenth century, horses were vehicles for other ideas. In the case of both Vassaf and Marco Polo, the fate of horses in South India offers each the opportu- nity to develop complex and contradictory discourses that attribute to horses an almost emblematic civilizational role. 76 See Nilakantha Sastri, Pandyan Kingdom, 192 (no. 161 of 1907). 76 See Nilakantha Sastri, Pandyan Kingdom, 192 (no. 161 of 1907). 0 eliZabeTh laMboURN 80 In Polo’s narrative, observations about horses are woven into a larger narra- tive fabric intent on pointing to Oriental, sometimes specifically Muslim, excess and ignorance. Although horses feature only briefly in his description of the main shipment point of Hormuz, the context in which they feature is highly significant: horses are included in a longer, disparaging description of the coir-sewn boats so typical of the western Indian Ocean. Because “they,” the Muslim merchants or perhaps the Indian shipmasters, do not have iron nails, the boats are sewn, and they are “very bad and many founder”; the loaded merchandise is simply covered with hides and onto this “they put the horses which they carry to India to sell.”77 According to Polo, Indian Ocean boat-building techniques are primitive, and understanding of lading methods is similarly backward. This passage sets the tone for the longest discussion of the bahrī trade, embedded in Polo’s description of the kingdom of the Pandyas. Having told his readers that this kingdom consumes most of its revenues on buying horses, and that these are good, valuable animals, he continues: they all die, because they [the Indians] do not have ostlers [marreschaus],78 nor know how to care for them, but they die though neglect. And I tell you that the merchants who carry these horses for sale do not allow to go, nor send, any ostlers, because they [the merchants] want these kings’ horses to die.79 Indian ignorance of horse care is here compounded by the exploitative practices of merchants from Muslim lands who deliberately impede knowledge circulation in order to ensure the early deaths of these animals. Eastern horses were rare luxuries in Europe, even into the sixteenth century, and to squander rare animals on such a scale—furthermore, transporting them in primitive ships—must have been shocking to Polo’s readers, perhaps to Polo himself. Polo’s narrative antici- pates many themes familiar from later Orientalist discourse, and this very famili- arity has perhaps aided an uncritical acceptance in later Western scholarship of the idea that the bahrī horse trade was impractical and even foolhardy, and that Indians did not know how to care for horses. Indian ignorance of horse care is here compounded by the exploitative practices of merchants from Muslim lands who deliberately impede knowledge circulation in order to ensure the early deaths of these animals. 0 eliZabeTh laMboURN Eastern horses were rare luxuries in Europe, even into the sixteenth century, and to squander rare animals on such a scale—furthermore, transporting them in primitive ships—must have been shocking to Polo’s readers, perhaps to Polo himself. Polo’s narrative antici- pates many themes familiar from later Orientalist discourse, and this very famili- arity has perhaps aided an uncritical acceptance in later Western scholarship of the idea that the bahrī horse trade was impractical and even foolhardy, and that Indians did not know how to care for horses. 77 Polo, Milione, 348: “Lor nes sunt mout mauves et ne perisent aseç”; “hi metent les cavaus qui portenbt en Yndie a vendre.” 78 At this period, the maréchal was responsible for all aspects of the care of horses, in- cluding the treatment of ailments and shoeing, and even their sale. 78 At this period, the maréchal was responsible for all aspects of the care of horses, in- cluding the treatment of ailments and shoeing, and even their sale. 79 Polo, Milione, 348: “tuit morent elz, por ce que il ne ont marreschaus, ne ne li sevent costoir, mes se morent por mal garde. E si voç di que les mercaant que portent cesti chavaus a vendre, ne[n] hi laisen ale[r] ne ne i moinent nul marescaus, por ce qu’il vuelt que les chavaus se morent aseç a cesti rois.” 77 Polo, Milione, 348: “Lor nes sunt mout mauves et ne perisent aseç”; “hi metent les cavaus qui portenbt en Yndie a vendre.” 77 Polo, Milione, 348: “Lor nes sunt mout mauves et ne perisent aseç”; “hi metent les cavaus qui portenbt en Yndie a vendre.” 78 At this period, the maréchal was responsible for all aspects of the care of horses, in- cluding the treatment of ailments and shoeing, and even their sale. 79 Polo, Milione, 348: “tuit morent elz, por ce que il ne ont marreschaus, ne ne li sevent costoir, mes se morent por mal garde. E si voç di que les mercaant que portent cesti chavaus a vendre, ne[n] hi laisen ale[r] ne ne i moinent nul marescaus, por ce qu’il vuelt que les chavaus se morent aseç a cesti rois.” ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 81 The Persian chronicler Vassaf offers a very different perspective on the prob- lem of horse mortality in South India and one that, again, must be situated within its original context. Vassaf’s focus is not the bahrī horse trade per se; rather, his discussion centers on the Tibi merchant family and is itself situated within a highly florid account of the “marvels and excellences” of the country of Hind—a discourse that betrays a clear lineage in the well-established tradition of the ‘ajā’ib, the “won- ders,” of non-Islamic lands.80 With the stock phrase “it is a strange thing,” he turns to peculiarly Indian practices of horse management, depicting what Finbarr Barry Flood has described elsewhere as a “tospy-turvy” Indian world where horses are neither fed, exercised, or ridden according to expected Middle Eastern practices.81 The result is that “in a short time the most strong, swift, fresh, and active horses become weak, slow, useless, and stupid.”82 Yet whereas Polo understands this to be a symptom of a wider Oriental backwardness, for Vassaf the fate of horses is ulti- mately explained as part of the divine natural order: it is predominantly “this cli- mate”—literally, “water and air” (īn āb va havā)—which renders horses “exceed- ingly weak and altogether worn out and unfit for riding,”83 and so explains the constant demand in South Asia. Vassaf concludes that their loss is not without its attendant advantages, for it is a providen- tial ordinance of God that the western [world] should continue to want eastern products, and the eastern world western products, and that the north should with labor procure the goods of the south, and the south be furnished in like manner with commodities brought in ships from the north.84 Horses are one of the commodities that mediate communication between the quarters of the earth, and the peculiar practices of their trade and “consumption” in south India are part of the “marvels and excellences” of the country of Hind. Horses are one of the commodities that mediate communication between the quarters of the earth, and the peculiar practices of their trade and “consumption” in south India are part of the “marvels and excellences” of the country of Hind. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia In both Polo and Vassaf, then, and yet in radically different ways, horses are vehicles for larger discourses about civilizational differences and values: about the encounter between Europe, the Middle East, and South Asia; and between Hindu, Muslim and Christian cultures. 80 Vassaf, Tazjiyatu-l amsar, 32. 81 Flood, Objects of Translation, 6. 82 Vassaf, Tazjiyatu-l amsar, 33. 83 Ibid., 34. 84 Ibid. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 80 Vassaf, Tazjiyatu-l amsar, 32. 81 Flood, Objects of Translation, 6. 82 Vassaf, Tazjiyatu-l amsar, 33. 83 Ibid., 34. 84 Ibid. 80 Vassaf, Tazjiyatu-l amsar, 32. Finding “Matters Equine” in South Indian Sources 86 This story comes from the sthalapurana of the Minakshi Temple in Madurai, Tamil Nadu, which depicts sixty-four stories about Siva, four of which are about Siva and Manikkavacakar. The story is paraphrased in multiple publications, but for a faithful summary of the Tamil text, see Cutler, Songs of Experience, 184. I am grateful to Leah Comeau for suggesting this story. 87 Ibid. eliZabeTh laMboURN 82 85 The Rasulid sultan al-Malik al-Mujahid (r. 1321–62) also authored a hippiatric text in which he noted that Arab horses did not breed in India and had a shorter lifespan; reported in Shehada, Mamluks and Animals, 266. 86 This story comes from the sthalapurana of the Minakshi Temple in Madurai, Tamil Nadu, which depicts sixty-four stories about Siva, four of which are about Siva and Manikkavacakar. The story is paraphrased in multiple publications, but for a faithful summary of the Tamil text, see Cutler, Songs of Experience, 184. I am grateful to Leah Comeau for suggesting this story. 87 Ibid. 85 The Rasulid sultan al-Malik al-Mujahid (r. 1321–62) also authored a hippiatric text in which he noted that Arab horses did not breed in India and had a shorter lifespan; reported in Shehada, Mamluks and Animals, 266. 86 This story comes from the sthalapurana of the Minakshi Temple in Madurai, Tamil Nadu, which depicts sixty-four stories about Siva, four of which are about Siva and Manikkavacakar. The story is paraphrased in multiple publications, but for a faithful summary of the Tamil text, see Cutler, Songs of Experience, 184. I am grateful to Leah Comeau for suggesting this story. 87 Ibid. Finding “Matters Equine” in South Indian Sources The seemingly obvious military destination of bahrī horses and their apparently brief Indian lives—mentioned by Polo, Vassaf, Rashid al-Din, and again in a later Yemeni treatise85—have hitherto limited the discussion of the reception and translation of horses into Peninsular India. Yet archaeological and visual evidence going back to the Megalithic period in South India indicates that, in the thirteenth century, these animals supplied already ancient regional equine cultures, even if the precise features and chronologies of these await further study. The problems that Indic sources pose for writing commodity histories of horses are understand- ably compounded in the attempt to move beyond this to broader equine histories. As noted above, epigraphic sources have not been systematically explored across the Peninsula, and little is known about the local circulation and eventual transla- tion of the classic Indian hippiatric treatises of SƵalihotra and Nakula, while liter- ary sources that might write the Indian lives of bahrī horses have similarly not been systematically sifted for equine references. Such a project would of neces- sity be collaborative, demanding at the very least expertise in Sanskrit, Kannada, Malayalam, Tamil, and Telugu. One example from the Tamil literature illustrates the potential of such mate- rial. A thirteenth-century narrative of the life of the ninth-century Tamil saint Man- ikkavacakar, the Tiruvilaiyatal puranam (The Divine Plays of Lord Shiva), sets the story of his entry into the religious life in a markedly contemporary, thirteenth- century context.86 Manikkavacakar was chief minister to the Pandya monarch and as such was sent to a port to purchase horses for the royal stables. En route, the future saint encountered SƵiva in the form of a guru and decided to devote himself to the SƵaiva cause. Returning to court without money or horses, Manikkavacakar was duly punished. Later, however, SƵiva arrived at court “in the guise of a foreign horseman leading a herd of fine horses to the Pantiyan’s stable.”87 The “horses,” however, were really wild jackals, and later they reverted to their original form and caused havoc within the palace. The story offers a subaltern view of both the wastefulness of the Pandya ruler’s spending on horses and the foreign dominance 85 The Rasulid sultan al-Malik al-Mujahid (r. 1321–62) also authored a hippiatric text in which he noted that Arab horses did not breed in India and had a shorter lifespan; reported in Shehada, Mamluks and Animals, 266. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 83 of that trade, but it also reprises the idea that horses were alien, terrifying crea- tures. Jackals in India are closely associated with death because of their scaveng- ing of corpses. Material sources are even more patchily documented and studied than such infor mative anecdotes. Surveys and excavations of elite architectural complexes and urban spaces have rarely looked for evidence of the material culture of horse riding and horse keeping (such as stables and training or parade areas). Also, because monsoon climate conditions complicate the survival of all but the most stable materials (such as stone and ceramics), faunal remains are rarely collected and researched during archaeological excavations. Compounding this problem, archaeologists have displayed a clear bias in favor of the Megalithic and Early Historic periods. Yet the work carried out at a regional or dynastic level provides models that might be transferable to the wider canvas of the Peninsular south. Although Deloche’s work has focused on using visual sources to index military- technological change, it has also traced the development of a unique visual cul- ture of equine representation among Hoysala craftsmen and, we must presume, their patrons. Further work on Hoysala hero stones and on equine references in Hoysala epigraphy would not only refine Deloche’s principal thesis but provide a working model for transfer to other regions and polities—in the process reveal- ing regional interactions, convergences, and divergences. Even more significant is the work of historian Cynthia Talbot on the Kakatiya polity, its military networks, and what she terms the “martial ethos” in medieval South Indian society. Together with the work of a small number of other historians, this work challenges prevail- ing models of medieval Indic kingship by emphasizing the place of military action and training in South Asian society.88 For Talbot, the models of so-called “dhar- mic kingship” favored by Western scholars fail to take account of the truism that “political power has a physical basis in armed might.”89 The central importance of cavalry units in the Kakatiya army and the elite status of those warriors inev- itably makes horses a recurring leitmotif of Talbot’s study. Although she works solely from Kakatiya epigraphic sources and does not engage with the rich body of equine representations on contemporary Kakatiya hero stones (Figure 6), Talbot provides valuable new models for the writing of broader equine histories across the Peninsular south. 88 See also the essay by Yadava, “Chivalry and Warfare.” 89 Talbot, Precolonial India, 144. From dharma (virtuous duty), this model sees the king as bound to protect his realm and ensure the peaceful co-existence of those living within it. 88 See also the essay by Yadava, “Chivalry and Warfare.” 89 Talbot, Precolonial India, 144. From dharma (virtuous duty), this model sees the king as bound to protect his realm and ensure the peaceful co-existence of those living within it. 4 eliZabeTh laMboURN 84 Horses, Horse Merchants, and the “Web of Military Associations” Talbot’s research has underlined the importance of thinking of polities as military networks: because a king’s own martial skills and his ability to recruit and retain the loyalties of other fighting men were essential to his survival, the web of military associations underlying every state system should be prominent in our models of […] medieval polity.90 Her analysis focuses on the personal networks forged among the Kakatiya ruler, regional governors, local chiefs, and their supporters, but it is clear that other human networks supported the maintenance of power, notably through access to essential military supplies. If understanding of the place of horses and horse merchants in the Kakatiya’s “web of military associations” must await further study, Talbot’s model translates well to the more abundant data we have for some of their contemporaries. The Indian nākhūdas who operated as middlemen in the trade between Rasulid Yemen and the Hoysalas can be seen as fundamental participants in the Hoysala’s own “web of military associations” through their participation in the purchase and transport of military supplies, notably horses. South Asian models of merchant activity (like models of kingship) have tended to neglect the prominence of armed conflict in merchant lives. The highly com- petitive and often violent character of Indian Ocean trade meant that ships regu- larly carried armed men or traveled in convoys with armed protection, while elite merchants also became courtiers and frequently fought with, or led, fighting forces—so it is entirely possible that some ship owners and merchants were also “fighting men.”91 We have much more precise information about the place of horse suppliers within the Pandyas’ “web of military associations.” According to Vassaf, one family of merchant-princes, the Tibi family of Kish, controlled both the export of horses from Il Khanid tributaries in the Gulf and their importation to the Pandya kingdom after 1292. In Iran, the Tibis paid tribute to the Il Khanids and bought the right to tax farm the province of Fars and its coastal areas more or less autonomously; later in that decade, the area under their control was extended to the head of the 91 On piracy and naval warfare along India’s western seaboard in the premodern period, see Margariti, “Mercantile Networks”; also Prange, “Trade of No Dishonor.” An example of a Hoysala merchant courtier with military experience, though not a trade in horses, is discussed in Ali, “Between Market and Court.” 90 Talbot, Precolonial India, 144. 90 Talbot, Precolonial India, 144. 91 On piracy and naval warfare along India’s western seaboard in the premodern period, see Margariti, “Mercantile Networks”; also Prange, “Trade of No Dishonor.” An example of a Hoysala merchant courtier with military experience, though not a trade in horses, is discussed in Ali, “Between Market and Court.” ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 85 Gulf and the port of Basra.92 Like many Iranian merchants under Il Khanid, rule the Tibis belonged to Mongol mercantile associations known as urtaqs which traded preferentially with and on behalf of the Mongol elite. What appears to have been unique about the Tibis—and perhaps explains Vassaf’s inclusion of their family history within his “wonders of India” discourse—is the fact that they controlled both ends of the network and derived enormous wealth from it. In India, members of the family occupied key administrative roles within the Pandya polity. Vassaf records that Jamal al-Din’s brother, Taqi al-Din ‘Abd al-Rahman ibn Muhammad al-Tibi, was deputy (nā’ib), minister (vazīr), adviser (mushīr), and administra- tor (sāhib tadbīr) to the Devar Sundar Bandi, likely the Pandya subregent Jata- varman Sundara Pandya III (r. 1276–92/93). In addition to this, the three prin- cipal ports of Fatan, Mali Fatan, and Qa’il “were made over to his [Taqi al-Din’s] possession,”93 and Vassaf reports that in 1292 the agents and factors of the Tibi brothers had the first choice of any merchandise arriving at Pandya ports.94 Vas- saf’s attribution of the ancient Sasanian title Marzubān al-hind or “Warden of the Marches of India” to Taqi al-Din reinforces the extent to which Il Khanid Iran regarded the Pandyan ports as an extension of its maritime frontier. If horses from other horse-breeding areas such as the Yemen were allowed to enter the Pandya market, it was patently only with Taqi al-Din’s permission. A list of gifts to influ- ential figures at Indian ports compiled at the port of Aden includes Taqi al-Din’s name, and Eric Vallet has noted that such gifting was probably intended to smooth Rasulid access to the Pandya market.95 The Tibis’ influence was sufficiently embedded in India to survive and even flourish after the death of Jatavarman Sun- dar Pandya in 692 AH/1292–93 CE, and, according to Vassaf, Malik Taqi al-Din “continued as prime minister, and, in fact, ruler of that kingdom, and his glory and magnificence were raised a thousand times higher.”96 The appointment of Middle Eastern merchants to administrative positions in Indian ports was already a well-established practice in South Asia, but possession of equine knowledge may have become an increasingly important prerequisite for appointment. 92 Vassaf, Tazjiyatu-l amsar, 33; on this particular system, see Aigle, Le Fārs sous la domina- tion mongole, 92–95. Horse merchants certainly feature prominently in the later political history of the Bahmani and Adil Shahi Sultanates in the fifteenth century, and in 94 Vassaf, Tazjiyatu-l amsar, 35; also Aubin, “Les Princes d’Ormuz,” 90. 93 Vassaf, Tazjiyatu-l amsar, 32–33. 92 Vassaf, Tazjiyatu-l amsar, 33; on this particular system, see Aigle, Le Fārs sous la domina- tion mongole, 92–95. 93 Vassaf, Tazjiyatu-l amsar, 32–33. 94 Vassaf, Tazjiyatu-l amsar, 35; also Aubin, “Les Princes d’Ormuz,” 90. 95 Vallet, L’Arabie marchande, 567. 96 Vassaf, Tazjiyatu-l amsar, 35. 97 Horse merchants may also have been encouraged to settle around ports. A Gurjarara Pratihara inscription, dated 736 CE, records the grant of a village in that district to a Brahmin from the sub-caste of the horse-trading Hetavukas; see Gupta, “Horse Trade in North India,” 197. eliZabeTh laMboURN 86 Mughal India in the seventeenth century; the example of the Tibis suggests that this may have been a much older practice and one that may have been particularly important in Peninsular India, away from terrestrial supply routes of barrī hors- es.97 The Tibis continued as key powerbrokers in South India right up until the conquest of the Pandya kingdom by the Delhi Sultanate in 1311.98 The consider- able political and economic influence allowed to the Tibi family is evidence for the Pandya polity’s ability to plan militarily on an international scale. This was not the exploitative relationship that Polo and even to some degree Vassaf suggest, but a symbiotic alliance that integrated Il Khanid merchants into the heart of the Pan- dya polity’s “web of military associations,” to the benefit of both parties. Horses were physical manifestations of the Pandyas’ international “web of military asso- ciations.” After centuries of importing horses, South Asian military and political elites would have been well aware of regional variations, and, in the Pandya king- dom, horses of different origins may have been recognized and understood within this framework of meaning as physical evidence of the king’s ability to command resources internationally. It remains to be seen how far this Pandya network of horse supply differed from that of their Chola predecessors and indeed from those of their Hoysala and Kakatiya contemporaries. The absence of horses from Pandya inscriptions con- trasts with frequent references in earlier Chola donative inscriptions to horse merchants from Malaimandalam, present-day Kerala, and with references in con- temporary Hoysala inscriptions to horses as traded items.99 While further analysis is needed, this pattern appears to suggest that the Tibis did indeed participate in a monopoly that largely bypassed earlier west-coast intermediaries. 99 In the Hoysala domains from 1167 CE onwards, horses are mentioned among lists of taxable commodities, although there is no mention of either their prices, their numbers, or their place of origin; see inscriptions listed in Epigraphia Carnatica, vol. 9, inscription no. BI 170; also inscription of 1209 CE Epigraphia Carnatica, vol. 7, inscription no. Hn.35 (cited in Appadurai, Economic Conditions, 119) and another dated 1261 CE listed in Epigraphia Carnatica, vol. 9, inscription no. BI 409 (Ibid., 129–30). 98 A full family history of the Tibis remains to be written, although a substantial effort, based on Persian sources, can be found in Aubin, “Les Princes d’Ormuz,” and in Aigle, Le Fārs sous la domination mongole. 99 In the Hoysala domains from 1167 CE onwards, horses are mentioned among lists of taxable commodities, although there is no mention of either their prices, their numbers, or their place of origin; see inscriptions listed in Epigraphia Carnatica, vol. 9, inscription no. BI 170; also inscription of 1209 CE Epigraphia Carnatica, vol. 7, inscription no. Hn.35 (cited in Appadurai, Economic Conditions, 119) and another dated 1261 CE listed in Epigraphia Carnatica, vol. 9, inscription no. BI 409 (Ibid., 129–30). Horses and the “Martial Ethos” in Medieval South India Another route into the South Indian reception and translation of bahrī horses is suggested by Cynthia Talbot’s work on the “martial ethos” of the Kakatiya dynas- ty’s court and army. In keeping with the importance of military action to the legiti- macy of kingship and a wider military society, Talbot is able to demonstrate that military action is also a major, if neglected, subject in the epigraphic repertoire. As she observes, “fierce fighting between mounted horsemen is the main theme, for elite warriors were inevitably found in the cavalry contingents.”100 Her analysis of the Kakatiya “martial ethos” as it emerges from the epigraphic record currently offers our best insight into how horses were received into medieval South India, and does so in ways that suggest fascinating opportunities for the comparative study of this South Asian martial ethos and contemporary European chivalric cul- tures. The inscriptions Talbot analyzes are extremely varied in imagery and con- tent, and here I discuss three examples, each one illustrating a different scale at which we might locate the translation of horses into Indic military culture. In large numbers, at the scale of a panoramic battle scene, cavalry adds to the terrifying sound and sight (drums and banners) of the advancing army, as the clouds of dust raised by the galloping horsemen obliterate the sun in an apoca- lyptic fashion. It is worth quoting at length a passage from an inscription of the Kakatiya ruler Ganapati (r. 100 Talbot, Precolonial India, 145. 101 Talbot, Precolonial India, 145–46; an English translation of the original inscription from Ganapapuram village (Nalgonda district) is reported and published in Corpus of Inscriptions, no. 22, vv. 3–5. Similar imagery appears in earlier Pala inscriptions from Bengal; see Chakra- varti, “Early Medieval Bengal,” 198. THIS DOCUMENT MAY BE PRINTED FOR PRIVATE USE ONLY AND MAY NOT BE DISTRIBUTED WITHOUT PERMISSION FROM THE PUBLISHER. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 87 Horses and the “Martial Ethos” in Medieval South India 1199–1260): When the thundering of the war-drums of his victorious army on the march pervaded the far corners, it was as if the echoes reverberating off the towering houses of his enemies were telling them, “Escape to the for- est quickly, for King Ganapati, a master in the battlefield, is approaching!” Held up high on tall poles and waving vigorously in the wind, his army’s battle colors seemed to signal to the many rival kings from a distance with the threat, “Run far away at once!” When the rays of the sun’s light had been totally extinguished by the clouds of dust that rose up from the ground as the rows of sharp hooves of his throngs of horses tore it asun- der, the astonished people thought the sun had gone away, observing the frightful heads of the hostile kings rise up [in the air] as they were cut off by his weapons and mistaking them for an army of Rahus.101 eliZabeTh laMboURN 88 In the closer focus of battle engagement, it is the speed of the horse that is fore- grounded and explicitly contrasted to that of elephants. An inscription celebrating a chief from the Telangana area of eastern India (r. 1262–89) describes an engage- ment featuring: warriors [as savage as] lions and tigers, whose horses became [as swift as] thoughts and winds, and whose elephant-troops became [as tower- ing as] mountains and clouds when Rudradeva’s army exhibited its fear- someness in the extermination, dispersal, or capturing of [enemy] kings without effort.102 Finally, at the most intimate scale, the generic horse becomes an individual horse, belonging to a named soldier and with its own name. A donative inscription dated 1235 records how “the king’s cavalier Vishaveli Masake Sahini,” in effect the Master of the Royal Stables103 “gave 25 cows for a perpetual lamp to the glorious lord Tripurantaka, having been victorious in the battle of Chintalapundi mounted on [the horse] Punyamurti.”104 One does not name an animal one does not value: Punyamurti means “Embodiment of Merits” and clearly was. If the first example seems to resonate with outsiders’ views of horses as sig- nificant only in, or because of, their large numbers, the second points to at least one of the qualities for which horses were prized above elephants—namely, their speed—while the third reminds us of the close bonds that developed between elite riders and their mounts. Horses may have lived brief lives, but they were no less important to their riders; and the close association between cavalryman and mount evoked in the third inscription is often illustrated on memorial stones. Talbot does not indicate whether Vishaveli Masake’s inscription was also carved, but Figure 6 shows a roughly contemporary memorial stone from the same site, Tripurantakam in Kurnool District, depicting a single-mounted rider viewed in profile with smaller foot soldiers around him. Single-mounted horsemen are more usually represented on hero stones from Rajasthan, Sindh, and Kutch, areas with indigenous horse breeds and strong local equine cultures; and yet, alone in the Peninsular south, Kakatiya Andhra Pradesh developed its own distinctive tradi- 102 Talbot, Precolonial India, 147; English translation of the original inscription reported and published in Corpus of Inscriptions, no. 38, v. 4. 103 Sahini was the Telugu equivalent of Sanskrit Sadhanika (Master of the Royal Stables); see Talbot, Precolonial India, 68 (citing Sircar’s Indian Epigraphical Glossary, 285). More generally, the term rautu designated a cavalry leader. 104 Talbot, Precolonial India, 69; English translation of an inscription at Tripurantakam (Markapur Taluk, Kurnool District) published in South Indian Inscriptions, inscription no. 283, v. 148. 102 Talbot, Precolonial India, 147; English translation of the original inscription reported and published in Corpus of Inscriptions, no. 38, v. 4. 103 Sahini was the Telugu equivalent of Sanskrit Sadhanika (Master of the Royal Stables); see Talbot, Precolonial India, 68 (citing Sircar’s Indian Epigraphical Glossary, 285). More generally, the term rautu designated a cavalry leader. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 89 tion of human and horse representation.105 The uniqueness of the Kakatiya ico- nography raises many questions about the deep embededness of equine cultures in the region and the place of the horse in the Kakatiya martial ethos. It remains to be seen whether Hoysala, Pandya, and other sources yield evi- dence for largely similar discourses and tropes, or whether the final picture will be of interrelated but nevertheless distinctive regional equine cultures, as dis- tinctive perhaps as the visual representation of horses and riders discussed thus far. Only with a better documentation of visual and textual sources about horses across South India will we begin to capture the more subtle differences between these regional or perhaps dynastic equine cultures and, in so doing, follow the Indian lives of bahrī and other imported horses. 105 See Reddy, Heroes, Cults and Memorials, 119–24. 106 English translation of Ibn Fadl Allah al-‘Umari in Masalik al-absar, 40. 107 Discussed in Shehada, Mamluks and Animals, 155. Indian Equine Knowledges in Circulation By the thirteenth century, Peninsular India had developed distinctive regional equine cultures born from a millennium or more of horse keeping and horse importation. Though a great deal of research remains to be carried out in this area, it is clear that South Asian equine cultures were already strong enough to impact the Middle East, a circular movement of equine cultures too rarely noted in the dominant focus on their export from the Middle East to South Asia. Vassaf’s less judgmental view of Indian horse-keeping skills resonates with evidence for well-developed equine knowledge in South Asia by the thirteenth century. In his account of India, the Damascene geographer Ibn Fadl Allah al-‘Umari (d. 1348) reports that one of his informants, a Bahrayni horse trader, said that the “people of India have got a knack to distinguish the horses of good breed. As [sic] they find the mark in a horse known to them, they may purchase it at any price.”106 Indian veterinary skills were another facet of this equine knowledge recognized out- side South Asia: an Arabic language veterinary treatise written around 1331 by the Rasulid ruler of the Yemen, al-Malik al-Mujahid (r. 1321–62), remarks on the expertise of Indian doctors in identifying early symptoms of a “horse plague” that affected animals across the Arabian Peninsula and at Indian ports in 728/1327.107 These examples point to a far greater interaction between Middle Eastern and South Asian traditions of equine knowledge than either Vassaf’s or Polo’s accounts eliZabeTh laMboURN 90 suggest, stimulated very directly by the horse trade across the western Indian Ocean and the further interactions this necessitated between specialists from both regions. The inclusion of this anecdote in al-Malik al-Mujahid’s treatise reminds us of the openness of the Arabic hippiatric tradition to other equine knowledges. Although South Asia was to become the major zone of encounter between Mid- dle Eastern and South Asian hippiatric traditions from the later fourteenth cen- tury onwards,108 horses also mediated this cultural encounter beyond South Asia, across the rest of the Islamic world. Several of the major Arabic hippiatric texts of the twelfth through fourteenth centuries, composed from Spain to the Yemen, include chapters on the interpreta- tion of the colors and markings of horses and attribute this knowledge directly to Indian authorities.109 As Housni Shehada observes, “the idea that markings or patches on the horse’s body indicate the good or bad luck that it may bring to its master is characteristic of the Indian tradition.”110 It is exactly this importance of markings (marks) that al-‘Umari highlights in his account of India.111 Yet attempts to map a textual chain of translation and transmission from South Asia to Spain via Abbasid Baghdad have largely failed, as these sources acknowledge only generic “Indian sages” or Indian viziers who cannot be traced historically. This vagueness contrasts with the care usually paid to chains of textual and/or oral transmission in Islamic scholarship and highlights the extent to which the hippiatric textual tra- dition emerged relatively late in the Middle East, and often from direct personal experience in the craft of horse care. The most likely vector for the transmission of this Indian knowledge to the Middle East, therefore, seems to have been not textual at all but oral, transmitted perhaps through the direct interactions of horse merchants, grooms, and buyers 108 With the Ghurid conquest of northern India in the late twelfth century, Indian equine treatises were quickly incorporated into Turkic and Persianate equine epistemologies. SƵalihotra’s Complete Ayurvedic System was first translated into Persian in the fourteenth century, and Persian translations of Sanskrit equine treatises were subsequently highly sought after and influential. 109 These include Ibn Awwam al-Ishbili’s twelfth century Kitab al-filaha, composed in Spain; the Kitab al-baytarah of the Mamluk vizier Sahib Taj al-Din (d. eliZabeTh laMboURN 1307 CE), which includes a “chapter on what the Indians said about the colors of beasts of burden, the round patches and the marks”; the Kashif of Abu Bakr al-Baytar, chief veterinarian to Sultan Qalawun of Egypt (r. 1285–1340); and the ca. 1331 treatise by the Rasulid ruler al-Malik al- Mujahid. See Shehada, Mamluks and Animals, 92–94 and 152. 111 Ibn Fadl Allah al-‘Umari, Masalik al-absar, 40. 108 With the Ghurid conquest of northern India in the late twelfth century, Indian equine treatises were quickly incorporated into Turkic and Persianate equine epistemologies. SƵalihotra’s Complete Ayurvedic System was first translated into Persian in the fourteenth century, and Persian translations of Sanskrit equine treatises were subsequently highly sought after and influential. 109 These include Ibn Awwam al-Ishbili’s twelfth century Kitab al-filaha, composed in Spain; the Kitab al-baytarah of the Mamluk vizier Sahib Taj al-Din (d. 1307 CE), which includes a “chapter on what the Indians said about the colors of beasts of burden, the round patches and the marks”; the Kashif of Abu Bakr al-Baytar, chief veterinarian to Sultan Qalawun of Egypt (r. 1285–1340); and the ca. 1331 treatise by the Rasulid ruler al-Malik al- Mujahid. See Shehada, Mamluks and Animals, 92–94 and 152. 110 Shehada, Mamluks and Animals, 94. 112 Talbot, “Inscribing the Other,” 710. 113 Ibid., 709. 114 Branfoot, “Expanding Form,” 206. Branfoot (203–06) offers the most comprehensive history of this column type. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 91 at horse fairs in the Gulf and Arabian ports, or indeed in India itself. An under- standing of South Asian equine cultures was essential for any merchant or court that maintained relations with South Asia, as Ibn Fadl Allah al-‘Umari’s Bahrayni horse-trader informant indicated, because in India horses were judged first on their color and the auspiciousness of their markings. These Arabic chapters on the Indian science of horse physiognomy, along with al-Malik al-Mujahid’s account of the horse plague, can thus be read as evidence for the circulation between South Asia and the Middle East of a very practical equine knowledge. But we might also reflect that, when they were read in places far away from this trade—for example, in Islamic Spain—such chapters also perpetuated an older Middle Eastern dis- course about the wonders of the non-Islamic world. Through the vehicle of horses, India emerges once more as a “topsy-turvy” world where Middle Eastern taxono- mies and practices are upended. An Agenda for “Matters Equine” in Medieval South Asia As Wendy Doniger has underlined, the horse was never perceived as an Indian animal in indigenous folklore, and even the boom in the bahrī horse trade in the thirteenth century may have had little impact on domesticating the horse in the Peninsular south, at least beyond the courts of elites. Perhaps most telling is the manner in which command over horses became intrinsic to certain South Indian conceptions of the geo-political universe, and yet how little horses were ever iden- tified as a South Indian animal. As Cynthia Talbot has demonstrated, from at least the early fifteenth century, in parts of South India, the idea developed that the subcontinent was in fact “divided into three realms, each ruled by a king laying claim to superiority in one contingent of an army.”112 The pattern that emerges over the centuries is that “the Lord of Horses was a designation that could signify any Muslim king,” but apparently only a Muslim king.113 Non-Muslim kings, by contrast, were lords of elephants, the quintessential royal mount of Indian kings and Indian war animal, or they were lords of men. Horse representations never became commonplace in the south of the Tamil country, and, as the art historian Crispin Branfoot has shown, it is only in the sixteenth century (and largely in the Tamil north, on the border with Andhra Pradesh) that horses enter the sculptural repertoire.114 Even then, the horse was somehow always a little fantastical and certainly a little terrifying: large rearing brutes with diminutive riders and strain- 114 Branfoot, “Expanding Form,” 206. Branfoot (203–06) offers the most comprehensive history of this column type. 112 Talbot, “Inscribing the Other,” 710. eliZabeTh laMboURN 92 ing grooms join older iconographies of rearing griffins and other mythical animals in a new type of temple support known as the “mounted cavalryman” composite column (see Figure 7). As many outsiders remarked of South India, there are no horses native to that place, and the horse never became an indigenous animal: and yet the complex history of its reception and translation to South India magnifies the rich geographical and cultural diversity of this area and the ways in which horses became vehicles for the expression of other ideas. The thirteenth century was not only a turning point in the diffusion of Middle Eastern horses to South Asia and beyond: by 1329, in northern Italy, the Gonzagas were cross-breeding Arab horses with local breeds, marking a symmetrical Euro- pean fascination with “eastern” horses that later exploded, as Lisa Jardine and Jerry Brotton have shown, during the European “Renaissance”.115 As Marco Polo’s account shows, horses were already a truly “global interest” in the late thirteenth century, and had been for some time. However, this case study has demonstrated that the ubiquity of the modern horse at a global level hides a complex history of diffusion, and one that is substantially unwritten for much of this period across much of the globe. Moreover, at least for medieval South India, this is still far from being a history that is possible to write: a huge volume of primary source material exists in old, untranslated editions; archaeological excavations have been slow to collect faunal remains, let alone analyze them; and much architectural and icono- graphic evidence is inconsistently documented and published. This situation is unlikely to change very quickly: there are few non-European medievalists compared to those specializing in Europe, fewer still being trained, and as long as this disproportion remains there will be tantalizing histories that can only be half told. Perhaps the frustration of not knowing how to tell these stories may spur more medievalists to study non-European history, or perhaps European medievalists will begin to see the possibilities for comparative and con- nected histories that these wider, global perspectives open. The medieval globe was always profoundly interconnected through the circulation of people, animals, things, and ideas, but its history can only be traced when seen from multiple per- spectives, through both textual and material sources, and through sources from many regions in many languages and media. 115 Jardine and Brotton, Global Interests, 148. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 93 93 Figure 7. Rearing Horse Depicted on Composite Columns at the Sesaraya Mandapa at Srirangam, Tiruchchirapalli in Tamil Nadu (Vijayanagara Period, Mid-Sixteenth Cen- tury CE). Photograph reproduced courtesy of the American Institute of Indian Studies, Accession No 101330. Figure 7. Rearing Horse Depicted on Composite Columns at the Sesaraya Mandapa at Srirangam, Tiruchchirapalli in Tamil Nadu (Vijayanagara Period, Mid-Sixteenth Cen- tury CE). Photograph reproduced courtesy of the American Institute of Indian Studies, Accession No 101330. Primary Sources Abu al-Fida’, ‘Imad al-Din Isma‘il ibn ‘Ali. Taqwim al-buldan. Translated by ‘Abd al-Muhammad Ayati as Taqwim al-Buldan. Teheran: Bunyad-i Farhang-i Iran, 1970. Chau Ju-Kua. Chu-Fan-Chi. Translated by Fredrich Hirth and W. W. Rockhill. Taipei: Cheng-Wen Publishing Comanpy, 1970. A Corpus of Inscriptions in the Telingana Districts of H.E.H. the Nizam’s Dominions. Part 2, Comprising Texts and Translations of Inscriptions. Hyderabad Archaeo- logical Series, 13. Hyderabad: Government of Andhra Pradesh, 1940. Cosmas Indicopleustes. The Christian Topography of Cosmas, an Egyptian Monk. Edited by J. W. McCrindle. London: Hakluyt Society, 1897. Epigraphia Carnatica. New Series. 17 vols to date. Bangalore: Mysore Govt. Central Press, 1934–. Ibn Fadl Allah al-ʻUmari, Ahmad ibn Yahya. Masalik al-absar fi mamalik al-amsar. Edited and translated by Iqtidar Husain Siddiqi and Qazi Mohammad Ahmad as A Fourteenth-Century Arab Account of India under Sultan Muhammad Bin Tughluq. Aligarh: Siddiqi, 1971. Ibn al-Mujawir, Yusuf ibn Ya‘qub. Ta’rikh al-mustabsir. Translated by G. Rex Smith as A Traveller in Thirteenth-Century Arabia: Ibn al-Mujawir’s “Tarikh al-Mustab- sir.” London: Hakluyt Society, 2008. Kautilya. Arthaśāstra. Translated by R. Shamasastry as Kautilya’s Arthaśāstra. 4th ed. Mysore: Sri Raghuveer, 1951. al-Malik al-Mujahid, ‘Ali. Al-aqwal al-kafiya wa-l-fusul al-shafiya fi al-khayl. Edited by Yayha Wahib al-Jaburi. Beirut: Dar al-Gharab al-Islami, 1987. Polo, Marco. Milione: il Milione nelle redazioni toscana e franco-italiana = Le divisa- ment dou monde. Edited by Gabriella Ronchi. Milan: Mondadori, 1982. Rashid al-Din Tabib, Fazl Allah. Ta’rīkh al-hind. Edited by Karl Jahn as Rashid al- Din’s History of India: Collected Essays with Facsimiles and Indices. London: Mouton, 1965. South Indian Inscriptions: Texts. Vol. 10, Telugu Inscriptions from the Madras Presi- dency. Edited by J. Ramayya Pantulu. Archaeological Survey of India 56. Delhi: Manager of Publications, 1948. Vassaf al-Hazrat, ʻAbd Allah ibn Fazl Allah. Tajziyat al-amsar va tazjiyat al-as‘ar. Lithograph by Muhammad Mahdi Isfahani. Teheran: Ibn Sina, 1959–60. —— . Tazjiyatu-l amsar. Partial English translation in The History of India as Told by its Own Historians, edited by H. M. Elliot and J. Dowson, 3:32–35. New Delhi: Low Price Publications, 1990. eliZabeTh laMboURN 94 ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 95 Secondary Studies Aigle, Denise. Le Fārs sous la domination mongole: politique et fiscalité (XIIIe–XIVe s.). Studia Iranica Cahier 31. Paris: Association pour l’avancement des études Iraniennes, 2005. Ali, Daud. “Between Market and Court: The Careers of Two Courtier-Merchants in the Twelfth-Century Deccan.” Journal of the Economic and Social History of the Orient 53 (2010): 185–211. Allsen, Thomas T. The Royal Hunt in Eurasian History. Philadelphia: Uni ver sity of Pennsylvania Press, 2006. Appadurai, A. Economic Conditions in Southern India (1000–1500 A.D.). 2 vols. Madras: Madras Uni ver sity, 1990. Asouti, Eleni, and Dorian Q. Fuller. Trees and Woodlands of South India: Archaeo- logical Perspectives. Walnut Creek, CA: Left Coast Press, 2007. Aubin, Jean. “Les Princes d’Ormuz du XIIIe au XVe siècle.” Journal Asiatique 241 (1953): 77–138. Bankoff, Greg and Sandra Swart, eds. Breeds of Empire: The “Invention” of the Horse in Southeast Asia and Southern Africa, 1500–1950. Copenhagen: NIAS Press, 2007. Blench, Robert. “Prehistory of African Ruminant Livestock, Horses, and Ponies.” In The Archaeology of Africa: Food, Metals, and Towns, edited by Thurston Shaw et al., 88–103. Abingdon: Routledge, 1993. Blinkhorn, Nicole Boivin, Paul S. C. Taçon, and Michael D. Petraglia. “Rock Art Re- search in India.” In A Companion to Rock Art, edited by Jo McDonald and Peter Veth, 179–96. Hoboken, NJ: Wiley Blackwell, 2012. Branfoot, Crispin. “‘Expanding Form’: The Architectural Sculpture of the South Indian Temple ca. 1500–1700.” Artibus Asiae 62, (2002): 189–245. Chakravarti, Ranabir. “Early Medieval Bengal and the Trade in Horses: A Note.” Jour nal of the Economic and Social History of the Orient 42, (1999): 194–211. —— . “Equestrian Demand and Dealers: the Early Indian Scenario (up to c. 1300).” In Pferde in Asien: Geschichte, Handel und Kultur, edited by Bert G. Fragner et al., 145–59. Vienna: Verlag der Oǆ sterreichischen Akademie der Wissenschaften, 2009. —— . “Horse Trade and Piracy at Tana (Thana Maharashtra, India): Gleanings from Marco Polo.” Journal of the Economic and Social History of the Orient 34, (1991): 159–82. Chauhan, Devising. “Arab Horses in India.” Annals of the Bhandarkar Oriental Re- search Institute 48–49 (1968): 391–94. Coelho, William. The Hoysala Vamsa. Bombay: Indian Historical Research Institute, St. Xavier’s College, 1950. Cutler, Norman. Songs of Experience: The Poetics of Tamil Devotion. Bloomington: Uni ver sity of Indiana Press, 1987. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia eliZabeTh laMboURN 96 Deloche, Jean. Le Cheval et son harnachement dans l’art indien. Lausanne: Caracole/ EFEO, 1986. Deloche, Jean. Le Cheval et son harnachement dans l’art indien. Lausanne: Caracole/ EFEO, 1986. Deloche, Jean. Le Cheval et son harnachement dans l’art indien. Lausanne: Caracole/ EFEO, 1986. Deloche, Jean. Le Cheval et son harnachement dans l’art indien. Lausanne: Caracole/ EFEO, 1986. —— . “EƵtudes sur la circulation en Inde: III. Le bateau de Tirupputaimarutur (sud de l’Inde).” Bulletin de l’École Française d’Extrême Orient 72 (1983): 1–11. —— . “EƵtudes sur la circulation en Inde: III. Le bateau de Tirupputaimarutur (sud de l’Inde).” Bulletin de l’École Française d’Extrême Orient 72 (1983): 1–11. —— . Military Technology in Hoysaḷa Sculpture : —— . Military Technology in Hoysaḷa Sculpture : —— . Military Technology in Hoysaḷa Sculpture : —— . “Techniques militaires dans les royaumes du Dekkan au temps des Hoysala (XIIe–XIIIe siècle), d’après l’iconographie.” Artibus Asiae 47 (1986): 147–232. —— . “Techniques militaires dans les royaumes du Dekkan au temps des Hoysala (XIIe–XIIIe siècle), d’après l’iconographie.” Artibus Asiae 47 (1986): 147–232. Digby, Simon. War-Horse and Elephant in the Delhi Sultanate: A Study in Military Supplies. Oxford: Orient Monographs, 1971. Digby, Simon. War-Horse and Elephant in the Delhi Sultanate: A Study in Military Supplies. Oxford: Orient Monographs, 1971. Doniger, Wendy. “The Deconstruction of Vedic Horselore in Indian Folklore.” In Ritual, State and History in South Asia: Essays in Honour of J. C. Heesterman, edited by A. W. van der Hoek, D. H. A. Kolff, and M. S. Oort, 76–101. Leiden: Brill, 1992. Doniger, Wendy. “The Deconstruction of Vedic Horselore in Indian Folklore.” In Ritual, State and History in South Asia: Essays in Honour of J. C. Heesterman, edited by A. W. van der Hoek, D. H. A. Kolff, and M. S. Oort, 76–101. Leiden: Brill, 1992. —— . “A Symbol in Search of an Object: The Mythology of Horses in India.” In A Co mmunion of Subjects: Animals in Religion, Science and Ethics, edited by Paul Waldau and Kimberley Patton, 335–50. New York: Columbia Uni ver sity Press, 2006. Edwards, Peter, Karl Enenkel, and Elspeth Graham, eds. The Horse as Cultural Icon: The Real and Symbolic Horse in the Early Modern World. Leiden: Brill, 2012. Flood, Finbarr Barry. Objects of Translation: Material Culture and Medieval “Hindu- Muslim” Encounter. Princeton: Princeton Uni ver sity Press, 2009. Fragner, Bert G. et al., eds. eliZabeTh laMboURN Pferde in Asien: Geschichte, Handel und Kultur. Vienna: Verlag der Oǆ sterreichischen Akademie der Wissenschaften, 2009. Goldgar, Anne. Tulipmania: Money, Honor, and Knowledge in the Dutch Golden Age. Chicago: Chicago Uni ver sity Press, 2007. Gommans, Joseph J. L. “The Horse Trade in Eighteenth Century South Asia.” Journal of the Economic and Social History of the Orient 37 (1994): 228–50. —— . The Rise of the Indo-Afghan Empire, 1710–1780. Leiden: Brill, 1995. —— . and Dirk H.A. Kolff, eds. Warfare and Weaponry in South Asia, 1000–1800. Oxford: Oxford Uni ver sity Press, 2001. Gupta, Chitrarekha. “Horse Trade in North India: Some Reflections on Socio- Economic Life.” Journal of Ancient Indian History 14 (1983–84): 186–206. Huda, M. Z. “Faras Namah-i-Hashimi and Shalihotra.” Journal of the Asiatic Society of Pakistan 14 (1969): 143–65. Jackson, Peter. “Wassaf.” In Encyclopaedia of Islam, Second Edition, edited by P. Bearman, Th. Bianquis, C. E. Bosworth, E. van Donzel, et al. Brill Online, 2015 <http://www.brillonline.nl/entries/encyclopaedia-of-islam-2/wassaf-SIM_7890> [accessed July 7, 2015]. Jardine, Lisa, and Jerry Brotton. Global Interests: Renaissance Art between East and West. London: Reaktion Books, 2000. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 97 Jazim, Muhammad ‘Abd al-Rahim. Nur al-ma‘arif: Lumière de la connaissance. Règles, lois et coutumes du Yemen sous le règne de sultan Rasoulide al-Muzaffar. 2 vols. Sanaa: Centre Français d’Archéologie et de Sciences Sociales de Sanaa, 2003–2005. Kauz, Ralph. “Horse Exports from the Gulf until the Arrival of the Portuguese.” In Pferde in Asien: Geschichte, Handel und Kultur, edited by Bert G. Franger et al., 129–36. Vienna: Verlag der Oǆ sterreichischen Akademie der Wissenschaften, 2009. —— . “Maritime Trade of Kish.” In Beyond the Legacy of Genghis Khan, edited by Linda Komaroff, 51–67. Leiden: Brill, 2006. Kinoshita, Sharon. “Animals and the Medieval Culture of Empire.” In Animal, Vegetable, Mineral: Ethics and Objects, edited by Jeffrey Jerome Cohen, 35–64. Washington, DC: Oliphaunt Books, 2012. Köppen, W., “Das geographisca System der Klimate.” In Handbuch der Klimatologie. Vol. 1, part C, edited by W. Köppen and R. Geiger, 1–44. Berlin: Gebrüder Born- traeger, 1936. Lambourn, Elizabeth. “Ali Akbar’s Red Horse: Collecting Arab Horses in the Early Modern Culture of Empire.” In Early Modern Merchants as Collectors, edited by Christina Anderson. Aldershot: Ashgate, forthcoming. Landry, Donna, Noble Brutes: How Eastern Horses Transformed English Culture. Baltimore: Johns Hopkins Uni ver sity Press, 2009. Law, Robin. The Horse in West African History: The Role of the Horse in the Societies of Pre-Colonial West Africa. Oxford: Oxford Uni ver sity Press, 1980. Leshnik, Lawrence S. “Some Early Indian Horse-Bits and Other Bridle Equipment.” American Journal of Archaeology 75 (1971): 141–50. Loureiro, Rui Manuel. “Portuguese Involvement in Sixteenth Century Horse Trade.” In Pferde in Asien: Geschichte, Handel und Kultur, edited by Bert G. Fragner et al., 203–30. Vienna: Verlag der Oǆ sterreichischen Akademie der Wissenschaften, 2009. 203–30. Vienna: Verlag der Oǆ sterreichischen Akademie der Wissenschaften, 2009. Levine, M. “Domestication and Early History of the Horse.” In The Domestic Horse: The Origins, Development and Management of its Behavior, edited by Daniel S. Mills and S. M. McDonnell, 5–22. Cambridge: Cambridge Uni ver sity Press, 2005. Levine, M. Domestication and Early History of the Horse. In The Domestic Horse: The Origins, Development and Management of its Behavior, edited by Daniel S. Mills and S M McDonnell 5–22 Cambridge: Cambridge University Press 2005 Mills and S. M. McDonnell, 5–22. Cambridge: Cambridge Uni ver sity Press, 2005. Majumdar, R. C., A. D. Pusalker, and A. K. Majumdar, eds. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia The Struggle for Empire: The History and Culture of the Indian People. Vol. 5. 4th ed. Bombay: Bharaiya Vidya Bhavan, 1989. Margariti, Roxani Eleni. “Mercantile Networks, Port Cities, and ‘Pirate’ States: Con- flict and Competition in the Indian Ocean World of Trade before the Sixteenth Cen tury.” Journal of the Economic and Social History of the Orient 51 (2008): 543–77. Mazars, George. “Traditional Veterinary Medicine in India.” Revue Scientifique et Technique (International Office of Epizootics) 13 (1994): 443–51. Michell, George. “Migrations and Cultural Transmissions in the Deccan: Evidence of Monuments at Vijayanagara.” In The Visual World of Muslim India: The Art, eliZabeTh laMboURN 98 Cul ture and Society of the Deccan in the Early Modern Era, edited by Laura Parodi, 79–85. London: I. B. Taurus, 2014. Cul ture and Society of the Deccan in the Early Modern Era, edited by Laura Parodi, 79–85. London: I. B. Taurus, 2014. Mitchell, Peter. Horse Nations: The Worldwide Impact of the Horse on Indigenous Societies Post–1492. Oxford: Oxford Uni ver sity Press, 2015. Mukherjee, Rila. “Introduction.” In Pelagic Passageways: The Northern Bay of Bengal before Colonialism, edited by Rila Mukherjee, 1–262. Delhi: Primus Books, 2011. Nilakantha Sastri, K. A. The Pandyan Kingdom: From the Earliest Times to the Six- teenth Century. London: Luzac, 1929. Peel, M. C., B. L. Finlayson, and T. A. McMahon. “Updated World Map of the Köppen- Geiger Climate Classification.” Hydrology and Earth Systems Sciences 11 (2007): 1633–44. Pfeiffer, Judith. “‘A Turgid History of the Mongol Empire in Persia’: Epistemological Reflections Concerning a Critical Edition of Vassaf’s Tajziyat al-amsar va tzaji- yat al-as’ar.” In Theoretical Approaches to the Transmission and Edition of Ori- ental Manuscripts, edited by Judith Pfeiffer and Manfred Kropp, 93–129. Würz- burg: Orient-Institut Beirut, 2007. Phillott, Douglas C. Faras-Nama-i Hashimi. Calcutta: Asiatic Society, 1910. Prange, Sebastian R. “A Trade of No Dishonor: Piracy, Commerce, and Community in the Western Indian Ocean, Twelfth to Sixteenth Century.” American Historical Review 116 (2011): 1269–93. Ptak, Roderich. “Pferde auf See: Ein vergessener Aspekt des maritimen chinesi- schen Handels im frühen 15. Jahrhundert.” Journal of the Economic and Social History of the Orient 34 (1991): 199–233. —— . “Yuan and Early Ming Notices on the Kayal Area in South India.” Bulletin de l’École Française d’Éxtrême Orient 80 (1993): 137–56. Raber, Karen, and Treva J. Tucker, eds. The Culture of the Horse: Status, Discipline, and Identity in the Early Modern World. New York: Palgrave Macmillan, 2005. Raychaudhuri, Tapan, Irfan Habib, and Dharma Kumar, eds. The Cambridge Eco- nomic History of India. Volume 1, c. 1200–c. 1750. Cambridge: Cambridge Uni- ver sity Press, 1982. Reddy, R. Chandrashekara. Heroes, Cults and Memorials: Andhra Pradesh, 300 A.D.– 1600 A.D. Madras: New Era Publications, 1994. Rubiés, Joan-Pau. Travel and Ethnology in the Renaissance: South India through European Eyes, 1250–1625. Cambridge: Cambridge Uni ver sity Press, 2000. Sainte-Palaye, M. de la Curne de, et al. Dictionnaire historique de l’ancien langage françois. 10 vols. Hildesheim: Olms, 1972. Sen, Tansen. eliZabeTh laMboURN “The Yuan Khanate and India: Cross-Cultural Diplomacy in the Thir- teenth and Fourteenth Centuries.” Asia Major 19 (2006): 299–326. Sethuraman, N. The Imperial Pandyas: Mathematics Reconstructs the Chronology. Kumbakonam: Sethuraman, 1977. ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia ToWaRdS a CoNNeCTed hiSToRy oF eQUiNe CUlTUReS iN SoUTh aSia 99 Settar, S., and Günther-Dietz Sontheimer, eds. Memorial Stones: A Study of their Origin, Significance, and Variety. Dharwad: Institute of Indian Art History, Karna taka Uni ver sity, 1982. Shehada, Housni Alkhateeb. Mamluks and Animals: Veterinary Medicine in Medieval Islam. Leiden: Brill, 2013. Sinopoli, Carla. “From the Lion Throne: Political and Social Dynamics of the Vija- yana gara Empire.” Journal of the Economic and Social History of the Orient 43 (2000): 364–98. Sircar, D.C. Indian Epigraphical Glossary. Delhi: Motilal Banarsidas, 1966. Talbot, Cynthia. “Inscribing the Other, Inscribing the Self: Hindu-Muslim Identities in Pre-Colonial India.” Comparative Studies in History and Society 37 (1995): 692–722. —— . Precolonial India in Practice: Society, Region, and Identity in Medieval Andhra. Oxford: Oxford Uni ver sity Press, 2001. Thinakaran, A. J. The Second Pandyan Empire (A.D. 1190–1312). Madurai: Thina- karan, 1987. Vallet, Eric. L’Arabie marchande: état et commerce sous les sultans rasulides du Yémen (626–858/1229–1454). Paris: Publications de la Sorbonne, 2010. Van der Geer, Alexandra. Animals in Stone: Indian Mammals Sculpted though Time. Leiden: Brill, 2008. Wagoner, Philip B. “Fortuitous Convergences and Essential Ambiguities: Trans- cultural Political Elites in the Medieval Deccan.” International Journal of Hindu Studies 3 (1999): 241–64. —— . “Harihara, Bukka and the Sultan: The Delhi Sultanate in the Political Imag- ination of Vijayanagara.” In Beyond Turk and Hindu: Rethinking Religious Iden- tities in Islamicate South Asia, edited by David Gilmartin and Bruce Lawrence, 300–26. Gainesville: Uni ver sity Press of Florida, 2000. Yadava, B. N. S. “Chivalry and Warfare.” In Warfare and Weaponry in South Asia, 1000–1800, edited by Jos J. L. Gommans and Dirk H. A. Kloff, 66–98. Oxford: Oxford Uni ver sity Press, 2001. Yang, Bin. “Horses, Silver, and Cowries: Yunnan in Global Perspective.” Journal of World History 15 (2004): 281–322. Yokkaichi, Yasuhiro. “Horses in the East-West Trade between China and Iran under Mongol Rule.” In Pferde in Asien: Geschichte, Handel und Kultur, edited by Bert G. Fragner et al., 87–97. Vienna: Verlag der Oǆ sterreichischen Akademie der Wissen- schaften, 2009. Yule, Henry. “An Endeavour to Elucidate Rashiduddin’s Geographcial Notices of India.” Journal of the Royal Asiatic Society of Great Britain and Ireland, n.s., 4 (1870): 340–56. THIS DOCUMENT MAY BE PRINTED FOR PRIVATE USE ONLY AND MAY NOT BE DISTRIBUTED WITHOUT PERMISSION FROM THE PUBLISHER. eliZabeTh laMboURN 100 Elizabeth Lambourn (ELambourn@dmu.ac.uk) trained in art history and now works at the interface of history and material culture. She specializes in the study of premodern Indian Ocean communities and societies and has published widely in this area. During 2011–13, she held a Leverhulme Major Research Fellowship for the project West Asia in the Indian Ocean, 500–1500 CE (from which this article derives). She is currently finishing her monograph Abraham’s Luggage: A Social Life of Things in the Indian Ocean World. Abstract This article explores ways that the concept of equine cultures, devel- oped thus far principally in European and/or early modern and colonial contexts, might translate to premodern South Asia. As a first contribution to a history of equine matters in South Asia, it focuses on the maritime circulation of horses from the Middle East to Peninsular India in the thirteenth century, examining the dif- ferent ways that this phenomenon is recorded in textual and material sources and exploring their potential for writing a new, more connected history of South Asia and the Indian Ocean world. Keywords Horse trade, equine culture, South Asia, bahr/bahrī, India, Il Khanid Iran, Pandya Kingdom, Kakatiya dynasty, Hoysala dynasty, China, Marco Polo, Vassaf al-Hazrat, Rashid ad-Din, martial ethos. Keywords Horse trade, equine culture, South Asia, bahr/bahrī, India, Il Khanid Iran, Pandya Kingdom, Kakatiya dynasty, Hoysala dynasty, China, Marco Polo, Vassaf al-Hazrat, Rashid ad-Din, martial ethos.
https://openalex.org/W2950542606	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0214583&type=printable	English	null	Development of a prognostic risk score to aid antibiotic decision-making for children aged 2-59 months with World Health Organization fast breathing pneumonia in Malawi: An Innovative Treatments in Pneumonia (ITIP) secondary analysis	PloS one	2,019	cc-by	6,774	RESEARCH ARTICLE Editor: Peter Collignon, Canberra Hospital, AUSTRALIA Due to increasing antimicrobial resistance in low-resource settings, strategies to rationalize antibiotic treatment of children unlikely to have a bacterial infection are needed. This study’s objective was to utilize a database of placebo treated Malawian children with World Health Organization (WHO) fast breathing pneumonia to develop a prognostic risk score that could aid antibiotic decision making. Received: September 13, 2018 Accepted: March 14, 2019 Published: June 20, 2019 Received: September 13, 2018 Accepted: March 14, 2019 Published: June 20, 2019 Copyright: © 2019 McCollum 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. Eric D. McCollumID1,2, Siobhan P. Brown3, Evangelyn Nkwopara4, Tisungane Mvalo5,6, Susanne May3, Amy Sarah Ginsburg4 Eric D. McCollumID1,2, Siobhan P. Brown3, Evangelyn Nkwopara4, Tisungane Mvalo5,6, Susanne May3, Amy Sarah Ginsburg4 1 Eudowood Division of Pediatric Respiratory Sciences, Department of Pediatrics, School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America, 2 Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America, 3 Department of Biostatistics, University of Washington Clinical Trial Center, Seattle, Washington, United States of America, 4 Save the Children, Fairfield, Connecticut, United States of America, 5 University of North Carolina Project, Lilongwe Medical Relief Fund Trust, Lilongwe, Malawi, 6 Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America OPEN ACCESS Citation: McCollum ED, Brown SP, Nkwopara E, Mvalo T, May S, Ginsburg AS (2019) Development of a prognostic risk score to aid antibiotic decision- making for children aged 2-59 months with World Health Organization fast breathing pneumonia in Malawi: An Innovative Treatments in Pneumonia (ITIP) secondary analysis. PLoS ONE 14(6): e0214583. https://doi.org/10.1371/journal. pone.0214583 * emccoll3@jhmi.edu Background Editor: Peter Collignon, Canberra Hospital, AUSTRALIA Editor: Peter Collignon, Canberra Hospital, AUSTRALIA Development of a prognostic risk score to aid antibiotic decision-making for children aged 2-59 months with World Health Organization fast breathing pneumonia in Malawi: An Innovative Treatments in Pneumonia (ITIP) secondary analysis a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Conclusion The model had inadequate discrimination to be appropriate for clinical application. Different strategies will likely be required for models to perform accurately among similar pediatric populations. Results funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Treatment failure or relapse occurred in 11.5% (61/526) of children included in this analysis. The final model incorporated the following predictors: heart rate terms, mid-upper arm cir- cumference, malaria status, water source, family income, and whether or not a sibling or other child in the household received childcare outside the home. The model’s area under the receiver operating characteristic score was 0.712 (95% confidence interval 0.66, 0.78) and it explained 6.1% of the variability in predicting treatment failure or relapse (R2, 0.061). For the model to categorize all children with treatment failure or relapse correctly, 77% of children without treatment failure or relapse would require antibiotics. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi Methods We conducted a secondary analysis of children randomized to the placebo group of the Innovative Treatments in Pneumonia (ITIP) fast breathing randomized, controlled, noninfer- iority trial. Participants were low-risk HIV-uninfected children 2–59 months old with WHO fast breathing pneumonia in Lilongwe, Malawi. Study endpoints were treatment failure, defined as either disease progression at any time on or before Day 4 of treatment or disease persistence on Day 4, or relapse, considered as the recurrence of pneumonia or severe dis- ease among previously cured children between Days 5 and 14. We utilized multivariable lin- ear regression and stepwise model selection to develop a model to predict the probability of treatment failure or relapse. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The Bill & Melinda Gates Foundation (OPP1105080) funded this study. EDM received support from the National Institutes of Health through the Fogarty International Center of the National Institutes of Health (K01TW009988). The 1 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Introduction According to the most recent global estimates, the greatest numbers of children under five years old who die each year live in sub-Saharan Africa [1]. Presumed bacterial pneumonia is the leading cause of child mortality with 490,000 annual deaths in Africa, and 921,000 annual deaths globally [1]. World Health Organization (WHO) guidelines define child pneumonia as a clinical syndrome with either fast breathing, lower chest wall indrawing, or general clinical danger signs in children aged 2–59 months with cough or difficult breathing [2]. Introduction of these pragmatic guidelines have markedly improved antibiotic access, contributing substan- tially to the decline in child pneumonia mortality over the past two decades in low- and mid- dle-income countries (LMICs) [3]. The more recent introduction of vaccines for Haemophilus influenzae and Streptococcus pneumoniae, two important etiologies for fatal bacterial pneumonia, to the sub-Saharan Afri- can region, have additionally accelerated this mortality decline [4]. However, these vaccines are also speculated to be driving the transition of pneumonia etiology away from more severe bacterial and viral co-infections toward less severe viral causes [5, 6]. This epidemiologic shift is suggested to be reducing the specificity of the WHO guidelines such that increasing num- bers of children with non-bacterial pneumonia are unnecessarily treated with antibiotics [7]. In parallel to this is the concerning rise of antimicrobial resistance (AMR) in LMICs [8]. Recent estimates indicate that high proportions of pathogens in sub-Saharan Africa have developed AMR to first-line antibiotics [9–12]. Innovative, pragmatic strategies that improve antibiotic stewardship among children at low risk of mortality with presumed pneumonia and are feasible to implement in LMICs are a priority [13]. In this secondary analysis of the Innovative Treatments in Pneumonia (ITIP) fast breathing randomized, controlled, noninferiority trial [14], we sought to develop a novel prognostic risk score to aid antibiotic decision making by leveraging a unique dataset that included blinded placebo treatment of children aged 2–59 months with WHO fast breathing pneumonia in the sub-Saharan African country of Malawi. The objective of this score would be to identify those children at initial clinical presentation who are at low risk of treatment failure or relapse with- out antibiotic treatment. Setting ITIP (ClinicalTrials.gov registration: NCT02760420) was a prospective, double-blind, random- ized, controlled, noninferiority trial of 1,126 HIV-uninfected children aged 2–59 months with WHO fast breathing pneumonia conducted between 2016–2017 in Lilongwe, Malawi. The tri- al’s primary aim was to determine if placebo treatment was noninferior to three days of amoxi- cillin treatment. This secondary analysis utilized data from children enrolled in the placebo group of this trial with outcome data. ITIP1 took place in the capital city of Lilongwe within the outpatient department of Bwaila District Hospital (BDH), which provides primary health- care, and the outpatient department and pediatric wards of Kamuzu Central Hospital (KCH), the tertiary referral hospital for the central region of Malawi. Introduction This score would serve to complement current WHO guidelines in 2 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi deciding whether or not to prescribe antibiotics to low risk children in LMICs with pneumonia characterized by fast breathing only and who are without other established risk factors associ- ated with poor outcomes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 https://doi.org/10.1371/journal.pone.0214583.t001 Participants All caregivers of participant children provided written informed consent. The ITIP1 fast breathing pneumonia case definition was consistent with WHO guidelines, and as shown in the Table 1, eligibility criteria included low risk children aged 2–59 months with cough or Table 1. Study eligibility criteria. Eligibility criteria Inclusion criteria • 2–59 months of age • Cough <14 days or difficulty breathing Fast-breathing for age, defined as a respiratory rate 50 breaths per minute for children 2 to <12 months old or 40 breaths per minute for children 12 months old Exclusion criteria • Severe respiratory distress (grunting, nasal flaring, head nodding, and/or chest-indrawing) • Hypoxemia, an arterial oxyhemoglobin saturation <90% in room air, as assessed non-invasively by a pulse oximeter • Resolution of fast breathing after bronchodilator challenge, if wheezing at screening examination • General danger signs (lethargy or unconsciousness, convulsions, vomiting everything, inability to drink or breastfeed, stridor when calm) • HIV-1 seropositivity • HIV-1 exposure (a child <24 months of age with a HIV-infected mother) • Severe acute malnutrition (weight for height/length < -3 SD, mid-upper arm circumference <11.5 cm, or peripheral edema) Possible tuberculosis (coughing 14 days) • Anemia with hemoglobin <8.0 g/dL • Severe malaria (positive malaria rapid diagnostic test with any general danger sign, stiff neck, abnormal bleeding, clinical jaundice, or hemoglobinuria) • Known allergy to penicillin or amoxicillin • Receipt of an antibiotic treatment in the 48 hours prior to the study • Hospitalized within 14 days prior to the study • Living outside the study area • Any medical or psychosocial condition or circumstance that, in the opinion of the investigators, would interfere with the conduct of the study or for which study participation might jeopardize the child’s health • Any non-pneumonia acute medical illness which requires antibiotic treatment per local standard of care • Participation in a clinical study of another investigational product within 12 weeks prior to randomization or planning to begin participation during this study • Prior participation in the study during a previous pneumonia diagnosis Table 1. Study eligibility criteria. https://doi.org/10.1371/journal.pone.0214583.t001 3 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi difficult breathing <14 days who also had fast breathing for age. Outcome definitions Treatment failure was defined as occurring anytime on or before study Day 4. For children without treatment failure on Day 4, they were considered to have relapsed if they were found to have recurrence of signs of pneumonia or severe disease on or before Day 14 (Table 2). Participants Enrolled children who did not meet all eligibility criteria were excluded from this secondary analysis, as were children missing the outcome or key variables. Data collection ITIP study clinicians, called clinical and medical officers, as well as nurses trained to a stan- dardized screening protocol under the supervision of an ITIP study pediatrician, were based at BDH and KCH, and assessed children aged 2–59 months with fast breathing pneumonia for trial eligibility. If children were found eligible, they were randomized double-blinded in a 1:1 ratio to three days of either placebo or amoxicillin treatment. To optimize child safety, all study children were monitored at KCH for 2–8 hours after enrollment. Study staff discontin- ued monitoring after two hours for children without fever who were breathing slower than at enrollment. Those children <6 months old, with a mid-upper arm circumference (MUAC) 11.5–13.5cm, or febrile with a negative malaria test were monitored overnight. At any point during monitoring, if a child clinically deteriorated, then parenteral antibiotics were initiated, and the child was classified as treatment failure (Table 2). Study staff contacted caregivers of children not under hospital monitoring on Days 1–3 by phone, and assessed children in person at the study clinic or at home on Days 2–4 and Day 14. At all in person assessments, whether scheduled or not, study staff evaluated children for treat- ment failure or relapse, and study drug adherence. Analysis Continuous data were summarized with means and standard deviations, and differences between children with and without treatment failure or relapse were tested with a student’s t- test. For categorical data, we gave the number and proportion falling into each category and used Pearson chi-squared tests to compare proportions. Table 2. Treatment failure and relapse criteria. Table 2. Treatment failure and relapse criteria. Treatment failure criteria Anytime on or before Day 4 • Severe respiratory distress (grunting, nasal flaring, head nodding, and/or chest- indrawing) • Hypoxemia (arterial oxyhemoglobin saturation <90% in room air, as assessed non- invasively by a pulse oximeter) • General danger sign (lethargy or unconsciousness, convulsions, vomiting everything, inability to drink or breastfeed, stridor while calm) Missing 2 study drug doses due to vomiting • Change in antibiotics prescribed by a study clinician • Hospitalization due to pneumonia (if not initially admitted) • Prolonged hospitalization or re-admission due to pneumonia (if initially admitted) • Death On Day 4 only • Axillary temperature 38˚C in the absence of a diagnosed co-infection with fever symptoms (e.g., malaria) Relapse criteria Anytime after Day 4 • Recurrence of signs of pneumonia • Signs of severe disease https://doi.org/10.1371/journal.pone.0214583.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Table 2. Treatment failure and relapse criteria. 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi We developed a prognostic model for the probability of treatment failure or relapse using multivariable linear regression through stepwise model selection [15]. Linear regression was used to allow direct estimate of the failure probability, with robust standard error to accommo- date the binary nature of the outcome, reasonable with the given sample size. The same model was used in the primary analysis of the study. Variables with a significance level of 0.10 or lower entered the model; those with a significance level below 0.20 remained in the final model. We chose cut-points on the high end of what is used in stepwise model building as we hoped to maximize the predictive performance of the model rather than focusing on the inter- pretation of the selected variables [16, 17]. Covariates to be considered for the prognostic model were selected a priori based on previous knowledge supported either by published liter- ature or expert input. For continuous variables, the functional form included in the model building was based on bivariate modeling of each factor with the outcome. For each, we con- sidered a linear form, a spline, and categorizations selected a priori; when the spline suggested a polynomial fit, one was considered as well. The form with the highest Akaike Information Criterion (AIC) was used in the model building [18]. The use of non-linear relationships such as splines and quadratics prioritized the development of the best fitting model regardless of interpretation complexity, under the assumption that the model could be implemented during care using electronic decision support aids. Missing covariates were minimal, with the excep- tion of family income, which was explicitly modeled. We used R2, AIC, and area under the receiver operating characteristic (AUROC) to evaluate the model performance. We also assessed the model under the presumption that the prognostic risk score would only be accept- able for implementation by policy makers, healthcare providers, and caregivers if all children with treatment failure or relapse were correctly identified for antibiotic treatment. Table 2. Treatment failure and relapse criteria. Therefore, we additionally evaluated the model’s performance by determining what percentage of chil- dren the model would falsely predict with likely treatment failure or relapse, and therefore receive antibiotic treatment, in order for the model to identify all children who would become a true treatment failure or relapse case without antibiotics. SAS 9.4 was used for data manage- ment and the creation of calculated variables, while baseline data summaries were produced in R 2.14.1. Predictive model building was done in STATA 14.2. Ethics The Western Institutional Review Board in Washington, USA, the College of Medicine Research and Ethics Committee in Blantyre, Malawi, and the Malawi Pharmacy, Medicines and Poisons Board approved this study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Pfailure or relapse PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Results In ITIP, 562 children with fast breathing were randomized to the placebo group. Of these, 27 were missing treatment outcome and were excluded from this secondary analysis. A further six were excluded because they did not meet inclusion/exclusion criteria, three others were dropped because of missing covariates (Fig 1, Table 3). Overall, 11.5% (61/526) of children treated with placebo met treatment failure or relapse criteria in this dataset. Placebo recipients with treatment failure or relapse, compared to those without treatment failure or relapse, were 5.0 months younger (17.1 vs 22.1 months, p-value = 0.008), their MUAC was 0.4 cm smaller on average (14.7 cm vs 15.1 cm, p-value = 0.009), and 11.3% more were malaria negative according to rapid antigen testing (98.4% vs 87.1%, p-value = 0.018, Table 3). In addition, there was weak evidence towards a higher proportion of children with a peripheral arterial oxyhemoglobin saturation 90–95% (6.6% vs 1.9%, p-value = 0.081), a higher average PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 5 / 13 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi Fig 1. Study schema. https://doi.org/10.1371/journal.pone.0214583.g001 https://doi.org/10.1371/journal.pone.0214583.g001 respiratory rate (51.7 vs 49.8 breaths/minute, p-value = 0.061), and a difference in categories of water source between children with and without treatment failure or relapse (p-value = 0.083). In Table 4 we report a model that predicts the probability of treatment failure or relapse among children receiving placebo. Heart rate terms, MUAC, malaria status, water source, fam- ily income, and whether or not a sibling or other child in the household received childcare out- side the home were all included in the final predictive model. Specifically, an increase in MUAC, testing malaria positive, the categories of other water source and well water, relative to the piped water referent category, and missing family income were all associated with a lower risk of treatment failure or relapse. On the other hand, increasing family income and having a sibling receive childcare outside the home were associated with a statistically significant higher risk of treatment failure or relapse. Failure rates were the highest for heart rates between 120 and 170, peaking near 145 beats per minute, and reduced for higher or lower baseline heart rates. Results The model equation is: Pfailure or relapse ¼ 1:691 0:010Iwell water 0:14Iother water 0:003xMUAC þ 0:227xheartrate=10 0:008x2 heartrate=10 0:082xmalaria þ 0:062xinðfamily incomeÞ 0:036Imissing income þ 0:045Isibling outside childcare ¼ 1:691 0:010Iwell water 0:14Iother water 0:003xMUAC þ 0:227xheartrate=10 0:008x2 heartrate=10 0:082xmalaria þ 0:062xinðfamily incomeÞ 0:036Imissing income þ 0:045Isibling outside childcare 6 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 CI indicates confidence interval; MUAC, mid-upper arm circumference. Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi Table 3. Baseline characteristics of placebo recipients according to outcome. No Treatment Failure or Relapse Treatment Failure or Relapse N = 465 N = 61 p-value Age in months, mean (SD) 22.1 (15.0) 17.1 (13.3) 0.008 Female, n (%) 255 (54.8%) 33 (54.1%) 0.978 PCV doses, n (%) 0 172 (37.0%) 19 (31.1%) 0.264 1 6 (1.3%) 0 (0.0%) 2 22 (4.7%) 6 (9.8%) 3 265 (57.0%) 36 (59.0%) Pentavalent vaccine doses, n (%) 0 171 (36.8%) 19 (31.1%) 0.351 1 7 (1.5%) 0 (0.0%) 2 25 (5.4%) 6 (9.8%) 3 262 (56.3%) 36 (59.0%) Mother’s education, n (%) None/Primary/Missing 214 (46.0%) 25 (41.0%) 0.544 Secondary/Tertiary 251 (54.0%) 36 (59.0%) Maternal HIV status, n (%) Negative 435 (93.5%) 57 (93.4%) 0.780 Positive 17 (3.7%) 3 (4.9%) Unknown 13 (2.8%) 1 (1.6%) Mother’s age in years, mean (SD) 25.6 (5.2) 25.7 (5.2) 0.923 Family income in thousands of Malawi kwacha, mean (SD) 59.8 (74.5) 87.9 (94.6) 0.118 Log family income in Malawi kwacha, mean (SD) 10.6 (0.8) 11.0 (0.8) 0.016 Missing family income, n (%) 271 (58.3%) 29 (47.5%) 0.146 Weight-for-height Z-score, mean (SD) 0.4 (1.2) 0.6 (1.3) 0.373 MUAC in cm, mean (SD) 15.1 (1.2) 14.7 (1.1) 0.009 Oxygen saturation, mean (SD) 98.7 (1.4) 98.6 (1.6) 0.681 Oxygen saturation 95%, n (%) 9 (1.9%) 4 (6.6%) 0.081 Dehydration, n (%) 9 (1.9%) 3 (4.9%) 0.312 Malaria rapid diagnostic test positive, n (%) 60 (12.9%) 1 (1.6%) 0.018 Respiratory rate in breaths per minute, mean (SD) 49.8 (7.3) 51.7 (7.2) 0.061 Respiratory rate category1, n (%) Fast 324 (69.7%) 41 (67.2%) 0.868 Very fast 131 (28.2%) 19 (31.1%) Extremely fast 10 (2.2%) 1 (1.6%) Temperature in˚C, mean (SD) 37.4 (1.0) 37.3 (1.0) 0.410 Diarrhea, n (%) 42 (9.0%) 7 (11.5%) 0.702 Heart rate in beats/min, mean (SD) 141.8 (17.8) 143.3 (13.6) 0.459 Hemoglobin in g/dL, mean (SD) 10.9 (1.2) 10.9 (1.3) 0.858 Lives with smoker, n (%) 45 (9.3%) 8 (12.7%) 0.540 Water source, n (%) 0.083 Other 17 (3.7%) 2 (3.3%) Piped 413 (88.8%) 59 (96.7%) Well 35 (7.5%) 0 (0.0%) Cooking smoke inside, n (%) 440 (94.6%) 58 (95.1%) 0.878 Table 3. Baseline characteristics of placebo recipients according to outcome. 7 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Sibling receives care out of home, n (%) SD indicates standard deviation; PCV, pneumococcal conjugate vaccine; HIV, human immunodeficiency virus. 1For ages 2–11 months: 50–59, 60–69, and 70 breaths/minute. For 12–59 months: 40–49, 50–59, and 60 breaths/min We found that this model explained just 6.1% of the variability in predicting treatment fail- ure or relapse (R2, 0.061) and achieved a goodness of fit, as measured by the c-statistic (AUROC), of 0.712 (95% confidence interval 0.66, 0.78). Both of these model fit indicators imply modest model performance overall. Furthermore, we found that in order to correctly classify all children who developed treatment failure or relapse in this dataset, we would need to incorrectly treat 77% of those children with antibiotics who did not develop treatment fail- ure or relapse without antibiotic treatment. While we considered applying less restrictive crite- ria to allow some children to develop treatment failure or relapse without antibiotics, the substantial overlap in prognostic risk scores across the two outcome groups meant–even with less restrictive criteria–the proportion of false positives would change little. A risk prediction score plot visually depicts the density of the relative overlap between children with and without treatment failure or relapse, again suggesting that the model inadequately discriminates between these two outcomes (Fig 2). Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi Table 3. (Continued) Table 3. (Continued) Table 3. (Continued) No Treatment Failure or Relapse Treatment Failure or Relapse N = 465 N = 61 p-value Sibling receives care out of home, n (%) 239 (51.4%) 38 (62.3%) 0.143 SD indicates standard deviation; PCV, pneumococcal conjugate vaccine; HIV, human immunodeficiency virus. 1For ages 2–11 months: 50–59, 60–69, and 70 breaths/minute. For 12–59 months: 40–49, 50–59, and 60 breaths/minute. https //doi org/10 1371/jo rnal pone 0214583 t003 1Missing compared to median known income. PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 tps://doi.org/10.1371/journal.pone.0214583.t004 https://doi.org/10.1371/journal.pone.0214583.t004 Discussion We leveraged a unique dataset of placebo treated Malawian children in an effort to develop a novel prognostic risk score to complement current WHO guidelines in deciding which 2–59 month olds with fast breathing do or do not require antibiotics in LMICs. Given incorrect non-treatment of a bacterial infection with antibiotics can be fatal in settings where many chil- dren may only access healthcare once, a prognostic risk score to facilitate a “watch and wait” antibiotic treatment approach requires high accuracy for it to be acceptable to caregivers, healthcare providers, or policy makers in LMICs. It is against this backdrop that we found our Table 4. Model predicting the risk of treatment failure or relapse among placebo recipients. Effect Estimated Risk Difference 95% CI P value Piped water Reference Other water source -0.014 -0.152, 0.125 0.846 Well water -0.100 -0.135, -0.065 <0.001 MUAC -0.003 -0.005, -0.001 0.008 Heart rate (10 beats per minute) 0.227 0.108, 0.346 <0.001 (Heart rate/10)2 -0.008 -0.012, -0.004 <0.001 Malaria -0.082 -0.128, -0.037 <0.001 Log family income 0.062 0.009, 0.116 0.022 Missing family income1 -0.036 -0.091, 0.018 0.192 Sibling receives childcare outside home 0.045 -0.008, 0.098 0.099 Intercept -1.691 -2.738, -0.643 0.002 Table 4. Model predicting the risk of treatment failure or relapse among placebo recipients. 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi Fig 2. Risk prediction score plot. Fig 2. Risk prediction score plot. https://doi.org/10.1371/journal.pone.0214583.g002 model to be inadequate for clinical application as the model performed only modestly, and did not have sufficient discrimination between outcomes. Application of our model would result in the incorrect treatment of 77% of fast breathing children with antibiotics in order to ensure no fast breathing child who would otherwise progress to treatment failure or relapse went without treatment. In other words, use of this model would theoretically reduce the number needed to treat to prevent one treatment failure or relapse episode from 33 to 26. Prognostic risk scores have been implemented in resource rich hospital settings to identify children at risk of death [19], and similarly designed scores are now emerging from LMICs [20]. Scores from hospitalized children have been developed using databases from Columbia, Ghana, India, Kenya, Malawi, Mexico, Pakistan, South Africa, Vietnam, and Zambia [21–25]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Discussion Only one score from South Africa has been successfully externally validated [21, 23], and only one score in Malawi has been implemented, with results suggesting a reduction in hospital mortality post-implementation [26]. Notably, the South African RISC score, which included one score for HIV-infected and another score for HIV-uninfected children with pneumonia, achieved a c-statistic of 0.77 (HIV-infected model) and 0.92 (HIV-uninfected model) during development, a c-statistic of 0.74 to 0.92 (HIV-uninfected) and 0.66 to 0.73 (HIV-infected) PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 9 / 13 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi during internal validation, and a c-statistic of 0.72 during external validation on a Malawi data- set [21, 23]. While our prognostic risk score achieved a similar c-statistic of 0.71, identifying children at low risk for poor outcomes requires much higher discrimination to be safely implementable. Interestingly, this same standard may not apply to adult pneumonia populations. In a large study of hospitalized adults in the United States a prediction score was developed and validated to stratify patients into categories of low or high risk of death [27]. The authors reported a c- statistic of 0.84 during score development and 0.83 during external validation, and concluded that their score accurately predicted adults with pneumonia who are at low risk of death and did not require hospitalization [27]. Despite achieving high accuracy, seven deaths were still included in the model’s low risk category [27]. Furthermore, to achieve this accuracy, the score was comprised of 20 characteristics including comprehensive medical histories, six invasive blood samples including an arterial blood gas, and chest radiography [27]. This level of diag- nostic complexity is not feasible in the vast majority of LMIC primary care settings where detailed medical histories are rarely available and laboratory and radiographic access is limited. Nevertheless, given that innovative approaches to improve antibiotic stewardship in LMICs remain a top priority, prognostic risk scores to identify low risk patients that can be managed without antibiotics warrants continued exploration in similar adult populations or other pedi- atric pneumonia patient groups. Children with an acute illness characterized by only fast breathing may be an especially challenging population to develop and validate an accurate prognostic risk score. PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Supporting information S2 File. Data discionary. (XLSX) S2 File. Data discionary. (XLSX) Discussion Fast breath- ing among children under the age of five years is non-specific with many possible causes, some of which are pathologic and some of which are benign. The spectrum of more pathologic causes includes bacterial lower respiratory tract infection, which requires antibiotics, while the spectrum of more benign causes includes anxiety, which does not require antibiotics. In addi- tion, counting respirations in a child can be a vexing task to even the most skilled clinicians, requiring one full minute of focused attention on the chest’s subtle rise and fall in an often moving, agitated, anxious, or crying child. Many caregivers are reluctant to fully expose the chest of their child and many time-constrained healthcare providers do not consistently count respirations for a full minute, both of which further reduce the reliability of respiratory rate counts [28, 29]. These issues pose substantial barriers to creating a sufficiently discriminative score from demographic variables and clinical markers. One previous study, also from Malawi, sought to develop a prognostic risk score to predict treatment failure among 2–59 month old children with fast breathing [30]. In this study trained community health workers diagnosed children with fast breathing and prescribed antibiotics [30]. While our model performed better in comparison, the previous study’s authors also concluded that their model had an unaccept- ably low discrimination (c-statistic 0.56) that prevented clinical implementation [30]. Taken together, these results suggest that a predictive score among a population of fast breathing chil- dren is unlikely to be sufficiently accurate without collecting invasive biospecimens or imaging. This secondary analysis has several limitations. First, the ITIP fast breathing trial excluded most children with known risk factors for death such that the dataset may not be representa- tive of all Malawian children with fast breathing. However, current WHO guidelines require hospitalization of high risk children with fast breathing, and this population was not our target group [2]. Second, the outcomes of treatment failure and relapse are themselves relatively non- specific for death, and many children meeting criteria for these endpoints improve without any intervention [31]. Given no placebo recipients participating in this trial died, these end- points were the only outcomes available. Acknowledgments We thank all study participant children, caregivers, and the local community in Lilongwe, Malawi for their participation. We also thank the University of North Carolina Project, Lilongwe Medical Relief Fund Trust study staff for providing dedicated patient care, Triclin- ium Clinical Development for providing data management and safety monitoring, and the Malawi Ministry of Health for their support. Discussion Additionally, treatment failure and relapse remain clinically relevant in real-world practice as current WHO guidelines recommend treatment PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 10 / 13 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi changes when they occur [2]. Third, treatment failure and relapse occurred relatively infre- quently, and this limited our ability to evaluate all potential risk factors in our model. Addi- tional studies from Pakistan that include placebo treatment of fast breathing children have been completed, and future data pooling for a meta-analysis may help to address this limita- tion [32, 33]. Lastly, since the primary aim of the project was to develop a predictive model and assess its accuracy, we did not adjust for multiple comparisons or use any other methods to reduce the risk of type I error. Indeed, the model building methods we utilized are known to result in biased point estimates and inflated p-values, so any direct interpretation of those met- rics should be done with caution [34]. In conclusion, while the outcomes of this study’s placebo group shed light on the natural history of children with WHO fast breathing pneumonia in a sub-Saharan African setting, model development to accurately predict treatment failure or relapse was unsuccessful. Next steps may include data pooling with other similarly unique datasets or development of prog- nostic risk scores based on minimally invasive biospecimens or imaging that could be feasible for implementation in LMICs. References 1. Liu L, Oza S, Hogan D, Chu Y, Perin J, Zhu J, et al. Global, regional, and national causes of under-5 mortality in 2000–15: an updated systematic analysis with implications for the Sustainable Development Goals. Lancet. 2017; 388:3027–35. 2. World Health Organization. Integrated Management of Childhood Illness: Chart Booklet. World Health Organization: Geneva. 2014. Available from: http://apps.who.int/iris/bitstream/handle/10665/104772/ 9789241506823_Chartbook_eng.pdf;jsessionid=186666A47ABF139612503B0572A61B43? sequence=16. Cited 3 September 2018. 3. Sazawal S, Black RE. Effect of pneumonia case management on mortality in neonates, infants, and pre- school children: a meta-analysis of community-based trials. Lancet Infect Dis. 2003; 3:547–56. PMID: 12954560 4. Wahl B, O’Brien KL, Greenbaum A, Majumder A, Liu L, Chu Y, et al. Burden of Streptococcus pneumo- niae and Haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000–15. Lancet Glob Health. 2018; 6:e744–e57. https://doi.org/ 10.1016/S2214-109X(18)30247-X PMID: 29903376 5. Madhi SA, Klugman KP, Vaccine Trialist G. A role for Streptococcus pneumoniae in virus-associated pneumonia. Nat Med. 2004; 10:811–3. https://doi.org/10.1038/nm1077 PMID: 15247911 6. McCollum ED, Nambiar B, Deula R, Zadutsa B, Bondo A, King C, et al. Impact of the 13-Valent Pneu- mococcal Conjugate Vaccine on Clinical and Hypoxemic Childhood Pneumonia over Three Years in Central Malawi: An Observational Study. PLoS ONE. 2017; 12:e0168209. https://doi.org/10.1371/ journal.pone.0168209 PMID: 28052071 7. McCollum ED, King C, Hollowell R, Zhou J, Colbourn T, Nambiar B, et al. Predictors of treatment failure for non-severe childhood pneumonia in developing countries—systematic literature review and expert survey—the first step towards a community focused mHealth risk-assessment tool? BMC Pediatr. 2015; 15:74. https://doi.org/10.1186/s12887-015-0392-x PMID: 26156710 8. World Health Organization. Antimicrobial Resistance Global Report on Surveillance. World Health Organization: Geneva. 2014. Available from: http://apps.who.int/iris/bitstream/handle/10665/112642/ 9789241564748_eng.pdf?sequence=1. Cited 3 September 2018. 9. Musicha P, Cornick JE, Bar-Zeev N, French N, Masesa C, Denis B, et al. Trends in antimicrobial resis- tance in bloodstream infection isolates at a large urban hospital in Malawi (1998–2016): a surveillance study. Lancet Infect Dis. 2017; 17:1042–52. https://doi.org/10.1016/S1473-3099(17)30394-8 PMID: 28818544 10. Dramowski A, Cotton MF, Rabie H, Whitelaw A. Trends in paediatric bloodstream infections at a South African referral hospital. BMC Pediatr. 2015; 15:33. https://doi.org/10.1186/s12887-015-0354-3 PMID: 25884449 11. Mandomando I, Sigauque B, Morais L, Espasa M, Valles X, Sacarlal J, et al. Antimicrobial drug resis- tance trends of bacteremia isolates in a rural hospital in southern Mozambique. Am J Trop Med Hyg. 2010; 83:152–7. https://doi.org/10.4269/ajtmh.2010.09-0578 PMID: 20595494 12. Author Contributions Author Contributions Conceptualization: Eric D. McCollum, Siobhan P. Brown, Evangelyn Nkwopara, Susanne May, Amy Sarah Ginsburg. Data curation: Tisungane Mvalo. Formal analysis: Eric D. McCollum, Siobhan P. Brown, Evangelyn Nkwopara, Susanne May, Amy Sarah Ginsburg. Funding acquisition: Amy Sarah Ginsburg. Methodology: Eric D. McCollum, Susanne May, Amy Sarah Ginsburg. Project administration: Evangelyn Nkwopara, Tisungane Mvalo, Amy Sarah Ginsburg. Supervision: Eric D. McCollum, Evangelyn Nkwopara, Tisungane Mvalo, Amy Sarah Ginsburg. Validation: Siobhan P. Brown, Susanne May. Writing – original draft: Eric D. McCollum. Conceptualization: Eric D. McCollum, Siobhan P. Brown, Evangelyn Nkwopara, Susanne May, Amy Sarah Ginsburg. Data curation: Tisungane Mvalo. Formal analysis: Eric D. McCollum, Siobhan P. Brown, Evangelyn Nkwopara, Susanne May, Amy Sarah Ginsburg. Formal analysis: Eric D. McCollum, Siobhan P. Brown, Evangelyn Nkwopara, Susanne May, Amy Sarah Ginsburg. Funding acquisition: Amy Sarah Ginsburg. Funding acquisition: Amy Sarah Ginsburg. Methodology: Eric D. McCollum, Susanne May, Amy Sarah Ginsburg. Methodology: Eric D. McCollum, Susanne May, Amy Sarah Ginsburg. Project administration: Evangelyn Nkwopara, Tisungane Mvalo, Amy Sarah Ginsburg. Supervision: Eric D. McCollum, Evangelyn Nkwopara, Tisungane Mvalo, Amy Sarah Ginsburg. Validation: Siobhan P. Brown, Susanne May. Writing – original draft: Eric D. McCollum. Writing – original draft: Eric D. McCollum. 11 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi Writing – review & editing: Eric D. McCollum, Siobhan P. Brown, Evangelyn Nkwopara, Tisungane Mvalo, Susanne May, Amy Sarah Ginsburg. References Ginsburg AS, Tinkham L, Riley K, Kay NA, Klugman KP, Gill CJ. Antibiotic non-susceptibility among Streptococcus pneumoniae and Haemophilus influenzae isolates identified in African cohorts: a meta- analysis of three decades of published studies. Int J Antimicrob Agents. 2013; 42:482–91. https://doi. org/10.1016/j.ijantimicag.2013.08.012 PMID: 24139883 13. McCollum ED, King C, Hammitt LL, Ginsburg AS, Colbourn T, Baqui AH, et al. Reduction of childhood pneumonia mortality in the Sustainable Development era. Lancet Resp Med. 2016; 4:932–3. 14. Ginsburg AS, Mvalo T, Nkwopara E, McCollum ED, Ndamala CB, Schmicker R, et al. Placebo versus amoxicillin for non-severe fast-breathing pneumonia in Malawian children aged 2–59 months: a double- blind, randomized controlled non-inferiority trial. JAMA Pediatr. 2018; In press. 15. Cheung YB. A modified least-squares regression approach to the estimation of risk difference. Am J Epidemiol. 2007; 166:1337–44. https://doi.org/10.1093/aje/kwm223 PMID: 18000021 16. Hocking RR. The Analysis and Selection of Variables in Linear Regression. Biometrics. 1976; 32:1–49. 17. Steyerberg EW, Eijkemans MJ, Habbema JD. Stepwise Selection in Small Data Sets: A Simulation Study of Bias in Logistic Regression Analysis. J Clin Epidemiol. 1999; 52:935–42. PMID: 10513756 18. Akaike H. A new look at the statistical model identification. IEEE Trans Automat Contr. 1974; 19:716– 23. 12 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 Prognostic risk score development for children 2-59 months old with fast breathing pneumonia in Malawi 19. Duncan H, Hutchison J, Parshuram CS. The Pediatric Early Warning System score: a severity of illness score to predict urgent medical need in hospitalized children. J Crit Care. 2006; 21:271–8. https://doi. org/10.1016/j.jcrc.2006.06.007 PMID: 16990097 20. Deardorff KV, McCollum ED, Ginsburg AS. Pneumonia Risk Stratification Scores for Children in Low- Resource Settings: A Systematic Literature Review. Pediatr Infect Dis J. 2018; 37:743–8. https://doi. org/10.1097/INF.0000000000001883 PMID: 29278608 21. Reed C, Madhi SA, Klugman KP, Kuwanda L, Ortiz JR, Finelli L, et al. Development of the Respiratory Index of Severity in Children (RISC) score among young children with respiratory infections in South Africa. PLoS ONE. 2012; 7:e27793. https://doi.org/10.1371/journal.pone.0027793 PMID: 22238570 22. Emukule GO, McMorrow M, Ulloa C, Khagayi S, Njuguna HN, Burton D, et al. Predicting mortality among hospitalized children with respiratory illness in Western Kenya, 2009–2012. PLoS ONE. 2014; 9:e92968. https://doi.org/10.1371/journal.pone.0092968 PMID: 24667695 23. Hooli S, Colbourn T, Lufesi N, Costello A, Nambiar B, Thammasitboon S, et al. References Predicting Hospitalised Paediatric Pneumonia Mortality Risk: An External Validation of RISC and mRISC, and Local Tool Devel- opment (RISC-Malawi) from Malawi. PLoS ONE. 2016; 11:e0168126. https://doi.org/10.1371/journal. pone.0168126 PMID: 28030608 24. Mamtani M, Patel A, Hibberd PL, Tuan TA, Jeena P, Chisaka N, et al. A clinical tool to predict failed response to therapy in children with severe pneumonia. Pediatr Pulmonol. 2009; 44:379–86. https://doi. org/10.1002/ppul.21014 PMID: 19330771 25. Olson D, Davis N, Milazi R, Lufesi N, Miller W, Preidis G, et al. Development of a severity of illness scor- ing system (inpatient triage, assessment and treatment) for resource-constrained hospitals in develop- ing countries. Trop Med Int Health. 2013; 18:871–8. https://doi.org/10.1111/tmi.12137 PMID: 23758198 26. Olson D, Preidis G, Milazi R, Spinler J, Lufesi N, Mwansambo C, et al. Task shifting an inpatient triage, assessment and treatment programme improves the quality of care for hospitalised Malawian children. Trop Med Int Health. 2013; 18:879–86. https://doi.org/10.1111/tmi.12114 PMID: 23600592 27. Fine MJ, Auble TE, Yealy DM, Hanusa BH, Weissfeld LA, Singer DE, et al. A prediction rule to identify low-risk patients with community-acquired pneumonia. N Engl J Med. 1997; 336:243–50. https://doi. org/10.1056/NEJM199701233360402 PMID: 8995086 28. Edward A, Dam K, Chege J, Ghee AE, Zare H, Chhorvann C. Measuring pediatric quality of care in rural clinics-a multi-country assessment-Cambodia, Guatemala, Zambia and Kenya. Int J Qual Health Care. 2016; 28:586–93. https://doi.org/10.1093/intqhc/mzw080 PMID: 27488477 29. Bjornstad E, Preidis G, Lufesi N, Olson D, Kamthunzi P, Hosseinipour M, et al. Determining the quality of IMCI pneumonia care in Malawian children. Paediatr Int Child Health. 2014; 34:29–36. https://doi.org/ 10.1179/2046905513Y.0000000070 PMID: 24091151 30. King C, McCollum ED, Mankhambo L, Colbourn T, Beard J, Hay Burgess DC, et al. Can We Predict Oral Antibiotic Treatment Failure in Children with Fast-Breathing Pneumonia Managed at the Commu- nity Level? A Prospective Cohort Study in Malawi. PLoS ONE. 2015; 10:e0136839. https://doi.org/10. 1371/journal.pone.0136839 PMID: 26313752 31. Hazir T, Qazi SA, Nisar YB, Maqbool S, Asghar R, Iqbal I, et al. Can WHO therapy failure criteria for non-severe pneumonia be improved in children aged 2–59 months? Int J Tuberc Lung Dis. 2006; 10:924–31. PMID: 16898379 32. Hazir T, Nisar YB, Abbasi S, Ashraf YP, Khurshid J, Tariq P, et al. Comparison of oral amoxicillin with placebo for the treatment of world health organization-defined nonsevere pneumonia in children aged 2–59 months: a multicenter, double-blind, randomized, placebo-controlled trial in pakistan. Clin Infect Dis. 2011; 52:293–300. https://doi.org/10.1093/cid/ciq142 PMID: 21189270 33. PLOS ONE \| https://doi.org/10.1371/journal.pone.0214583 June 20, 2019 34. Hurvich CM, Chih-Ling T. The Impact of Model Selection on Inference in Linear Regression. Am Stat. 1990; 44:214–7. References Jehan F, Nisar MI, Kerai S, Brown N, Balouch B, Hyder Z, et al. A double blind community-based ran- domized trial of amoxicillin versus placebo for fast breathing pneumonia in children aged 2–59 months in Karachi, Pakistan (RETAPP). BMC Infect Dis. 2016; 16:13. https://doi.org/10.1186/s12879-015- 1334-9 PMID: 26758747 34. Hurvich CM, Chih-Ling T. The Impact of Model Selection on Inference in Linear Regression. Am Stat. 1990; 44:214–7. 13 / 13
https://openalex.org/W4319840126	http://uhra.herts.ac.uk/bitstream/2299/26131/1/10.1177_03080226221148414.pdf	English	null	Evidence for 24-hour posture management: A scoping review	The British journal of occupational therapy/British journal of occupational therapy	2,023	cc-by	10,407	Research Article British Journal of Occupational Therapy 2023, Vol. 86(3) 176–187 © The Author(s) 2023 https://doi.org/10.1177/03080226221148414 British Journal of Occupational Therapy 2023, Vol. 86(3) 176–187 © The Author(s) 2023 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/03080226221148414 journals.sagepub.com/home/bjot Keywords Keywords 24-hour postural management, assistive technology, postural care, national guidelines Received: 10 December 2021; accepted: 1 December 2022 Received: 10 December 2021; accepted: 1 December 2022 To reduce the negative effects of gravity and avoid the development of such complications, ‘24-hour posture man- agement’ is regarded as necessary (Hill and Goldsmith, 2010) by many therapists. Farley et al. (2003) define 24-hour postural management as utilisation of a range of interven- tions to reduce postural asymmetry and improve function, Evidence for 24-hour posture management: A scoping review https://doi.org/10.1177/030802262211484 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/03080226221148414 journals.sagepub.com/home/bjot Lauren Julia Osborne1 , Rosemary Joan Gowran2,3,4, Jackie Casey5,6 Lauren Julia Osborne1 , Rosemary Joan Gowran2,3,4, Jackie Casey5,6 Abstract Introduction: People with complex physical disabilities unable to change their position independently are at risk of developing postural deformities and secondary complications. 24-hour posture management is needed to protect body structure. With inconsistencies in current service provision, this research aimed to scope the evidence for a 24-hour posture management approach. Method: A scoping review was conducted using four health and social science databases. Inclusion and exclusion criteria were applied; further papers were included through citation chaining. Results: The evidence for 24-hour posture management was often low quality due to the complications of completing robust research studies in this complex specialty. However, many professionals in the field agree that a 24-hour approach to postural care is essential. Conclusion: There is a need for clear national policy and guidance relating to postural care and scope for development of dedicated posture management services. Current NHS service provision is variable and inconsistent. Lack of postural care is a safeguarding and human rights issue. Specialist training and research in postural care within the Occupational Therapy profession is required to raise awareness of the role Occupational Therapists can play in preventing postural deformities and other secondary complications through providing good postural care. Introduction For people with complex physical and sensory disabilities, who are unable to independently change their body position, posture and positioning become a very important issue. Their body structures are influenced by both postural alignment and the forces of gravity, which in turn affect body functions and the ability to participate in everyday activities. Posture is defined as the alignment of body segments relative to each other (Casey et al., 2020; Stinson et al., 2021). If an indi- vidual adopts an asymmetrical position, is not able to inde- pendently change position and is submitted to the forces of gravity they can develop secondary complications such as tissue damage, muscle contractures, pain and discomfort, constipation and infections (Pope, 2007); characteristics which are typically not a direct consequence of the diag- nosed impairment (Pope, 2007). Often the body structures distort because of an inability to change position and an asymmetric posture, coupled with the influence of gravita- tional forces, which compress and restrict the function of the internal organs. If left uncorrected, this can lead to further health and participation complications and even premature death (Changing our Lives, 2018; Clayton et al., 2017). 1University of Hertfordshire, Hatfield, Hertfordshire, England 2Discipline Occupational Therapy, Faculty of Education and Health Sciences, School of Allied Health, Health Research Institute, Health Implementation Science and Technology, University of Limerick, Limerick, Ireland 3School of Health and Sports Science, University of the Sunshine Coast, Queensland, Australia 4Assisting Living and Learning (ALL) Institute Maynooth University, Maynooth, Co. Kildare, Ireland 5Advanced Practitioner Occupational Therapist-Specialised Seating, Regional Rehabilitation Engineering Centre, Belfast Health & Social Care Trust, Belfast, Northern Ireland 6Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, UK Corresponding author: Corresponding author: Lauren Julia Osborne, University of Hertfordshire, College Lane, Hatfield, Hertfordshire. AL10 9AB, UK. Email: l.osborne2@herts.ac.uk 177 Osborne et al. and includes all lying, sitting and standing positions that occur within any 24-hour period. Assessment of posture throughout the day and night needs to be examined within a multi-disciplinary framework to ensure appropriate provi- sion of postural advice and supports (Castle et al., 2014; Hill and Goldsmith, 2009; Wright et al., 2010) that match the needs of both the individual and their caregivers. Techniques include the use of lying supports, standing supports and spe- cialised seating (Agustsson and Jonsdottir, 2018) to sustain comfort and symmetry across multiple orientations that a person can spend significant lengths of time over a 24-hour period. If posture is not examined collectively in each orien- tation of sitting, lying and standing, it can be counter productive to the provision of postural supports in only one orientation. short periods of time in a wheelchair that has been custom made for their complex needs and frequently have no provi- sion to address their postural needs outside of the wheelchair. There is often no funding for alternative seating or lying sup- ports. Provision is patchy and inconsistent throughout the UK; it is not typically coordinated to ensure that postural supports work in harmony to cover the whole 24-hour period. Failure to provide appropriate wheelchair seating and static seating leads to individuals being unable to reach their occu- pational potential and often not able to participate in life as an equal citizen (Gowran, 2013). At this point, posture manage- ment provision becomes an issue of safeguarding and human rights as it is seen as ‘a pre-requisite for survival and personal mobility’ (Gowran et al., 2020: 9). Therefore, the primary objective of this study is to examine the evidence for 24-hour posture management. Secondary objectives were to explore the current provision of postural care and consider recommen- dations that can improve postural management practice within the Occupational Therapy profession in the UK. From an Occupational Therapy perspective, taking a ‘whole-person approach’ to support both mental and physi- cal health and wellbeing is essential to enable individuals the opportunity to achieve their full potential (RCOT, 2019). Therefore, it is important for the profession to consider posture management as a pre-requisite (Agustsson and Jonsdottir, 2018) and fundamental to occupational perfor- mance. Method A scoping review methodology was used to synthesise the breadth of available literature and study designs employed. This method of scoping an emerging topic area that is broad, ensures that all available literature is included, including policy as well as intervention and exploratory research papers. Munn et al. (2018) suggested that scoping reviews offer a different technique for scrutinising theories and could therefore be more beneficial for practice development. As the aim of this study is to explore the use and merits of 24-hour posture management to inform practice, it is more appropriate to adopt a scoping method that captures rationale from across a range of sources. In the United Kingdom, NHS provision of postural man- agement assistive technology is ad hoc due to complicated and fragmented commissioning procedures and funding across varying models of postural management services (Aldersea, 1999; Darzi, 2018). Integration of these services as well as a joined-up approach to commissioning has long been recommended to improve efficiency and cost-effec- tiveness of equipment provision for posture management (Aldersea, 1999). Today, wheelchair services are well-estab- lished at meeting mobility and postural needs within wheeled mobility, but often there is no consideration given to the remainder of the 24-hour period, including no provision for static seating. Often, persons who need specialist static seat- ing provision outside of a wheelchair, or who do not meet the criteria for a wheelchair, will fall into a gap in service provision with their needs going unmet. In these instances, individuals and families are left to purchase static seating independently, which can often lead to expensive mistakes of purchasing ill-fitting or inappropriate seating (Collins, 2001). Further, Anderson et al. (2008) stated that scoping studies are a crucial research methodology for guiding policy mak- ers and can provide the opportunity to develop further research, policy and evidence-based practice (Colquhoun et al., 2014). Global literature was included since this is an emerging topic with patchy provision in the UK. It was felt there could be potential learning from the international community for implementing 24-hour posture management that might be relevant to the development of recommenda- tions for enhanced practice within the UK. This scoping review was structured using the PRISMA (Preferred Reporting Items for Systematic review and Meta Analyses) guidelines. The concept of posture management continues to evolve, so published research studies, policies, clinical frameworks and emerging unpublished studies and reports were included in the search. Corresponding author: For example, if an individual is unable to maintain sitting balance because their trunk is not stable, their arms will naturally seek to provide the stability required; leaving their hands unavailable to engage in task performance. They may uneconomically use energy attempting to maintain an upright position, rather than engaging in an activity. Optimal postural care often relies on therapists’ knowledge and experience (Humphreys and Pountney, 2006; Pountney et al., 2002) leading to a postcode lottery of expertise, fund- ing and timely provision of appropriate 24-hour postural management intervention. Table 1. PICO framework. Population Any person with complex and/or multiple disabilities, which result in the person being unable to independently transfer their weight, reposition themselves or move out of their base of support and back again without assistance. Any person with postural asymmetries and/or contractures. All diagnoses included. All ages included. Intervention 24-hour Posture Management Programme Provision of wheelchairs, postural armchairs, static seating, sleep systems, night-time positioning systems and/ or standing frames for the use of 24-hour posture management. Context A person’s own home. Residential and nursing homes. Schools. Outcome Reduction of postural deformities. Reduction of pressure ulcers. Improved pain, breathing, digestion, interaction, swallow, quality of life. Any person with complex and/or multiple disabilities, which result in the person being unable to independently transfer their weight, reposition themselves or move out of their base of support and back again without assistance. Any person with postural asymmetries and/or contractures. the search terms and names of known authors in the field. They were then reviewed and scanned in the same way as the retrieved published peer-reviewed literature and the same inclusion/exclusion criteria were applied. and Medline covering the range of scientific literature pub- lished in educational, medical, psychological and social sci- ences journals. An initial search of PubMed was used to test out the preliminary search terms. A PICO framework was used to identify a list of 33 keywords based on the authors’ (LO and JC) experience of the field relating to posture and types of posture management devices, such as ‘custom moulded seating’. An additional 21 keywords describing pos- tural deformities, such as ‘contracture’ and ‘hip subluxation’ were also identified (Table 1). Titles and abstracts were independently reviewed by two authors (LO and JC), then full texts obtained if the abstract appeared relevant or if it seemed unclear. The following inclusion and exclusion criteria were applied to focus the search into meaningful results. Each of the keywords listed under ‘posture management’ were searched using the Boolean operator ‘OR’ to capture all possible terms; this yielded 101,434 hits. These were then searched in association with the keywords against ‘postural deformities’ using the Boolean operator ‘AND’, yielding a total of 4387 hits. The Boolean phrase ‘AND’ was used to combine the search results, resulting in only five papers. Inclusion •• Scientific and grey literature relating to 24-hour posture management •• Literature relating to use of wheelchairs, specialised seating, night-time positioning and standing frame use within the context of 24-hour posture management •• Literature worldwide •• Written in English language only •• Dated from 2003 to 2019 •• Scientific and grey literature relating to 24-hour posture management •• Literature relating to use of wheelchairs, specialised seating, night-time positioning and standing frame use within the context of 24-hour posture management •• Literature worldwide •• Literature worldwide •• Written in English language only •• Dated from 2003 to 2019 Table 1. PICO framework. Therefore, it was decided to remove the second column of postural deformity terms and widen the search by concen- trating on the posture management keywords; using the dis- ability and diagnosis terms did not enhance the search strategy. As it is an emerging topic, scoping the breadth of the literature, including such refined terms narrowed the search results. Research that is completed on this topic is catalogued using the main heading of postural terms, rather than being diagnosis-specific. This also ensured that there was no bias towards one particular diagnosis, as postural deformity can affect anyone who is unable to move their position independently and is not determined by their medi- cal diagnosis. Method The literature review conducted by Farley et al. (2003) was used as the starting point for this review examining the evidence since 2003 for posture management interventions. There is growing evidence that supports the notion that positioning in lying has a direct relationship on the success of postural alignment in sitting. Many individuals spend Relevant scientific literature was identified using four electronic databases: CINAHL, Psych INFO, Web of Science, 178 British Journal of Occupational Therapy 86(3) Table 1. PICO framework. Exclusion •• Non-English language literature •• Dated pre-2003 •• Literature detailing product testing or development •• Literature relating to wheelchairs, specialist seating, night- time positioning and standing frames specifically, without applying the context of 24-hour posture management. As the papers from each database were reviewed, so too were their reference lists; a method referred to as ‘citation chain- ing’ (Greenhalgh, 2010). Typically, a scoping review does not critically appraise the evidence presented (Colquhoun et al., 2010; Peters et al., 2015). However, it was felt necessary to comment on the strength of the research available to demon- strate the challenges of conducting research in this field, and to consider the reasons for this and hence the importance of considering other types of literature. However, with a result of over 100,000 papers, this needed to be refined. In consultation with the health librarians all generic terms such as ‘chair’ and ‘position*’ were removed as these were felt to be too broad. Papers not relevant to the research question were excluded. The final search terms were ‘posture management’, ‘night-time positioning’, ‘seat- ing assessment’ and ‘postural seating’; these were truncated to include similar terms such as ‘posture’ and ‘postural’. The grey literature was retrieved via a Google search engine using 179 Osborne et al. Idenﬁcaon Records Excluded: Did not meet criteria (n = 55) Records Idenﬁed through database searching (n = 263) Addional records idenﬁed through other sources (n = 5) Total Arcles Idenﬁed (n = 268) Duplicates Removed (n = 87) Records Excluded: Title and abstract not relevant (n = 91) Records screened (n = 181) Inclusion/Exclusion Criteria Applied to Full Texts (n = 90) Addional papers from reference lists Included (n = 9) Total papers Included (n = 44) Screening Eligibility Included Figure 1. PRISMA Diagram showing search results. Addional records idenﬁed through other sources Total Arcles Idenﬁed Records Excluded: Did not meet criteria Addional papers from reference lists Included Total papers Included (n = 44) Total papers Included Total papers Included Figure 1. PRISMA Diagram showing search results. The papers were read multiple times and the primary author was fully immersed in the literature. Exclusion A simple catego- risation system by Wallace and Wray (2006), cited in Aveyard (2014) was used to categorise the literature type and then an interpretive phenomenological approach was used to identify common themes arising across all of the literature. Types of papers Of the 44 papers included, 4 were classified as theoretical literature, 25 as research literature, 13 as practice literature, and 2 as policy literature. See Table 2. Study design 1. Theoretical literature – developing theories through examining evidence. There were no RCTs and only one quasi-experimental design amongst the included research papers, supporting the view these research methodologies are not appropriate for this population and clinical field (see Table 3). 2. Research literature – reporting a specific enquiry into a topic or treatment method. 3. Practice literature – expert opinion, debate, discussion papers from industry experts. Most papers were systematic literature reviews, con- cluding that the research available was weak in design and of low quality (Blake et al., 2015; Gmelig Meyling et al., 2017; Humphreys et al., 2019; O’Connor et al., 2006) and stipulated that further research was recommended such as development of a consensus statement of expert opinion and regular publishing of case studies from clinical practice (Farley et al., 2003; Wynn and Wickham, 2009). 4. Policy literature – national or local policies and guide- lines that inform practice. (Wallace and Wray, 2006 cited in Aveyard, 2014: 44). Results (2017) Prevention and postural management of residents in care homes. RCOT Annual Conference, pp. 109. Practice Daly, O., Casey, J. and Martin, S. (2013) The effectiveness of specialist seating provision for nursing home residents. A report for Seating Matters Ltd. Knowledge Transfer Partnership (KTP) Sept 2011-Sept 2013. Research Day, E. and Lockwood, D. (2012) The impact of posture management on two adults with learning disabilities. RCOT Annual Conference, pp. 49. Practice Farley, R., Clark, J., Davidson, C., Evans, G., Maclennan, K., Michael, S., Morrow, M. and Thorpe, S. (2003) What is the evidence for the effectiveness of postural management? International Journal of Therapy and Rehabilitation. Vol 10, No 10. Pg 449-455. Research Fields, F. & McGrath, M. (2008) 24 hour postural management: a new direction in therapeutic care. Physiotherapy Ireland, 2008 Jun; 29(1): 75-75. Practice Gmelig Meyling, C., Ketelaar, M., Kuijper, M., Voorman, J. and Buizer, A. (2017) Postural management to reduce or prevent hip migration in children with cerebral palsy: a systematic review. Developmental Medicine & Child Neurology. Pediatric Physical Therapy vol. 30,2: 82-91. Research Goodwin, J., Lecouturier, J., Basu, A., Colver, A., Crombie, S., Smith, J., Howel, D., McColl, E., Parr, J. R., Kolehmainen, N., Roberts, A., Miller, K. and Cadwgan, J. (2018a) Standing frames for children with cerebral palsy: a mixed-methods feasibility study. NIHR Health Technology Assessment. Vol 22, Issue 50. Research Goodwin, J., Lecouturier, J., Crombie, S., Smith, J., Basu, A., Colver, A., Kolehmainen, N., Parr, J. R., Howel, D., McColl, E., Roberts, A., Miller, K. and Cadwgan, J. (2018b) Understanding frames: A qualitative study of young people's experiences of using standing frames as part of postural management for cerebral palsy. Child: care, health and development, 44(2), pp. 203-211. Research Goodwin, J., Lecouturier, J., Smith, J., Crombie, S., Basu, A., Parr, J. R., Howel, D., McColl, E., Roberts, A., Miller, K. and Cadwgan, J. (2019) Understanding frames: A qualitative exploration of standing frame use for young people with cerebral palsy in educational settings. Child: Care, Health and Development, 45(3), pp. 433-439. Research Gough, M. (2009) Continuous postural management and the prevention of deformity in children with cerebral palsy: An appraisal. Developmental Medicine & Child Neurology, Vol 51(2), Feb, 2009 pp. 105-110. Theoretical Hill, C. M., Parker, R. C., Allen, P., Paul, A. and Padoa, K. A. (2009) Sleep quality and respiratory function in children with severe cerebral palsy using night-time postural equipment: a pilot study. Results A total of 268 papers were retrieved, with 44 meeting the inclusion criteria. Of these, half were centred around a view of 24-hour posture management, or posture management in general, 10 were focused on lying postures and night-time positioning, 8 on sitting posture and seating assessment and 4 on standing programmes (see Figure 1). Whilst the evidence body is small, it is growing and does appear to support 24-hour posture management (Wynn and Wickham, 2009). The systematic review by Blake et al. (2015) also noted that many papers included in their review were not indexed on electronic databases and were retrieved British Journal of Occupational Therapy 86(3) 180 Table 2. Included papers: Categorised according to Literature category. Casey, J., Hoffman, L., Hutson, J. and Kittelson-Aldred, T. (2019) Supporting the occupation of sleep through night-time positioning equipment. SIS Quarterly Practice Connections, 4 (2), pp. 7-9. Practice Casey, J., Hoffman, L., Hutson, J. and Kittelson-Aldred, T. (2019) Supporting the occupation of sleep through night-time positioning equipment. SIS Quarterly Practice Connections, 4 (2), pp. 7-9. Practice Castle, D., Stubbs, B., Clayton, S. and Soundy, A. (2014) A 24-hour postural care service: Views, understanding and training needs of referring multidisciplinary staff. International Journal of Therapy and Rehabilitation, 21(3), pp. 132-139. Research Castle, D., Stubbs, B., Clayton, S. and Soundy, A. (2014) A 24-hour postural care service: Views, understanding and training needs of referring multidisciplinary staff. International Journal of Therapy and Rehabilitation, 21(3), pp. 132-139. Research Clayton, S. (2013) Living Local Postural Care Project Evaluation. Postural Care CIC. ayton, S. (2013) Living Local Postural Care Project Evaluation. Postural Care CIC. Prac Collins, F. (2008) An essential guide to managing seated patients in the community. British Journal of Community Nursing, 13(3), pp. 39-40. Practice Collins, F. (2008) An essential guide to managing seated patients in the community. British Journal of Community Nursing, 13(3), pp. 39-40. Practice Crawford, S. and Curran, A. (2014) 24 Hour Postural Management for Community Dwelling Adults with Learning Disabilities. The Journal of Posture and Mobility Group, 31, pp. 15-19. Research Crawford, S. and Curran, A. (2014) 24 Hour Postural Management for Community Dwelling Adults with Learning Disabilities. The Journal of Posture and Mobility Group, 31, pp. 15-19. Research Crawford, S. and Stinson, M. (2015) Management of 24-h-Body Positioning. In: Söderback, I. (ed.) International Handbook of Occupational Therapy Interventions. Cham: Springer International Publishing, pp. 189-203. T Daly, G. (Continued) Results RCOT Annual Conference, pp. 72. Practic Lyons, E. et al. (2017) An exploration of comfort and discomfort amongst children and young people with intellectual disabilities who depend on postural management equipment. Journal of Applied Research in Intellectual Disabilities, Vol 30(4), Jul, 2017 pp. 727-742. Research Lyons, E. et al. (2017) An exploration of comfort and discomfort amongst children and young people with intellectual disabilities who depend on postural management equipment. Journal of Applied Research in Intellectual Disabilities, Vol 30(4), Jul, 2017 pp. 727-742. Research Macias-Merlo, L. et al. (2016) Effects of the standing program with hip abduction on hip acetabular development in children with spastic diplegia cerebral palsy. Disability And Rehabilitation Volume: 38 Issue: 11 Pages: 1075-1081 Published: MAY 21 2016 Research Maher, C. et al. (2011) Factors influencing postural management for children with cerebral palsy in the special school setting. Disability and Rehabilitation: An International, Multidisciplinary Journal, Vol 33(2), 2011 pp. 146-158. Research McDonald, R., Surtees, R. and Wirz, S. (2004) The International Classification of Functioning, Disability and Health provides a Model for Adaptive Seating Interventions for Children with Cerebral Palsy. British Journal of Occupational Therapy, 67(7):293-302. Practice O'Connor, B., Boyd, R. N. and Shields, N. (2006) A systematic review of postural management of hip displacement in children with cerebral palsy. Developmental Medicine & Child Neurology, (48), pp. 42-43. Research Owens, K. & Daly, G. (2016) A study into the effectiveness of 24 hour postural care in the management of contractures in Care Homes. Middlesborough Council. Research Owens, K. & Daly, G. (2016) A study into the effectiveness of 24 hour postural care in the management of contractures in Care Homes. Middlesborough Council. Research Polak, F., Clift, M. and Clift, L. (2009) Buyers' Guide. Night-time postural management equipment for children CEP08030.: London: NHS Centre for Evidence-based Purchasing. Policy Polak, F., Clift, M. and Clift, L. (2009) Buyers' Guide. Night-time postural management equipment for children CEP08030.: London: NHS Centre for Evidence-based Purchasing. Policy Pountney, T. et al. (2009) Hip subluxation and dislocation in cerebral palsy – a prospective study on the effectiveness of postural management programmes. Physiotherapy Research International: The Journal for Researchers And Clinicians In Physical Therapy, 2009 Jun; Vol. 14 (2), pp. 116-27. Research Pountney, T. et al. (2009) Hip subluxation and dislocation in cerebral palsy – a prospective study on the effectiveness of postural management programmes. Results Journal of Applied Research in Intellectual Disabilities, Vol 30(4), Jul, 2017 pp. 727-742. Research Macias-Merlo, L. et al. (2016) Effects of the standing program with hip abduction on hip acetabular development in children with spastic diplegia cerebral palsy. Disability And Rehabilitation Volume: 38 Issue: 11 Pages: 1075-1081 Published: MAY 21 2016 Research Maher, C. et al. (2011) Factors influencing postural management for children with cerebral palsy in the special school setting. Disability and Rehabilitation: An International, Multidisciplinary Journal, Vol 33(2), 2011 pp. 146-158. Research McDonald, R., Surtees, R. and Wirz, S. (2004) The International Classification of Functioning, Disability and Health provides a Model for Adaptive Seating Interventions for Children with Cerebral Palsy. British Journal of Occupational Therapy, 67(7):293-302. Practice O'Connor, B., Boyd, R. N. and Shields, N. (2006) A systematic review of postural management of hip displacement in children with cerebral palsy. Developmental Medicine & Child Neurology, (48), pp. 42-43. Research Owens, K. & Daly, G. (2016) A study into the effectiveness of 24 hour postural care in the management of contractures in Care Homes. Middlesborough Council. Research Polak, F., Clift, M. and Clift, L. (2009) Buyers' Guide. Night-time postural management equipment for children CEP08030.: London: NHS Centre for Evidence-based Purchasing. Policy Pountney, T. et al. (2009) Hip subluxation and dislocation in cerebral palsy – a prospective study on the effectiveness of postural management programmes. Physiotherapy Research International: The Journal for Researchers And Clinicians In Physical Therapy, 2009 Jun; Vol. 14 (2), pp. 116-27. Research Robertson, J. et al. (2018) Postural care for people with intellectual disabilities and severely impaired motor function: A scoping review. Research in Intellectual Disabilities, Vol 31(Suppl 1), Jan, 2018 pp. 11-28. Research Seabrook, R. (2017) Specialist seating - a model to increase efficiencies in provision and meet a growing demand. RCOT Annual Conference, pp. 114. Practice Stephens, M., Bartley, C. and Priestley, C. (2018) Night-time Positioning for Care Home Residents. [Online} URL: https://pdfs.semanticscholar.org/fae9/f3c7670c8226384c266aa5562f0e24249d49.pdf (Accessed 18/01/20). Research Wright, C., Casey, J. and Porter-Armstrong, A. (2010) Establishing best practice in seating assessment for children with physical disabilities using qualitative methodologies. Disability and Rehabilitation: Assistive Technology, 5(1), pp. 34-47. Research Wynn, N. and Wickham, J. (2009) Night-time positioning for children with postural needs: What is the evidence to inform best practice? The British Journal of Occupational Therapy., 40(12), pp. 543. Research Table 2. Results (Continued) Paper Literature Type Humphreys, G., King, T., Jex, J., Rogers, M., Blake, S., Thompson-Coon, J. and Morris, C. (2019) Sleep positioning systems for children and adults with a neurodisability: A systematic review. British Journal of Occupational Therapy, 82(1), pp. 5-14. Research Hutton, E. and Coxon, K. (2011) ‘Posture for Learning’: meeting the postural care needs of children with physical disabilities in mainstream primary schools in England – a research into practice exploratory study. Disability and Rehabilitation, 33:19-20. 1912-1914. Research Innocente, R. (2014) Night-time positioning equipment: A review of practices. New Zealand Journal of Occupational Therapy, 61(1), pp. 13-20. Practice Jones, C., Underhill, M. and Baylis, M. (2015) Service transformation through integration: specialist seating model for children. RCOT Annual Conference, pp. 77. Practice Kent, F. (2015) 24-hour postural management: all about seating 1 – practical considerations. National Association of Equipment Providers, Oct 2015; 98-103. Practice Lansdown, K. and Dawson, K. (2019) Are you sitting comfortably: An evaluation of a postural management masterclass for occupational therapy staff. RCOT Annual Conference, pp. 72. Practice Lyons, E. et al. (2017) An exploration of comfort and discomfort amongst children and young people with intellectual disabilities who depend on postural management equipment. Journal of Applied Research in Intellectual Disabilities, Vol 30(4), Jul, 2017 pp. 727-742. Research Macias-Merlo, L. et al. (2016) Effects of the standing program with hip abduction on hip acetabular development in children with spastic diplegia cerebral palsy. Disability And Rehabilitation Volume: 38 Issue: 11 Pages: 1075-1081 Published: MAY 21 2016 Research Maher, C. et al. (2011) Factors influencing postural management for children with cerebral palsy in the special school setting. Disability and Rehabilitation: An International, Multidisciplinary Journal, Vol 33(2), 2011 pp. 146-158. Research McDonald, R., Surtees, R. and Wirz, S. (2004) The International Classification of Functioning, Disability and Health provides a Model for Adaptive Seating Interventions for Children with Cerebral Palsy. British Journal of Occupational Therapy, 67(7):293-302. Practice O'Connor, B., Boyd, R. N. and Shields, N. (2006) A systematic review of postural management of hip displacement in children with cerebral palsy. Developmental Medicine & Child Neurology, (48), pp. 42-43. Research Owens, K. & Daly, G. (2016) A study into the effectiveness of 24 hour postural care in the management of contractures in Care Homes. Middlesborough Council. Research Polak, F., Clift, M. and Clift, L. (2009) Buyers' Guide. Night-time postural management equipment for children CEP08030.: London: NHS Centre for Evidence-based Purchasing. Results Policy Pountney, T. et al. (2009) Hip subluxation and dislocation in cerebral palsy – a prospective study on the effectiveness of postural management programmes. Physiotherapy Research International: The Journal for Researchers And Clinicians In Physical Therapy, 2009 Jun; Vol. 14 (2), pp. 116-27. Research Robertson, J. et al. (2018) Postural care for people with intellectual disabilities and severely impaired motor function: A scoping review. Research in Intellectual Disabilities, Vol 31(Suppl 1), Jan, 2018 pp. 11-28. Research Seabrook, R. (2017) Specialist seating - a model to increase efficiencies in provision and meet a growing demand. RCOT Annual Conference, pp. 114. Practice Stephens, M., Bartley, C. and Priestley, C. (2018) Night-time Positioning for Care Home Residents. [Online} URL: https://pdfs.semanticscholar.org/fae9/f3c7670c8226384c266aa5562f0e24249d49.pdf (Accessed 18/01/20). Research Wright, C., Casey, J. and Porter-Armstrong, A. (2010) Establishing best practice in seating assessment for children with physical disabilities using qualitative methodologies. Disability and Rehabilitation: Assistive Technology, 5(1), pp. 34-47. Research Wynn, N. and Wickham, J. (2009) Night-time positioning for children with postural needs: What is the evidence to inform best practice? The British Journal of Occupational Therapy., 40(12), pp. 543. Research able 2. (Continued) Paper Humphreys, G., King, T., Jex, J., Rogers, M., Blake, S., Thompson-Coon, J. and Morris, C. (2019) Sleep positioning systems for children and adults with a neurodisability: A systematic review. British Journal of Occupational Therapy, 82(1), pp. 5-14. Research Hutton, E. and Coxon, K. (2011) ‘Posture for Learning’: meeting the postural care needs of children with physical disabilities in mainstream primary schools in England – a research into practice exploratory study. Disability and Rehabilitation, 33:19-20. 1912-1914. Research Innocente, R. (2014) Night-time positioning equipment: A review of practices. New Zealand Journal of Occupational Therapy, 61(1), pp. 13-20. Practice Jones, C., Underhill, M. and Baylis, M. (2015) Service transformation through integration: specialist seating model for children. RCOT Annual Conference, pp. 77. Practice Kent, F. (2015) 24-hour postural management: all about seating 1 – practical considerations. National Association of Equipment Providers, Oct 2015; 98-103. Practice Kent, F. (2015) 24-hour postural management: all about seating 1 – practical considerations. National Association of Equipment Providers, Oct 2015; 98-103. Practic Lansdown, K. and Dawson, K. (2019) Are you sitting comfortably: An evaluation of a postural management masterclass for occupational therapy staff. RCOT Annual Conference, pp. 72. Practice Lansdown, K. and Dawson, K. (2019) Are you sitting comfortably: An evaluation of a postural management masterclass for occupational therapy staff. Results Acta Paediatrica, 98, pp. 1809-1814. Research Hill, S. (2011) A one year postural care training programme for the workforce supporting the needs of those with complex and continuing healthcare needs: Project Evaluation. Postural Care CIC. Practice Hill, S. and Goldsmith, J. (2010) Biomechanics and prevention of body shape distortion. Tizard Learning Disability Review, 15(2), pp. 15-32. Theoretical Hill, S. and Goldsmith, L. (2009) Mobility, Posture and Comfort. In: Pawlyn, J. and Carnaby, S. (eds.) (2009) Profound Intellectual and Multiple Disabilities: 9781444301533: Wiley-Blackwell Publishing, Chichester, pp 328-346. Theoretical Hotham, S. et al. (2017) A study into the effectiveness of a postural care training programme aimed at improving knowledge, understanding and confidence in parents and school staff. Child: Care, Health and Development, Vol 43(5), Sep, 2017 pp. 743-751. Research Humphreys, G. and Pountney, T. (2006) The development and implementation of an integrated care pathway for 24-hour postural management: a study of the views of staff and carers. Physiotherapy, 92(4), pp. 233-239. Research Humphreys, G. and Pountney, T. (2006) The development and implementation of an integrated care pathway for 24-hour postural management: a study of the views of staff and carers. Physiotherapy, 92(4), pp. 233-239. Research (Continued) (Continued) Osborne et al. 181 Table 2. (Continued) Paper Literature Type Humphreys, G., King, T., Jex, J., Rogers, M., Blake, S., Thompson-Coon, J. and Morris, C. (2019) Sleep positioning systems for children and adults with a neurodisability: A systematic review. British Journal of Occupational Therapy, 82(1), pp. 5-14. Research Hutton, E. and Coxon, K. (2011) ‘Posture for Learning’: meeting the postural care needs of children with physical disabilities in mainstream primary schools in England – a research into practice exploratory study. Disability and Rehabilitation, 33:19-20. 1912-1914. Research Innocente, R. (2014) Night-time positioning equipment: A review of practices. New Zealand Journal of Occupational Therapy, 61(1), pp. 13-20. Practice Jones, C., Underhill, M. and Baylis, M. (2015) Service transformation through integration: specialist seating model for children. RCOT Annual Conference, pp. 77. Practice Kent, F. (2015) 24-hour postural management: all about seating 1 – practical considerations. National Association of Equipment Providers, Oct 2015; 98-103. Practice Lansdown, K. and Dawson, K. (2019) Are you sitting comfortably: An evaluation of a postural management masterclass for occupational therapy staff. RCOT Annual Conference, pp. 72. Practice Lyons, E. et al. (2017) An exploration of comfort and discomfort amongst children and young people with intellectual disabilities who depend on postural management equipment. Results Research design No of papers Research design No of papers Research design No of papers Action Research 2 Evaluation 2 Scoping Review 1 Case Study 2 Exploratory 4 Sequential 2 Cohort 1 Observation 1 Systematic Review 6 Comparison 1 Pilot Study 1 Cross-sectional Descriptive 1 Quasi-Experimental 1 Table 3. Classification of scientific papers by research design. Table 4. Included papers: Categorised according to emerging themes. Paper Service provision Education Posture management Occupational Performance Aburto (2016) X Birth Defects Foundation Newlife (2007) X Castle et al. (2014) X Clayton (2013) X Collins (2008) X Crawford and Curran (2014) X Crawford and Stinson (2015) X Daly (2017) X X Daly et al. (2013) X Day and Lockwood (2012) X X Fields and McGrath (2008) X Goodwin et al. (2018) X X Hill et al. (2009) X Hill (2011) X Hill and Goldsmith (2010) X Hutton and Coxon (2011) X Innocente (2014) X Jones et al. (2015) X Kent (2015) X Lansdown and Dawson (2019) X McDonald et al. (2004) X Polak et al. (2009) X Seabrook (2017) X Stephens et al. (2018) X Wright et al. (2010) X Wynn and Wickham (2009) X Table 4. Included papers: Categorised according to emerging themes. 3. Impact on occupational performance and participation 4. Service provision 3. Impact on occupational performance and participation 4. Service provision National Association of Equipment Providers and Postural Care CIC, exploring relevant evidence, policy and guide- lines, then making recommendations for future service pro- vision (Clayton, 2013; Hill, 2011; Kent, 2015). Education on posture management. Literature identified similar definitions of posture management as the use of a range of techniques that can be used to preserve body shape to prevent secondary complications, promoting a symme trical body shape and enhance function and participation, whilst recognising that its use is mainly directed at individu- als who are unable to change their position independently (Castle et al., 2014; Crawford and Curran, 2014; Crawford and Stinson, 2015). These benefits to health and wellbeing will have an impact on the individual’s quality of life. This demonstrates that clinicians on the front line have recognised a need for postural management and are begin- ning to implement interventions and seek out an evidence base for this. Results Physiotherapy Research International: The Journal for Researchers And Clinicians In Physical Therapy, 2009 Jun; Vol. 14 (2), pp. 116-27. Research Robertson, J. et al. (2018) Postural care for people with intellectual disabilities and severely impaired motor function: A scoping review. Research in Intellectual Disabilities, Vol 31(Suppl 1), Jan, 2018 pp. 11-28. Research Robertson, J. et al. (2018) Postural care for people with intellectual disabilities and severely impaired motor function: A scoping review. Research in Intellectual Disabilities, Vol 31(Suppl 1), Jan, 2018 pp. 11-28. Research Seabrook, R. (2017) Specialist seating - a model to increase efficiencies in provision and meet a growing demand. RCOT Annual Conference, pp. 114. Practice Stephens, M., Bartley, C. and Priestley, C. (2018) Night-time Positioning for Care Home Residents. [Online} URL: https://pdfs.semanticscholar.org/fae9/f3c7670c8226384c266aa5562f0e24249d49.pdf (Accessed 18/01/20). Research Wright, C., Casey, J. and Porter-Armstrong, A. (2010) Establishing best practice in seating assessment for children with physical disabilities using qualitative methodologies. Disability and Rehabilitation: Assistive Technology, 5(1), pp. 34-47. Research gy, ( ), pp nn, N. and Wickham, J. (2009) Night-time positioning for children with postural needs: What is the dence to inform best practice? The British Journal of Occupational Therapy., 40(12), pp. 543. Resea 2017; Day and Lockwood, 2012; Lansdown and Dawson, 2019). Whilst the other half presented their own develop- ment of multidisciplinary working towards a posture man- agement service in their geographical area (Fields and McGrath, 2008; Jones et al., 2015; Seabrook, 2017). Four of the practice papers were from clinicians writing to review an area of their practice or providing their own guidance from experience within the field (Casey et al., 2019; Collins, 2008; Innocente, 2014; McDonald et al., 2004). The remain- ing three were projects from the specialist interest groups through directly contacting manufacturers and authors, illus- trating that literature on this topic is hard to locate. An expla- nation for this may be due to strict submission criteria and that many journals will not publish research with lower lev- els of research design. Of the 13 papers classified as practice literature, 6 were conference abstracts of presentations given by clinicians working within the posture management field. Half of those directly stated that Occupational Therapists have a direct role in assessing and delivering posture management (Daly, British Journal of Occupational Therapy 86(3) 182 Table 3. Classification of scientific papers by research design. 2. Posture management across the three primary positions 1. Education on posture management Osborne et al. In addition, there is a need for further training for health- care professionals in prescribing postural care techniques and associated devices as well as for families and carers at how best to apply those techniques and devices. Castle et al. (2014) found that most staff within the multidiscipli- nary team had no understanding of the need for postural management and they did not have any assessment tool for identifying need. Instilling staff and family knowledge and understanding of the need for posture management and the benefits it can produce, will help to alleviate any fear or anxieties of not applying postural supports appropriately and reduce the risk of causing harm (Goodwin et al., 2019; Hutton and Coxon, 2011). often overlooked as an occupation, plus having good sleep can have additional positive benefits to daytime occupa- tional engagement. Service provision. McDonald et al. (2004) referred to the Audit Commission (2000) report ‘Fully Equipped’ as empha- sising poor strategic and operational methods for delivery of mobility services. The disbanding of healthcare services has subsequently resulted in a postcode lottery of service provi- sion, with much confusion not only amongst members of the public, but also health and social care professionals about where and what funding is provided, and often with insuffi- cient budgets allocated (Birth Defects Foundation Newlife, 2007). The lack of providing adequate individualised pos- tural support devices, whether it is an armchair, night-time positioning system or a standing frame can lead to more complex postural deformities developing and therefore ulti- mately present a higher cost, requiring more specialised care and equipment for long-term care plans, which could be pre- vented. Also, adopting a pro-active intervention at the outset would often reduce the likelihood of the individual having pain and developing preventable secondary complications (Daly, 2017). The numbers of people requiring posture man- agement equipment are a relatively small proportion of an overall clinical group. However, their needs are more com- plex and therefore use a greater proportion of the limited resources (Aburto, 2016). It could be assumed that if their postural needs were addressed early before they deteriorate and become non-reducible requiring complex care, more resources would be available to share. On a positive note, dedicated posture management services are starting to emerge in different areas across the UK (Aburto, 2016; Crawford and Curran, 2014; Fields and McGrath, 2008; Robertson et al., 2018; Seabrook, 2017). Osborne et al. Further, several cli- nicians are now presenting on this topic at conferences (Daly, 2017; Day and Lockwood, 2012; Fields and McGrath, 2008; Jones et al., 2015; Lansdown and Dawson, 2019; Seabrook, 2017), which shows there is some innovation of service development across the professional field. With wheelchair services in much of the UK often being tendered to private providers, competition for contracts could be an opportunity to drive pioneering service provision to offer an advantage over competitors (Daly et al., 2013). This could present an opportunity for posture management to become embedded into wheelchair services and enable a more rounded and coordinated 24-hour postural management approach to be adopted. Posture management across the three primary positions. Posture management in lying: Posture management in a lying position is a crucial part of 24-hour posture manage- ment, owing to the length of time spent in bed (Clayton, 2013; Hill, 2011; Hill et al., 2009; Hill and Goldsmith, 2010; Innocente, 2014; Polak et al. 2009; Wynn and Wickham, 2009) and the body being more receptive to specific posi- tioning when muscle tone is more relaxed (Polak et al., 2009; Stephens et al., 2018). Positioning during the day can be influenced by the position adopted overnight (Polak et al., 2009). Symmetrical supine lying is reported to be the opti- mum position to aid balanced functioning of the muscles (Hill and Goldsmith, 2010; Polak et al., 2009). Posture management in sitting: Seating is only one aspect of 24-hour posture management and therefore should not be viewed exclusively without considering the other ori- entations of posture (Kent, 2015). People sit for several hours per day (Daly et al., 2013; Wright et al., 2010), so seat- ing must be correctly fitting and provide adequate pressure distribution (Collins, 2008). One client group who are par- ticularly in need of specialist seating are older people living in care homes. Despite demonstrated benefits, there is a lack of clinicians with specialised seating knowledge and very lit- tle advice in national guidelines to inform the assessment and prescription process, with no national care pathway for seating (Collins, 2008; Wright et al., 2010). Posture management in standing: The benefits of standing as part of a posture management programme have been reported as improved bone mineral density, hip stabil- ity, joint range of movement, reduced pain, improved muscle stretch and increased participation (Goodwin et al., 2018). Impact on occupational performance and participation. Osborne et al. It is recognised that provision of appropriate postural seating can increase participation in activities through improving the accessibility and awareness of the environment, as well as enabling individuals to be more functional in a better supported position (Daly, 2017). The involvement of Occu- pational Therapists in the provision of seating offers a dis- tinctive approach in how positioning affects a person’s ability to participate (Day and Lockwood, 2012). Sleep is Results In addition, this suggests that they are searching for policy or guidance to steer them and to share their findings with peers, indicating that posture manage- ment is a tangible issue that is increasingly part of stand- ard Occupational Therapy practice. The team discussed the themes and further analysis con- densed these into four over-arching themes which were then linked back to the primary objective (see Table 4): There was also a clear, defined need for 24-hour posture management, recognising that many of the secondary com- plications that can occur due to lack of posture management are avoidable and therefore, clinicians have a duty of care to prevent their development (Castle et al., 2014). 1. Education on posture management 2. Posture management across the three primary positions 183 Osborne et al. Discussion and recommendations Posture management equipment is often very expensive and therefore, not invest- ing in training families and carers in how to use it effectively, is a waste of resources and can have a negative effect for the individual through insufficient or incorrect posture manage- ment (Polak et al., 2009). There are multiple authors from this field with similar conclusions; namely that 24-hour posture management can prevent the development of secondary complications, pres- sure ulcer development and improve health and wellbeing outcomes so that people are able to engage in occupation to live a more meaningful and comfortable life. Considering Humphreys et al.’s (2019) conclusions, a consensus of expert opinion on the subject would be beneficial to drive this agenda forwards into development of policy to guide assess- ment of need and provision of intervention. Therefore, it is recommended that a consensus paper on 24-hour posture management is produced, leading to development of national policy and guidance for Clinicians, individuals and their car- ers in the delivery of postural care for all people that have complex physical needs and who are at risk to the effects of gravity on their posture. It is important to recognise that this is an issue that does not only affect one client group and this work presents an opportunity to guide the creation and delivery of postural care services for all who need it. At pre- sent, there are guidelines around pressure ulcer prevention (NICE, 2014) and spasticity management in under 19’s (NICE, 2012) but these do not go far enough to recognise that habitual asymmetric postures can affect any person with complex physical disabilities, all age groups, all positions, and those unable to change their position independently. A limitation of this scoping review could be the exclusion of standing for which there is already an extensive body of published literature, particularly within physiotherapy. The authors recognise that standing is a vital strand of 24-hour postural care programmes and it is important that physio- therapy colleagues are part of the overall pathway. Another limitation is, there was no analysis on the complexity of postural supports used. For example, night-time positioning did not differentiate between using simple off-the-shelf pillows and wedges or a custom-moulded system. It was also acknowledged that none of the literature explored the per- spective of the person using the assistive devices, although some did comment on the carer’s perspective. Discussion and recommendations There currently is no global interest group or platform for influencing postural care on a wider scale. Therefore, initial recommendations are tailored to improve the provision of postural care within UK practice. Many papers identify a lack of robust evidence for posture management techniques British Journal of Occupational Therapy 86(3) 184 and interventions, all recognising there was a lack of ran- domised control trials (RCTs). Although often perceived as the ‘gold standard’ of research design, complex rehabilita- tion does not easily lend itself to RCTs. It would be unethical to withhold posture management interventions when the harmful effect of gravity on body-structures is well recog- nised. Further, blinding of the researchers on whether pos- tural supports are used or not would be difficult depending upon the type of outcome measures employed; thereby mak- ing it very difficult to have a treatment group versus control group. Much of the literature cited in this scoping review is based on specialist clinical knowledge and expertise gained from real-life experience of working with the complex popu- lation who require posture management. expert, preventative services including proactive postural care support’. It has been reported that current service provi- sion is patchy and inconsistent. With postural care being so devolved and postural supports for the three orientations of lying, sitting and standing being provided in isolation, or not at all, the current provision falls far short of the joined-up service that is advocated. To address this, development of a dedicated posture management service where all postural care needs and equipment is assessed for and provided as part of a holistic, 24-hour approach is recommended. This provides an opportunity to reduce duplication and ensure that all prescribed devices and recommendations work collectively. For postural care to be recognised and delivered appropri- ately, it is proposed that universities in the UK and Ireland must include posture management training for undergradu- ate students and that training is multidisciplinary (Collins, 2012, cited in Kent, 2015). Additionally, training is required for families and carers of people who need postural care to ensure they are competent and confident to transfer and posi- tion the individual using the equipment so that it is used effectively and does not cause more harm (Blake et al., 2015; Hill et al., 2009; Polak et al., 2009). Discussion and recommendations Funding is a key barrier for preventing access to postural care. According to the Human Rights Act (1998), everyone has a ‘right to life’ and the secondary complications caused by poor posture management have been presented as a cause of premature death. Therefore, access to postural manage- ment services is a human right. The Audit Commission (2002: 4) stated that commissioning was a real problem for the provision of equipment and that ‘service commissioners and providers generally have no idea about the underlying level of demand for equipment services. Unmet need repre- sents a major cause of social exclusion’. This is reflected in the findings from the Birth Defects Foundation Newlife (2007) campaign report; and in the Guidance for Postural Care and People with Learning Disabilities, Public Health England (2018), it is stated ‘It is recommended that clinical commissioning groups should ensure they commission As highlighted by Blake et al. (2015), it is important to be mindful when reviewing literature in this field of any potential bias and ethical concerns regarding objectivity when manufacturers may have sponsored research or pro- vided assistive technology devices. Research ethics Casey J, Hoffman L, Hutson J, et al. (2019) Supporting the occu- pation of sleep through night-time positioning equipment. SIS Quarterly Practice Connections 4: 7–9. Ethics approval was not required for this study. What the study has added This review identifies the need for best-practice clinical guidelines on 24-hour posture management; scope for dedi- cated posture management services and increased training throughout the Occupational Therapy profession on 24-hour postural management intervention. Anderson S, Allen P, Peckham S, et al. (2008) Asking the right questions: Scoping studies in the commissioning of research on the organisation and delivery of health services. Health Research Policy and Systems 6: 7. Audit Commission (2000) Fully Equipped: The provision of equip- ment to older or disabled people by the NHS and social services in England and Wales. Audit Commission (2002) Fully Equipped 2002: Assisting Independence (update) [Online] URL: http://www.nsoc.org.uk/ evidence/auditcommissionupdate2002.pdf (accessed 18 January 2020). ORCID iDs Lauren Julia Osborne https://orcid.org/0000-0002-1240-8439 Jackie Casey https://orcid.org/0000-0001-9408-9293 Conclusion A scoping review was conducted to examine the evidence for 24-hour posture management, which is regarded as nec- essary to reduce the effects of postural asymmetries and gravity on people that are unable to change their position independently and prevent the development of postural deformities and pressure ulcers. Whilst the evidence for Osborne et al. 185 24-hour posture management may not be considered robust or of sufficient research quality, there is evidence none-the- less that holds weight and is valuable in guiding future prac- tice. There are clear health and wellbeing benefits from the provision of 24-hour posture management for people who are unable to change their position independently. Acknowledgements The primary author expresses sincere gratitude to Millbrook Healthcare and to the Oxford Brookes Healthcare Librarians. Casey J, Rosenblad A and Rodby-Bousquet E (2020) Postural asymmetries, pain, and ability to change position of children with cerebral palsy in sitting and supine: A cross-sectional study. Disability and Rehabilitation 44: 2363–2371. Authors’ note Jackie Casey is now affiliated with Advanced Occupational Therapist Practitioner, Regional Wheelchair: Training, Research & Service Development, Belfast Health & Social Care Trust, Belfast, Northern Ireland; Discipline Occupational Therapy, Faculty of Education and Health Sciences, School of Allied Health, Health Research Institute, Health Implementation Science and Technology, University of Limerick, Limerick, Ireland. Aveyard H. (2014) Doing a Literature Review in Health and Social Care: A Practical Guide, 3rd edn. Maidenhead: Open University Press. Birth Defects Foundation Newlife (2007) It’s not too much to ask. Campaign Report. Cannock: BDF Newlife. Blake SF, Logan S, Humphreys G, et al. (2015) Sleep position- ing systems for children with cerebral palsy. The Cochrane Database of Systematic Reviews 11: CD009257. Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was conducted as part of an MSc, which was funded by Millbrook Healthcare. Current NHS service provision for posture management and commissioning is weak and insufficient. The resulting postcode lottery of service provision is inadequate (Birth Defects Foundation Newlife, 2007) and failure to provide adequate posture management can be considered a safe- guarding issue and a breach of human rights (Gowran, 2020). Therefore, to advance the posture management agenda, we urgently need to develop national guidance and a posture care pathway informing future practice. There are opportuni- ties for the development of a dedicated posture management service through the expansion of current wheelchair services to include the provision of night-time positioning equip- ment, standing frames and postural static seating. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Key findings Aburto N (2016) Sleep System Service Evaluation. Hounslow Wheelchair and Posture Management Service. NHS Hounslow and Richmond Community Healthcare Trust. •• Current NHS provision of posture management is inade- quate, resulting in unmet needs. •• Current NHS provision of posture management is inade- quate, resulting in unmet needs. Agustsson A and Jonsdottir G (2018) Chapter 7: Posture man- agement 24/7. In Lange ML and Minkel JL (eds) Seating and Wheeled Mobility: A Clinical Resource Guide. SLACK Incorporated, pp.121–136. •• Research in this field is challenging; subsequently, greater credibility should be attributed to specialist expertise. •• National guidelines are needed. Aldersea P (1999) NHS wheelchairs and seating for disabled children. British Journal of Therapy and Rehabilitation 6: 408–412. Contributorship LO researched literature and conceived the study. LO and JC devel- oped search strategy. LO completed literature analysis. LO wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript. Patient and public involvement data Humphreys G and Pountney T (2006) The development and implementation of an integrated care pathway for 24-hour postural management: a study of the views of staff and carers. Physiotherapy 92: 233–239. Daly G (2017) Prevention and postural management of residents in care homes. RCOT annual conference, pp. 109. Hutton E and Coxon K (2011) ‘Posture for Learning’: Meeting the postural care needs of children with physical disabilities in mainstream primary schools in England – A research into prac- tice exploratory study. Disability and Rehabilitation 33: 19–20, 1912–1914. Daly O, Casey J and Martin S (2013) The Effectiveness of Specialist Seating Provision for Nursing Home Residents. A report for Seating Matters Ltd.; Knowledge Transfer Partnership (KTP). Darzi A (2018) The Lord Darzi Review of Health and Care Interim Report. IPPR. https://www.ippr.org/files/2018-04/1524670994_ lord-darzi-review-interim-report.pdf (accessed 18 January 2020). Innocente R (2014) Night-time positioning equipment: A review of practices. New Zealand Journal of Occupational Therapy 61: 13–20. Day E and Lockwood D (2012) The impact of posture manage- ment on two adults with learning disabilities. RCOT Annual Conference, pp. 49. Jones C, Underhill M and Baylis M (2015) Service transformation through integration: Specialist seating model for children. In: RCOT annual conference, pp. 77. Farley R, Clark J, Davidson C, et al. (2003) What is the evidence for the effectiveness of postural management? International Journal of Therapy and Rehabilitation 10: 449–455. Kent F (2015) 24-hour Postural Management: All About Seating 1 – Practical Considerations. National Association of Equipment Services, pp. 98–103. Fields F and McGrath M (2008) 24 hour postural management: A new direction in therapeutic care. Physiotherapy Ireland 2008; 29: 75–75. Lansdown K and Dawson K (2019) Are you sitting comfortably: An evaluation of a postural management masterclass for occu- pational therapy staff. RCOT annual conference, pp. 72. Gmelig Meyling C, Ketelaar M, Kuijper M, et al. (2017) Postural management to reduce or prevent hip migration in children with cerebral palsy: A systematic review. Developmental Medicine & Child Neurology 30: 82–91. McDonald R, Surtees R and Wirz S (2004) The international classification of functioning, disability and health provides a model for adaptive seating interventions for children with cerebral palsy. British Journal of Occupational Therapy 67: 293–302. Goodwin J, Lecouturier J, Crombie S, et al. (2018) Understanding frames: A qualitative study of young people’s experiences of using standing frames as part of postural management for cer- ebral palsy. Child: Care, Health and Development 44: 203–211. Patient and public involvement data Castle D, Stubbs B, Clayton S, et al. (2014) A 24-hour postural care service: Views, understanding and training needs of referring multidisciplinary staff. International Journal of Therapy and Rehabilitation 21: 132–139. During the development, progress, and reporting of the submitted research, Patient and Public Involvement in the research was not included at any stage of the research. included at any stage of the research. British Journal of Occupational Therapy 86(3) 186 Changing our Lives (2018) Postural care strategy. Changing Our Lives. night-time postural equipment: A pilot study. Acta Paediatrica 98: 1809–1814. Hill S (2011) A one year postural care training programme for the workforce supporting the needs of those with complex and continuing healthcare needs: Project Evaluation. Postural Care CIC. Clayton S (2013) living local postural care project evaluation. Postural Care CIC. Clayton S, Goldsmith L and Ellis T (2017) 24 hour postural care: The journey so far in the UK. Postural Care CIC. The journey so far in the UK. Postural Care CIC. Hill S and Goldsmith L (2009) Mobility, posture and comfort. In: Pawlyn J and Carnaby S (eds.) (2009) Profound Intellectual and Multiple Disabilities. Chichester: Wiley-Blackwell Publishing, pp.328–346. Collins F (2001) An adequate service? Specialist seating provision in the UK. Journal of Wound Care 10: 333–337. in the UK. Journal of Wound Care 10: 333–337. Collins F (2008) An essential guide to managing seated patients in the community. British Journal of Community Nursing 13: 39–40. Colquhoun H, Levac D and O’Brien K (2010) Scoping studies: Advancing the methodology. Implementation Science 5: 1–9. Hill S and Goldsmith J (2010) Biomechanics and prevention of body shape distortion. Tizard Learning Disability Review 15: 15–32. Colquhoun H, Levac D, O’Brien K, et al. (2014) Scoping reviews: Time for clarity in definition, methods and reporting. Journal of Clinical Epidemiology 67: 1291–1294. Human Rights Act (1998) [Online]. www.legislation.gov.uk/ukpga /1998/42/contents (accessed 18 January 2020). Crawford S and Curran A (2014) 24 Hour postural management for community dwelling adults with learning disabilities. The Journal of Posture and Mobility Group 31: 15–19. Humphreys G, King T, Jex J, et al. (2019) Sleep positioning sys- tems for children and adults with a neurodisability: A systematic review. British Journal of Occupational Therapy 82: 5–14. Crawford S and Stinson M (2015) Management of 24-h-Body Positioning. In: Söderback I (ed.) International Handbook of Occupational Therapy Interventions. Cham: Springer Inter national Publishing, pp. 189–203. Patient and public involvement data Munn Z, Peters MDJ, Stern C, et al. (2018) Systematic review or scoping review? Guidance for authors when choosing between a systematic or scoping review approach. BMC Medical Research Methodology 18: 143. Goodwin J, Lecouturier J, Smith J, et al. (2019) Understanding frames: A qualitative exploration of standing frame use for young people with cerebral palsy in educational settings. Child: Care, Health and Development 45: 433–439. National Institute of Clinical Excellence (2012) Spasticity in under 19s: Management. [Online] Available at: https://www.nice.org. uk/guidance/cg145 (accessed 22 October 2019). Gowran R (2013) Sustainable solutions for wheelchair and seat- ing assistive technology provision: presenting a cosmopolitan narrative with rich pictures. In: Encarnação P, Azevedo L and Gelderblom GJ (eds) Assistive Technology: From Research to Practice. Amsterdam: IOS Press. National Institute of Clinical Excellence (2014) Pressure ulcers: Prevention and management. Available at: www.nice.org.uk/ guidance/cg179 (accessed 9 October 2019). O’Connor B, Boyd RN and Shields N (2006) A systematic review of postural management of hip displacement in children with cerebral palsy. Developmental Medicine & Child Neurology 48: 42–43. Gowran R, Clifford A, McKee J, et al. (2020) Wheelchair and seating assistive technology provision: A gateway to freedom. Disability and Rehabilitation 44: 370–381. Peters MDJ, Godfrey CM, Khalil H, et al. (2015) Guidance for con- ducting systematic scoping reviews. International Journal of Evidence-Based Healthcare 13: 141–146. Greenhalgh T (2010) How to Read a Paper: The Basics of Evidence- Based Medicine, 4th edn. Chichester, West Sussex, UK: Wiley- Blackwell. Polak F, Clift M and Clift L (2009) Buyers’ Guide. Night Time Postural Management Equipment for Children CEP08030: London: NHS Centre for Evidence-based Purchasing. Hill CM, Parker RC, Allen P, et al. (2009) Sleep quality and respiratory function in children with severe cerebral palsy using 187 Osborne et al. about-occupational-therapy/what-is-occupational-therapy (accessed 18 January 2020). Pope PM (2007) Severe and Complex Neurological Disability: Management of the Physical Condition. Edinburgh: Butterworth- Heinemann/Elsevier. Seabrook R (2017) Specialist seating – A model to increase effi- ciencies in provision and meet a growing demand. RCOT annual conference, pp. 114. Pountney T, Mandy A, Green E, et al. (2002) Management of hip dislocation with postural management. Child: Care, Health and Development 28: 179–185. Stephens M, Bartley C and Priestley C (2018) Night-time Positioning for Care Home Residents. Available at: https://pdfs.semantic scholar.org/fae9/f3c7670c8226384c266aa5562f0e24249d49. pdf (accessed 18 January 2020). Public Health England (2018) Postural Care and People with Learning Disabilities: Guidance. Available at: https://www. Patient and public involvement data gov.uk/government/publications/postural-care-services- making-reasonable-adjustments/postural-care-and-people- with-learning-disabilities?fbclid=IwAR3b_Ub6pW6VJEfZ g9mNcHB3SXj2Xe7CWwBnzDe03Rh2Mgjhn5DQKQUO BMo#national-guidance-and-reports (accessed 22 January 2020). Stinson M, Crawford S and Madden E (2021) Current clinical prac- tice in 24-hour postural management and the impact on carers and service users with severe neurodisability. British Journal of Occupational Therapy 84: 355–365. Wright C, Casey J and Porter-Armstrong A (2010) Establishing best practice in seating assessment for children with physical disabilities using qualitative methodologies. Disability and Rehabilitation: Assistive Technology 5: 34–47. Robertson J, Baines S, Emerson E, et al. (2018) Postural care for people with intellectual disabilities and severely impaired motor function: A scoping review. Journal of Applied Research in Intellectual Disabilities 31(Suppl. 1): 11–28. Wynn N and Wickham J (2009) Night-time positioning for chil- dren with postural needs: What is the evidence to inform best practice? The British Journal of Occupational Therapy 40: 543. Royal College of Occupational Therapists (2019) What is Occupational Therapy? Available at: https://www.rcot.co.uk/
https://openalex.org/W4280631030	https://www.mdpi.com/2072-6694/14/10/2478/pdf?version=1652851463	English	null	How Phosphofructokinase-1 Promotes PI3K and YAP/TAZ in Cancer: Therapeutic Perspectives	Cancers	2,022	cc-by	10,898	Luca Simula 1 , Marco Alifano 2,3 and Philippe Icard 3,4,* Luca Simula 1 , Marco Alifano 2,3 and Philippe Icard 3,4,* Luca Simula 1 , Marco Alifano 2,3 and Philippe Icard 3,4,* 1 Department of Infection, Immunity and Inﬂammation, Cochin Institute, INSERM U1016, CNRS UMR8104, University of Paris, 75014 Paris, France; luca.simula@inserm.fr 1 Department of Infection, Immunity and Inﬂammation, Cochin Institute, INSERM U1016, CNRS UMR8104, U i i f P i 75014 P i F l i l @i f 1 Department of Infection, Immunity and Inﬂammation, Cochin Institute, INSERM U1016, CNRS UMR8104, University of Paris, 75014 Paris, France; luca.simula@inserm.fr p , y , , , University of Paris, 75014 Paris, France; luca.simula@inserm.fr y 2 INSERM U1138, Integrative Cancer Immunology, University of Paris, 75006 Paris, France; marco.alifano@aphp.fr y 2 INSERM U1138, Integrative Cancer Immunology, University of Paris, 75006 Paris, France; marco.alifano@aphp.fr 3 Service de Chirurgie Thoracique, Hôpital Cochin, Hôpitaux Universitaires Paris Centre, APHP, Paris-Descartes University, 75014 Paris, France 3 Service de Chirurgie Thoracique, Hôpital Cochin, Hôpitaux Universitaires Paris Centre, APHP, Paris-Descartes University, 75014 Paris, France y 4 UNICAEN, INSERM U1086 Interdisciplinary Research Unit for Cancer Prevention and Treatment, y 4 UNICAEN, INSERM U1086 Interdisciplinary Research Unit for Cancer Prevention and Treatment, Normandie Université, 14000 Caen, France * Correspondence: philippe.icard@aphp.fr Simple Summary: We propose that PFK1 promotes a positive feedback loop with PI3K/AKT and YAP/TAZ signaling pathways in cancer cells. Therefore, targeting PFK1 (or its product F-1,6-BP) could improve the efﬁcacy of PI3K and YAP/TAZ inhibitors currently tested in clinical trials. To this aim, we suggest the use of citrate, which is a physiologic and potent inhibitor of PFK1. Abstract: PI3K/AKT is one of the most frequently altered signaling pathways in human cancers, sup- porting the activation of many proteins sustaining cell metabolism, proliferation, and aggressiveness. Another important pathway frequently altered in cancer cells is the one regulating the YAP/TAZ transcriptional coactivators, which promote the expression of genes sustaining aerobic glycolysis (such as WNT, MYC, HIF-1), EMT, and drug resistance. Of note, the PI3K/AKT pathway can also regulate the YAP/TAZ one. Unfortunately, although PI3K and YAP inhibitors are currently tested in highly resistant cancers (both solid and hematologic ones), several resistance mechanisms may arise. Resistance mechanisms to PI3K inhibitors may involve the stimulation of alternative pathways (such as RAS, HER, IGFR/AKT), the inactivation of PTEN (the physiologic inhibitor of PI3K), and the expression of anti-apoptotic Bcl-xL and MCL1 proteins. Citation: Simula, L.; Alifano, M.; Icard, P. How Phosphofructokinase-1 Promotes PI3K and YAP/TAZ in Cancer: Therapeutic Perspectives. Cancers 2022, 14, 2478. https:// doi.org/10.3390/cancers14102478 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Keywords: PFK1; F-1,6-BP; PI3K; YAP/TAZ; drug resistance; citrate cancers cancers cancers Luca Simula 1 , Marco Alifano 2,3 and Philippe Icard 3,4,* Therefore, it is important to improve current therapeutic strategies to overcome these limitations. Here, we want to highlight how the glycolytic enzyme PFK1 (and its product F-1,6-BP) promotes the activation of both PI3K/AKT and YAP/TAZ pathways by several direct and indirect mechanisms. In turn, PI3K/AKT and YAP/TAZ can promote PFK1 activity and F-1,6-BP production in a positive feedback loop, thus sustaining the Warburg effect and drug resistance. Thus, we propose that the inhibition of PFK1 (and of its key activator PFK2/PFKFB3) could potentiate the sensitivity to PI3K and YAP inhibitors currently tested. Awaiting the development of non-toxic inhibitors of these enzymes, we propose to test the administration of citrate at a high dosage, because citrate is a physiologic inhibitor of both PFK1 and PFK2/PFKFB3. Consistently, in various cultured cancer cells (including melanoma, sarcoma, hematologic, and epithelial cancer cells), this “citrate strategy” efﬁciently inhibits the IGFR1/AKT pathway, promotes PTEN activity, reduces Bcl-xL and MCL1 expression, and increases sensitivity to standard chemotherapy. It also inhibits the development of sarcoma, pancreatic, mammary HER+ and lung RAS-driven tumors in mice without apparent toxicities. Citation: Simula, L.; Alifano, M.; Icard, P. How Phosphofructokinase-1 Promotes PI3K and YAP/TAZ in Cancer: Therapeutic Perspectives. Cancers 2022, 14, 2478. https:// doi.org/10.3390/cancers14102478 Academic Editors: Ángel Luis García-Otín and Patricia Sancho Received: 22 April 2022 Accepted: 17 May 2022 Published: 18 May 2022 1. Introduction For ex- ample, resistance to PI3K/AKT and YAP/TAZ inhibitors can be supported by multiple mechanisms involving their direct activation or the inactivation of upstream negative regulators, such as: (i) gain function mutations, in particular of PI3K or membrane receptors, such as receptor tyrosine kinases (RTKs), promoting PI3K activity [2,14]; (ii) compensatory activation of pathways able to bypass the PI3K inhibition, such as the activation of JAK2/STAT5 [15] and/or insulin dependent pathways (promoted by insulin-like growth factor 1 (IGF1), insulin-like growth factor receptor 1 (IGFR1), and insulin receptor substrate 2 (IRS2)) [7,16]; (iii) upregulation of key pro-survival factors, such as anti-apoptotic Bcl-xL and MCL1 proteins [17]; (iv) activationof keymutatedoncogenes(gain offunction), particularly EGFR[8], HER/ERK [18], Kirsten Rat Sarcoma homolog (RAS) [8,19], promoting constitutive activation of PI3K/AKT signaling; (v) loss of function or epigenetic silencing of suppressor genes, such as Phosphatase and TENsin homolog (PTEN) protein, the key physiologic inhibitor of PI3K [2,20,21]; (vi) inactivation of the Hippo pathway that inhibits YAP/TAZ, a deregulation promoting resistance to inhibitors of V-raf murine sarcoma viral oncogene homolog B (BRAF) and mitogen-activated protein kinase (MAPK) in BRAF mutant cancer cells [4,5,22]; (vii) ampliﬁcation of the WWTR1 (encoding TAZ) and YAP1 genes or mutations and deletions of the FAT1 gene (a tumor suppressor, whose inactivation favors TAZ nuclear translocation), as reported for head and neck squamous cell carcinoma (HNSCC) [23]; (viii) YAP1 gene fusions, as observed in different tumors (for a review see [24]). YAP1 fusion proteins (normally constituted by N-terminal YAP1 and C-terminal part of another protein) can retain a TEAD-dependent YAP activity and can show resistance to inhibition by the Hippo pathway; (ix) activating point mutations in TAZ gene, which can transform TAZ into an oncogene, as observed in muscle cells [25]. Consequently, to increase the effectiveness of PI3K/AKT and YAP inhibitors, there is an urgent need to target these various resistance mechanisms possibly through a miss- ing link with PI3K/AKT and YAP/TAZ, as recently suggested [3]. To this aim, we here propose to consider the role of phosphofructokinase-1 (PFK1), the key regulatory en- zyme of glycolysis. As we will see, PFK1 and its product fructose-1,6-bisphosphate (F-1,6-BP) can activate both PI3K and/or YAP/TAZ by independent mechanisms. In turn, PI3K/AKT and YAP/TAZ can promote PFK1 and/or glycolysis in a feedback loop [26]. 1. Introduction The Phosphatidylinositol-3-Kinase (PI3K)/ Protein Kinase B (AKT) pathway is one of the most frequently altered pathways in human cancers [1,2]. Two recently identiﬁed down- https://www.mdpi.com/journal/cancers Cancers 2022, 14, 2478. https://doi.org/10.3390/cancers14102478 Cancers 2022, 14, 2478 2 of 13 stream targets of PI3K/AKT are the transcriptional coactivators Yes-associated protein (YAP) and its homolog WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ) (thereafter indicated as YAP/TAZ), which promote multiple transcription factors regulating cell development [3–5]. In a wide variety of cancers, the deregula- tion and activation of PI3K/AKT and YAP/TAZ signaling is nowadays considered as a key event supporting mechano-transduction (shear forces, elasticity, and tissue stretch- ing) [3,6], metabolism, cell cycle progression, survival, epithelial-mesenchymal transition (EMT), metastasis, and drug resistance (including immunotherapy) [3–5,7–11]. Therefore, there is an increasing interest in the development of potent PI3K and YAP inhibitors, especially to target various highly resistant cancers (both solid tumors and hematologic malignancies) [2,7,12,13]. However, the occurrence of resistance mechanisms to PI3K and YAP inhibitors may reduce their therapeutic effectiveness. Additionally, PI3K inhibitors have principally cytostatic effect (tumor stabilization rather than tumor regression) [7,12], whereas the efﬁciency and toxicities of YAP inhibitors need further research [4]. 2. The Activation of PI3K/AKT and YAP/TAZ Pathways PI3K is a key transducer of extracellular stimuli, such as growth factors and mechanical stress [3]. After activation by RTKs, G-protein-coupled receptors (GPCR) sensible to various factors (including hormones, metabolites such as purine, adenosine), or by proto-oncogenes RAS-GTPases proteins (Figure 1), PI3Ks are activated and catalyze the phosphorylation of phosphatidylinositol-4,5-bisphosphate-2 (PIP2) into phosphatidylinositol-4,5-bisphosphate-3 (PIP3). Then, PI3P recruits and activates AKT, a kinase that regulates various down- stream pathways sustaining cancer cells metabolism, especially the mammalian target of rapamycin (mTOR), sustaining protein translation [35], cell cycle progression, survival, and EMT (for a review, see [1,2]). Importantly, AKT can be considered as “the Warburg ki- nase” [36] because it activates the mitochondrial pyruvate dehydrogenase kinase-1 (PDK1), which inhibits the pyruvate dehydrogenase (PDH) enzyme [37]. Consequently, pyruvate does not feed the Krebs cycle and is oriented towards lactate production by lactate dehy- drogenase A (LDHA). Importantly, lactate secreted in a tumor microenvironment (TME) inhibits the immune response [38] and favors the establishment of an acidic pH that coun- teracts the efﬁcacy of many anti-cancer agents (including immunotherapies and mTOR inhibitors) [33,34,38,39]. Among the three class of PI3Ks, class IA and IB isoforms have been the most extensively studied. They are heterodimers formed of a catalytic subunit (a p110α and p110β, respectively), associated with a regulatory subunit (most often p85α isoform) [7]. Schematically, class IA isoforms mediate signaling downstream of RTKs, and class IB isoforms mediate signaling downstream of GPCRs [7,12]. Importantly, the PI3K/AKT signaling pathway can be activated even in the absence of extracellular stimuli. This activation can be driven by genetic alterations (gain of function mutations or ampli- ﬁcation), in particular of the PI3K p110α catalytic subunit (also named PIK3CA [14]) or of membrane receptors promoting PI3K activity, such as RTKs, including EGFR [2,8,14]. Also, the frequent loss of function or epigenetic silencing of the suppressor PTEN promotes PI3K because PTEN physiologically counteracts the activity of the catalytic subunit of PI3K by transforming PIP3 into PIP2 [2,20]. Similarly, the frequent upregulation of the proto-oncogene KRAS protein promotes the activation of PI3K/AKT signaling [8]. The KRAS proto-oncogene is a small G-protein with guanosine triphosphatase (GTPase) activity, which is involved in the transmission of the signal of most RTKs, including EGFR [8,40]. This small GTPase catalyzes the hydrolysis of guanosine triphosphate (GTP) into guano- sine biphosphate (GDP). This reaction ensures the shutdown of the signaling because the GDP-bound isoform of KRAS is inactive [41]. 1. Introduction Thus, a “Gordian Knot” of interconnections is created between PFK1/F-1,6-BP, PI3K/AKT, and YAP/TAZ pathways that sustains the Warburg effect (i.e., aerobic glycolysis even in the presence of oxygen) [27], and thus proliferation, aggressiveness, and drug resis- tance [28]. Therefore, targeting PFK1, as well as its key activator fructose-2,6-bisphosphate (F-2,6-BP) produced by phosphofructokinase-2 (PFK2) (also called 6-phosphofructo-2- Cancers 2022, 14, 2478 3 of 13 kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) in cancer cells), could be a novel and efﬁcient strategy to increase the sensitivity to PI3K and YAP inhibitors. Awaiting the devel- opment of non-toxic inhibitors of PFK1 and PFKFB3, we will emphasize how in cultured cancer cells of diverse origins, the targeting of PFK1 has been achieved by the adminis- tration of high dosages of citrate, a physiologic inhibitor of PFK1. This “citrate strategy” efﬁciently inhibits the IGFR1/AKT pathway, promotes PTEN activity, reduces Bcl-xL and MCL1 expression, and increases sensitivity to standard chemotherapy. Oral administra- tion of sodium citrate inhibits the development of sarcoma, pancreatic, HER+ mammary and RAS-driven lung tumors in mice models without apparent toxicities [29–31]. It also neutralizes the acidity of the tumor microenvironment [32], a condition that decreases the efﬁciency and penetration of many anti-cancer agents [33,34]. 2. The Activation of PI3K/AKT and YAP/TAZ Pathways Also, F-1,6-BP activates PI3K/AKT through Ras induction. PI3K/AKT can be also activated by tensile stresses from stress receptors. Once activated, PI3K/AKT promote transcription of glycolytic genes (though mTOR and low active PKM2), β-catenin stabilization (through PKM2, also promoted by ACLY), activation of glycolytic enzymes (favoring glycolysis), and YAP/TAZ activation (also induced by mevalonate pathway and β-catenin). Once translocated in the nucleus, YAP/TAZ mediate the transcription of genes promoting cell cycle progression and glycolysis. In parallel, (i) F-1,6-BP and AKT inhibit OXPHOS, thus increasing lactate production, which in turn inhibits the immune response and promotes extracellular acidiﬁcation and drug resistance, and (ii) EMT is promoted by β-catenin and YAP/TAZ. Therapeutic strategies increasing citrate levels in cancer cells can inhibit glycolysis and F-1,6-BP production. Therefore, they may be useful to disrupt the Gordian Knot orchestrated by PI3K/AKT, YAP/TAZ, and PFK1/F-1,6-BP. Numbers (1–5) and uppercase letters (A–D) refer to regulatory mechanisms as described in Section 3 (numbers) and Section 4 (letters). Figure created with Biorender. Fi 1 PI3K/AKT YAP/TAZ d PFK1/F 1 6 BP t “G di K t” ti Figure 1. PI3K/AKT, YAP/TAZ, and PFK1/F-1,6-BP create a “Gordian Knot”, supporting cancer pro- gression. In cancer cells, F-1,6-BP promotes glycolysis and activates ACLY, reducing citrate levels (which could inhibit PFK1 and PFK2) and increasing cytosolic acetyl-CoA (promoting lipid synthe- sis, protein and histone acetylation, and mevalonate pathway) and oxalacetate (OAA, sustaining nucleotide synthesis). Also, F-1,6-BP activates PI3K/AKT through Ras induction. PI3K/AKT can be also activated by tensile stresses from stress receptors. Once activated, PI3K/AKT promote tran- scription of glycolytic genes (though mTOR and low active PKM2), β-catenin stabilization (through PKM2, also promoted by ACLY), activation of glycolytic enzymes (favoring glycolysis), and YAP/TAZ activation (also induced by mevalonate pathway and β-catenin). Once translocated in the nucleus, YAP/TAZ mediate the transcription of genes promoting cell cycle progression and glycol- ysis. In parallel, (i) F-1,6-BP and AKT inhibit OXPHOS, thus increasing lactate production, which in turn inhibits the immune response and promotes extracellular acidification and drug resistance, and (ii) EMT is promoted by β-catenin and YAP/TAZ. Therapeutic strategies increasing citrate levels in cancer cells can inhibit glycolysis and F-1,6-BP production. Therefore, they may be useful to disrupt the Gordian Knot orchestrated by PI3K/AKT, YAP/TAZ, and PFK1/F-1,6-BP. Numbers (1–5) and uppercase letters (A–D) refer to regulatory mechanisms as described in sections 3 (numbers) and 4 (letters). Figure created with Biorender. 2. The Activation of PI3K/AKT and YAP/TAZ Pathways Importantly (as we see later), the conversion from an inactive GDP state into an active GTP state needs KRAS to bind to Son of sevenless homolog 1 (SOS1), a protein activated by PFK1 [42]. Once RAS is activated, its downstream targets are engaged in a cascade, such as the PI3K/AKT and the RAS/RAF/MEK/ERK (MAPK) pathways. Both of them promote cancer cell development and resistance [40], partly by inducing YAP/TAZ activity by several independent mechanisms (see below). 4 of 13 cancer ral in- Cancers 2022, 14, 2478 cell development and resistance [40], partly by inducing YAP/TAZ activity by several in- dependent mechanisms (see below). Figure 1. PI3K/AKT, YAP/TAZ, and PFK1/F-1,6-BP create a “Gordian Knot”, supporting cancer pro- gression. In cancer cells, F-1,6-BP promotes glycolysis and activates ACLY, reducing citrate levels (which could inhibit PFK1 and PFK2) and increasing cytosolic acetyl-CoA (promoting lipid synthe- sis, protein and histone acetylation, and mevalonate pathway) and oxalacetate (OAA, sustaining nucleotide synthesis). Also, F-1,6-BP activates PI3K/AKT through Ras induction. PI3K/AKT can be also activated by tensile stresses from stress receptors. Once activated, PI3K/AKT promote tran- scription of glycolytic genes (though mTOR and low active PKM2), β-catenin stabilization (through PKM2, also promoted by ACLY), activation of glycolytic enzymes (favoring glycolysis), and YAP/TAZ activation (also induced by mevalonate pathway and β-catenin). Once translocated in the nucleus, YAP/TAZ mediate the transcription of genes promoting cell cycle progression and glycol- ysis. In parallel, (i) F-1,6-BP and AKT inhibit OXPHOS, thus increasing lactate production, which in turn inhibits the immune response and promotes extracellular acidification and drug resistance, and (ii) EMT is promoted by β-catenin and YAP/TAZ. Therapeutic strategies increasing citrate levels in cancer cells can inhibit glycolysis and F-1,6-BP production. Therefore, they may be useful to disrupt the Gordian Knot orchestrated by PI3K/AKT, YAP/TAZ, and PFK1/F-1,6-BP. Numbers (1–5) and uppercase letters (A–D) refer to regulatory mechanisms as described in sections 3 (numbers) and 4 (letters). Figure created with Biorender. YAP and TAZ are transcriptional coactivators encoded by paralogous genes that reg- ulate transcription by binding to the members of the transcriptional enhancer factor Figure 1. PI3K/AKT, YAP/TAZ, and PFK1/F-1,6-BP create a “Gordian Knot”, supporting cancer progression. In cancer cells, F-1,6-BP promotes glycolysis and activates ACLY, reducing citrate levels (which could inhibit PFK1 and PFK2) and increasing cytosolic acetyl-CoA (promoting lipid synthesis, protein and histone acetylation, and mevalonate pathway) and oxalacetate (OAA, sustaining nu- cleotide synthesis). 2. The Activation of PI3K/AKT and YAP/TAZ Pathways In case of an active control by the Hippo pathway, YAP/TAZ are inactivated by LATS. Phosphorylated YAP/TAZ proteins are retained in the cytoplasm, and then degraded by proteasomal ligase [43,45,46]. In contrast, lack of inhibition by the Hippo pathway favors the translocation of YAP/TAZ into the nucleus, and their further binding to TEAD proteins to form YAP/TAZ-TEAD complexes. Of note, this mechanism has been reported to promote the loss of contact inhibition of cell growth in cancer cells in a PI3K-dependent way [47]. Indeed, activa- tion of PI3K and phosphoinositide-dependent kinase-1 downstream of EGF receptor can induce the dissociation of the Hippo core complex and the consequent inactivation of LATS, dephosphorylation of YAP, and YAP nuclear accumulation and transcriptional acti- vation [47]. In addition to PI3K, also AKT can increase the activity of YAP proteins through phosphorylation of MST1/2, which inhibits their dimerization and activation [44]. Once in the nucleus, YAP/TAZ-TEAD complexes cooperate with other transcription factors (including AP-1, E2F, MYC) to regulate gene transcription [48]. In addition, YAP/TAZ can bind to other transcriptional factors, including β-catenin, to upregulate the expression of genes favoring EMT (such as Slug, Snail, RhoA) and cell survival (such as Bcl-xL, survivin). Also, YAP downregulates PTEN by inducing miR-29, which inhibits PTEN translation [49]. Of note, in diabetic mice with nephropathy, YAP protein can promote its activation in a feedback loop by inhibiting the Hippo pathway, thus promoting its nuclear accumulation and transcriptional effect resulting in an increased proliferation of glomerular mesangial cells [50]. YAP/TAZ activation can promote cancer development in several ways (for a review see [51]). First, YAP/TAZ can suppress apoptotic pathway of cell death by upregulating Bcl-2 family members [52]. Second, sustained activation of YAP/TAZ can promote aberrant cell cycle proliferation, partly through the activation of proto-oncogenes, such as cMYC [53], or through loss of contact inhibition of cell growth [45–47]. Third, YAP/TAZ activation has been associated with the reprogramming of tumor cells into tumor cancer stem cells (CSCs) [54]. A positive correlation between YAP/TAZ levels and cancer malignancy (including lower response to therapy), or a higher YAP/TAZ expression level compared to normal tissue, has been observed in several human cancer or murine models, including colorectal, liver, lung, pancreatic, gastric, and breast cancer (see [51] for a review). As stated before, genetic alterations have been observed in YAP/TAZ genes in cancer cells, such as gene ampliﬁcation [23] and gene fusions [24]. 2. The Activation of PI3K/AKT and YAP/TAZ Pathways YAP and TAZ are transcriptional coactivators encoded by paralogous genes that reg- ulate t a i tio by bi di to the e be of the t a i tio al e ha e fa to Figure 1. PI3K/AKT, YAP/TAZ, and PFK1/F-1,6-BP create a “Gordian Knot”, supporting cancer progression. In cancer cells, F-1,6-BP promotes glycolysis and activates ACLY, reducing citrate levels (which could inhibit PFK1 and PFK2) and increasing cytosolic acetyl-CoA (promoting lipid synthesis, protein and histone acetylation, and mevalonate pathway) and oxalacetate (OAA, sustaining nu- cleotide synthesis). Also, F-1,6-BP activates PI3K/AKT through Ras induction. PI3K/AKT can be also activated by tensile stresses from stress receptors. Once activated, PI3K/AKT promote transcription of glycolytic genes (though mTOR and low active PKM2), β-catenin stabilization (through PKM2, also promoted by ACLY), activation of glycolytic enzymes (favoring glycolysis), and YAP/TAZ activation (also induced by mevalonate pathway and β-catenin). Once translocated in the nucleus, YAP/TAZ mediate the transcription of genes promoting cell cycle progression and glycolysis. In parallel, (i) F-1,6-BP and AKT inhibit OXPHOS, thus increasing lactate production, which in turn inhibits the immune response and promotes extracellular acidiﬁcation and drug resistance, and (ii) EMT is promoted by β-catenin and YAP/TAZ. Therapeutic strategies increasing citrate levels in cancer cells can inhibit glycolysis and F-1,6-BP production. Therefore, they may be useful to disrupt the Gordian Knot orchestrated by PI3K/AKT, YAP/TAZ, and PFK1/F-1,6-BP. Numbers (1–5) and uppercase letters (A–D) refer to regulatory mechanisms as described in Section 3 (numbers) and Section 4 (letters). Figure created with Biorender. p y g p (TEA)-domain (TEAD) family. Then, YAP/TAZ-TEAD complexes promote expression of YAP and TAZ are transcriptional coactivators encoded by paralogous genes that regulate transcription by binding to the members of the transcriptional enhancer factor (TEA)-domain (TEAD) family. Then, YAP/TAZ-TEAD complexes promote expression of various genes sustaining cell proliferation, migration, and aggressiveness [4,5]. YAP/TAZ Cancers 2022, 14, 2478 5 of 13 5 of 13 are activated downstream of oncogenic pathways such as PI3K/AKT and WNT, and are also promoted by inactivation of suppressors, such as their key regulator known as the Hippo pathway [5,10,43,44]. Indeed, in normal tissues, YAP/TAZ signaling is downreg- ulated by the core components of the Hippo pathway formed by a cascade of kinases (MST1/2 and LATS1/2) that coordinates organ growth and homeostasis together with nutrition and metabolism [45]. Mechanistically, LATS kinases are activated by MST1/2 (drosophila Hippo) or MAP4K4 through phosphorylation [45]. 2. The Activation of PI3K/AKT and YAP/TAZ Pathways Of note, YAP/TAZ may also upregulate PD-L1 in human breast cancer, mesothelioma, melanoma, and non-small cell lung cancer (NSCLC) cells, thus favoring cancer immune evasion [55]. Additional effects of YAP/TAZ on tumoral microenvironment favoring tumor growth or immune escape have also been reported (see [51] for a review). To summarize, PI3K/AKT and YAP/TAZ pathways are closely interconnected, and their deregulation can drive cell cycle progression, anchorage-independent cell growth, altered metabolism, and cancer cell survival. 3. PFK1 and F-1,6-BP Activates PI3K/AKT and YAP/TAZ Signaling Pathways In this paragraph, we will examine several mechanisms by which PFK1 and F-1,6-BP can activate or modulate the PI3K/AKT and YAP/TAZ signaling pathways (Figure 1): (1) F-1,6-BP promotes the activation of PI3K/AKT signaling by binding to SOS1, the key guanine nucleotide exchange factor that promotes strong activation of RAS protein [42]. Subsequently, PI3K/AKT is activated downstream of RAS [40]. Importantly, the binding Cancers 2022, 14, 2478 6 of 13 F-1,6-BP/SOS1 is an evolutionary conserved mechanism that links glycolysis to RAS activation in yeast, as well as in mammalian and cancer cells [42]. F-1,6-BP/SOS1 is an evolutionary conserved mechanism that links glycolysis to RAS activation in yeast, as well as in mammalian and cancer cells [42]. F-1,6-BP/SOS1 is an evolutionary conserved mechanism that links glycolysis to RAS activation in yeast, as well as in mammalian and cancer cells [42]. y (2) Platelet isoform of PFK1 (PFKP) mediates PI3K activation downstream of EGF receptor [56]. Mechanistically, EGFR activation resulted in acetylation (on K395), plasma membrane translocation, and phosphorylation (on Y64) of PFKP. Phosphorylated PFKP binds to the N-terminal SH2 domain of p85α, which in turn results in PI3K activation [56]. Of note, PFKP isoform is frequently highly expressed in lung cancer tissues and cell lines, and it is associated with tumor size and patient prognosis [57]. (3) As shown in several human breast cancer cell lines, PFK1 can bind the transcrip- tional cofactors TEADs, a binding promoting the functional and biochemical cooperation of TEADs with YAP/TAZ [58] and resulting in gene transcription sustaining proliferation and aggressiveness. gg (4) In addition to promoting YAP/TAZ through activation of SOS1/RAS/PI3K/AKT axis, F-1,6-BP can also promote YAP through PI3K/AKT independent pathways, by promot- ing the β-catenin/WNT pathway, for example. Indeed, F-1,6-BP can bind to the epidermal growth factor receptor (EGFR), as shown in triple negative breast cancer cells, thus enhanc- ing EGFR activity [42,59]. p Once activated, both PI3K/AKT and YAP/TAZ pathways can activate PFK1 and favor the accumulation of F-1,6-BP by several mechanisms (Figure 1): 2. The Activation of PI3K/AKT and YAP/TAZ Pathways The EGFR/ERK pathway induces the translocation of the low active dimeric pyruvate kinase M2 (PKM2) isoform into the nucleus [60], with subsequent β-catenin transactivation and further stimulation of the WNT signaling pathway [61]. In turn, the WNT/ β-catenin pathway promotes YAP/TAZ transcription [61,62]. Remark- ably, the WNT/β-catenin pathway also promotes the activation of c-MYC, cyclin D1, and, through hypoxia-inducible factor-1alpha (HIF-1α) and signal transducer and activator of transcription-3 (STAT3), the transcription of many glycolytic enzyme genes (PFK1, PFKFB3, PKM2, LDHA) [61,63,64]. Thus, aerobic glycolysis is enhanced in a hypoxia-independent manner. Of note, alternative WNT signaling (i.e., not involving β-catenin) can also induce YAP/TAZ activation [65]. (5) F-1,6-BP and AKT can cooperate to induce YAP/TAZ activation by promoting the activity of ATP citrate lyase (ACLY) [66,67]. This enzyme transforms cytosolic citrate into oxaloacetate (sustaining nucleotide synthesis) and acetyl-CoA (sustaining histone acetylation and lipid synthesis). Although no formal studies show that ACLY can induce YAP/TAZ activation, we would like to propose that ACLY may promote YAP/TAZ by two indirect mechanisms (but further studies are required to show whether they really occur in cancer cells). First, ACLY can promote the WNT/β-catenin pathway by affecting the stability of β-catenin, and this is known to promote YAP/TAZ transcription [68]. Second, ACLY could sustain YAP/TAZ by providing the acetyl-CoA required for the mevalonate pathway (MVP), which not only controls cholesterol synthesis but can also promote YAP/TAZ activation [69]. Mechanistically, the mevalonate cascade produces the geranylgeranyl-pyrophosphate that is required for activation of Rho GTPases that, in turn, activates YAP/TAZ by inhibiting their phosphorylation, thus promoting their nuclear accumulation, as shown in breast cancer cells [69]. Of note, the transcription of ACLY and MVP genes is induced by the nuclear translocation and activation of the sterol regulatory element-binding protein-1 (SREBP1) (as shown in hepatocarcinoma) [70]. This transcription is promoted by AKT that upregulates SREBP1 at both the transcriptional and post-translational levels (as shown in lung adenocarcinoma) [71]. YAP/TAZ can reinforce the lipogenic effects of PI3K/AKT by interacting with SREBP-1c and SREBP-2 on the promoters of the fatty acid synthase (FAS) and hydroxyl methyl glutaryl coenzyme A reductase (HMGCR), the regulatory enzyme of the MVP which is also inhibited by statins [71–73]. To summarize, PFK1 and F-1,6-BP can activate PI3K/AKT and YAP/TAZ signaling pathways through different mechanisms. 4. 5. Discussion As we have seen, PI3K/AKT and YAP/TAZ signaling, which are frequently activated in resistant cancers, are deeply interconnected. Efﬁciently targeting these pathways by speciﬁc inhibitors alone or in combination is a great challenge, frequently due to different resistance mechanisms. Furthermore, YAP/TAZ can be activated even downstream of a RAS/MAPK or EGFR blockade, thus allowing the inhibitors of these pathways to be bypassed. In this setting, as we have seen, PI3K/AKT and YAP/TAZ can also be activated by PFK1/F-1,6-BP, either through PI3K-dependent or PI3K-independent mechanisms. Thus, targeting the PFK1/F-1,6-BP axis could be fundamental to increase the effectiveness of PI3K/AKT and YAP/TAZ inhibitors, which currently appear modest [7,12]. Of note, a high expression of PFK1 isoform PFKP is related to tumor size, histological grade, lymph node metastasis, and poor patient survival in NSCLC [57,81], which represents the most frequent cause of cancer death in 2020 [82]). Also, a high expression of PFKFB3 is a prognostic factor of poor prognosis in human HER2+ breast cancer and it correlates with TNM in lung adenocarcinoma [83]. As we have seen, many studies argue for the presence of a positive feedback loop between PFK1/F-1,6-BP, PI3K/AKT, and YAP/TAZ. This interconnection can conspire to support cancer cell development and resistance to therapeutics agents, particularly to PI3K and YAP inhibitors. In this loop, PFK1/F-1,6-BP appears as an essential link to couple the enhancement of glycolysis with the activation of PI3K/AKT and YAP/TAZ signaling. All these activations join forces to promote the Warburg effect, proliferation, EMT, and drug resistance. Of note, the interaction of RAS with p110α should be required for RAS-driven tumor formation, as shown in experimental studies in mice [84]. In this context, decreasing the production of F-1,6-BP could attenuate SOS1/RAS binding, and thus slowdown the development of cancers driven by RAS proto-oncogene or RAS mutations (this later form being highly drug resistant) [85]. In addition, the acidiﬁcation of the extracellular environment promoted by the Warburg effect [28] contributes to the exhaustion of the immune response [38] and to drug resistance [33,34,38]. This occurs because the vast majority of drugs (including conventional chemotherapeutics and more recent biological agents) are weak bases that are neutralized in acidic environments [39]. Thus, targeting the Warburg effect could increase drug sensitivity and could be applied to PI3K and YAP/TAZ inhibitors as well. 2. The Activation of PI3K/AKT and YAP/TAZ Pathways In a Feedback Loop, PI3K/AKT and YAP/TAZ can Promote PFK1/F-1,6-BP Once activated, both PI3K/AKT and YAP/TAZ pathways can activate PFK1 and favor the accumulation of F-1,6-BP by several mechanisms (Figure 1): Once activated, both PI3K/AKT and YAP/TAZ pathways can activate PFK1 and favor the accumulation of F-1,6-BP by several mechanisms (Figure 1): 7 of 13 Cancers 2022, 14, 2478 7 of 13 (A) PI3K activates AKT, which in turn promotes the activity of PFK1 [26] (as shown in glioblastoma), and also of PFKFB3 (as showed in HeLa cells). The latter is the enzyme producing F-2,6-BP, the key activator of PFK1. (A) PI3K activates AKT, which in turn promotes the activity of PFK1 [26] (as shown in glioblastoma), and also of PFKFB3 (as showed in HeLa cells). The latter is the enzyme producing F-2,6-BP, the key activator of PFK1. (B) The transcription factor HIF-1α [74] is a key driver of aerobic glycolysis, promoting the expression of many glycolytic genes, including PFK1 [64]. Of note, both PI3K/AKT and YAP can activate HIF-1α (and thus PFK1 transcription) through increasing transcription [75] or protein stabilization [74], respectively. (C) YAP/TAZ can promote PFK1 activity via a cross-talk with a Hedgehog signaling pathway. This cross-talk induces the transcription of PFKFB3 (producing F-2,6-BP) and hexokinase 2 (HK2), thus increasing the carbon ﬂux towards PFK1 [76]. g (D) Both PI3K/AKT and YAP/TAZ favor the glucose uptake by increasing the expres- sion of glucose transporters GLUT1 and/or GLUT3 [77–80], thus increasing further the carbon ﬂux towards PFK1 (and so F-1,6-BP production). g (D) Both PI3K/AKT and YAP/TAZ favor the glucose uptake by increasing the expres- sion of glucose transporters GLUT1 and/or GLUT3 [77–80], thus increasing further the carbon ﬂux towards PFK1 (and so F-1,6-BP production). p Overall, by taking into consideration all the cross-regulations so far described, a posi- tive feedback loop can be established between PFK1/F-1,6-B, PI3K/AKT, and YAP/TAZ. 6. Therapeutic Implications We suggest that targeting PFK1 activity and F-1,6-BP production could help disrupt the here identiﬁed Gordian Knot between PFK1, PI3K/AKT, and YAP/TAZ. Of note, this strategy could enhance the efﬁcacy of PI3K and YAP/TAZ inhibitors currently tested in several aggressive solid cancers and hematologic malignancies (listed in [12]). Particularly, Cancers 2022, 14, 2478 8 of 13 this could be important in those cases in which resistance mechanisms to PI3K and/or YAP/TAZ inhibitors arise in cancer cells. In addition, although PFK1 likely acts as a priming factor in this interconnection between metabolism and PI3K/AKT and YAP/TAZ, the inhibition of the PFK1 activator PFK2/PFKFB3 could also be an efﬁcient strategy. gy Awaiting the development of specific and non-toxic inhibitors of PFK1 and PFKFB3 [86,87], we propose to test strategies increasing the citrate level in cancer cells, since citrate is a well-known physiologic and potent inhibitor of PFK1 and PFK2 (Figure 1) [88,89]. Citrate is an intermediate metabolite of the TCA cycle. It is produced by citrate synthase (CS) from acetyl-CoA and oxalacetate, and it is subsequently transformed into isocitrate by the enzyme aconitase. Alternatively, citrate can be exported into the cytosol through the mitochondrial citrate carrier (SLC25A1/CIC) [90]. In the cytosol, citrate can be consumed by the ATP Citrate Lyase (ACLY) enzyme to generate acetyl-CoA (which sustains pro- tein and DNA acetylation, as well as lipid synthesis) and oxalacetate (which can sustain gluconeogenesis and nucleotide synthesis). In addition to its involvement in metabolic reactions, citrate can also exert some regulatory functions. Indeed, as previously said, citrate can suppress glycolysis and favor gluconeogenesis, since it is a potent inhibitor of glycolytic enzymes PFK1 and PFK2 [88,89] and enhances the activity of the gluconeogenesis enzyme fructose-1,6-biphosphatase (FBPase, an enzyme counteracting PFK1 activity) [91]. In hepatocytes, a citrate-mediated inhibition of glycolysis can sustain the gluconeogenesis induced by glucagon [30]. We previously proposed that in proliferative cancer cells, a low citrate level should be established [30]. Indeed, in cells relying on aerobic glycolysis (Warburg effect), PI3K/AKT and the glycolytic intermediate F-1,6-BP cooperate to inhibit mitochondrial OXPHOS [37,92], and thus the production of citrate, which is also rapidly consumed by ACLY [30]. This would prevent the potential inhibition of PFK1 and PFK2 by citrate, sustaining aerobic glycolysis further. Of note, according to in vitro studies, dichloroacetate restores sensitivity to Paclitaxel in resistant lung adenocarcinoma cells by inducing citric acid accumulation [93]. 6. Therapeutic Implications g By taking into account these considerations, we propose that administration of citrate at high doses could reinforce the efﬁcacy of PI3K and YAP/TAZ inhibitors currently used against cancer cells since (by inhibiting PFK1) it can contribute to disrupt the positive feedback loop between these two signaling pathways and PFK1/F-1,6-BP. Remarkably, as shown in cancer cell lines of various origins (hematologic, sarcoma, melanoma, and epithelial cancers) (for recent review see [30]), the direct administration of sodium citrate at a high concentration (>10 mM) shows various anti-cancer effects. Indeed, citrate at a high concentration efﬁciently inhibits PFK1 [94], inactivates PI3K/AKT/mTOR [95] and IGFR1/RAS/AKT pathways [29], reduces expression of anti-apoptotic factors (Bcl-xL, MCL1 and survivin) [96–98], reduces β-catenin levels [99], attenuates HIF-1α expres- sion [100], induces PTEN expression [29,101], and increases the sensitivity to cisplatin treatment [29,31,96]. Coherently, in A549 cells, ACLY knockdown (increasing citrate level) inhibits PI3K/AKT and reverses cancer stemness and EMT, especially by reducing Snail expression [66,102]. ACLY knockdown also attenuated cisplatin resistance by inhibiting the PI3K-AKT pathway in ovarian cancer cells [103] and in hepatocarcinoma cells. Addi- tionally, it remarkably suppresses stemness properties, migration, and invasion [68]. These results are also corroborated in in vivo studies by oral administration of sodium citrate at high dosages (around 500 mg/kg). Indeed, in mice models, including osteosarcoma and ﬁbrosarcoma [31], prostate cancer [95], RAS-driven lung tumor, breast HER2+ cancer, and also pancreatic cancer xenografts [29], this “citrate strategy” administered orally regressed tumor growth in all cases without remarkable toxicity. Furthermore, it enhanced cisplatin anti-tumor effects [31], reversed EMT, increased intra-tumoral lymphocytes inﬁltration [29], and also neutralized the TME acidity, enhancing the therapeutic effect of 5-ﬂuoro-uracil derivative in a pancreatic cancer xenograft mice model [32]. Lastly, it should be mentioned that citrate has a very low toxicity because it is an endogenous metabolite with a rapid metabolism and a short half-life. Although excessive administration of citrate could lead to muscle spasms, convulsions, hemorrhage, and hypocalcemia, these adverse effects can Cancers 2022, 14, 2478 9 of 13 9 of 13 be treated and prevented by administration of calcium chloride. In addition, it should be remarked that the active dose showing anti-cancer effects in humans should be much lower than the one causing these adverse effects [30]. However, clinical trials should determine the mode and duration of citrate administration to optimize its efﬁcacy while avoiding any side effects on patients. 7. Conclusions In conclusion, we propose to target PFK1/F-1,6-BP to disrupt the “Gordian Knot” linking PFK1/F-1,6-BP, PI3K/AKT and YAP/TAZ, as well as to increase the sensitivity to current PI3K/AKT and YAP/TAZ inhibitors. To this aim, we encourage to test the “citrate strategy” above described in clinical trials given its promising results in preclinical models obtained without remarkable toxicities. Author Contributions: Conceptualization, L.S. and P.I.; writing—original draft preparation, L.S. and P.I.; writing—review and editing, M.A. All authors have read and agreed to the published version of the manuscript. Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska Curie grant agreement No 945298 “ParisRe- gionFP” (to L.S.). L.S. was a Fellow of the Paris Region Fellowship Programme supported by the Paris Region. Conﬂicts of Interest: The authors have no conﬂict of interest to declare. Conﬂicts of Interest: The authors have no conﬂict of interest to declare. References 1. Cantley, L.C. The Phosphoinositide 3-Kinase Pathway. Science 2002, 296, 1655–1657. [CrossRef] [PubM 1. Cantley, L.C. The Phosphoinositide 3-Kinase Pathway. Science 2002, 296, 1655–1657. [CrossRef] [PubMed] 2. Yuan, T.L.; Cantley, L.C. PI3K Pathway Alterations in Cancer: Variations on a Theme. Oncogene 2008, 27, 5497–5510. [CrossRef] [PubMed] 2. Yuan, T.L.; Cantley, L.C. PI3K Pathway Alterations in Cancer: Variations on a Theme. Oncogene 2008, 27, 5497–5510. [CrossRef] [PubMed] 3. Di-Luoffo, M.; Ben-Meriem, Z.; Lefebvre, P.; Delarue, M.; Guillermet-Guibert, J. PI3K Functions as a Hub in Mechanotransduction. Trends Biochem. Sci. 2021, 46, 878–888. [CrossRef] D.K.; Yi, C. YAP/TAZ Signaling and Resistance to Cancer Therapy. Trends Cancer 2019, 5, 283–296. [CrossRef 4. Nguyen, C.D.K.; Yi, C. YAP/TAZ Signaling and Resistance to Cancer Therapy. Trends Cancer 2019, 5, 2 5. Zhang, X.; Zhao, H.; Li, Y.; Xia, D.; Yang, L.; Ma, Y.; Li, H. The Role of YAP/TAZ Activity in Cancer Metabolic Reprogramming. Molecular Cancer 2018, 17, 134. [CrossRef] 6. Krajnik, A.; Brazzo, J.A.; Vaidyanathan, K.; Das, T.; Redondo-Muñoz, J.; Bae, Y. Phosphoinositide Signaling and Mechanotrans- duction in Cardiovascular Biology and Disease. Front. Cell Dev. Biol. 2020, 8, 595849. [CrossRef] 7. Thorpe, L.M.; Yuzugullu, H.; Zhao, J.J. PI3K in Cancer: Divergent Roles of Isoforms, Modes of Activation and Therapeutic Targeting. Nat. Rev. Cancer 2015, 15, 7–24. [CrossRef] 8. Rotow, J.; Bivona, T.G. Understanding and Targeting Resistance Mechanisms in NSCLC. Nat. Rev. Cancer 2017, 17, 637–658. [CrossRef] en, Y.; Nie, J.; Chen, C. Activation of PI3K/AKT/MTOR Pathway Causes Drug Resistance in Breast Cancer 21, 12, 628690. [CrossRef] 9. Dong, C.; Wu, J.; Chen, Y.; Nie, J.; Chen, C. Activation of PI3K/AKT/MTOR Pathway Causes Drug Front. Pharmacol. 2021, 12, 628690. [CrossRef] 10. Zeng, R.; Dong, J. The Hippo Signaling Pathway in Drug Resistance in Cancer. Cancers 2021, 13, 318. [CrossRef] 11 Li E C L i ti C A C tl L C M t b li R i b th PI3K Akt MTOR P th i C R t R lt 10. Zeng, R.; Dong, J. The Hippo Signaling Pathway in Drug Resistance in Cancer. Cancers 2021, 13, 318. [CrossRef] 11. Lien, E.C.; Lyssiotis, C.A.; Cantley, L.C. Metabolic Reprogramming by the PI3K-Akt-MTOR Pathway in Cancer. Recent Results Cancer Res. 2016, 207, 39–72. [CrossRef] [PubMed] Cancer Res. 2016, 207, 39–72. [CrossRef] [PubMed] ong, D. Class I Phosphatidylinositol 3-Kinase Inhibitors for Cancer Therapy. Acta Pharm. Sin. B 2017, 7, 27–37 d] 12. Zhao, W.; Qiu, Y.; Kong, D. References Caffeine Citrate Enhanced Cisplatin Antitumor Effects in Osteosarcoma and Fibrosarcoma in Vitro and in Vivo. BMC Cancer 2019, 19, 689. [CrossRef] [PubMed] Abe, K.; Yamamoto, N.; Hayashi, K.; Takeuchi, A.; Tsuchiya, H. Caffeine Citrate Enhanced Cisplatin Osteosarcoma and Fibrosarcoma in Vitro and in Vivo. BMC Cancer 2019, 19, 689. [CrossRef] [PubMed] 32. Ando, H.; Eshima, K.; Ishida, T. Neutralization of Acidic Tumor Microenvironment (TME) with Daily Oral Dosing of Sodium Potassium Citrate (K/Na Citrate) Increases Therapeutic Effect of Anti-Cancer Agent in Pancreatic Cancer Xenograft Mice Model. Biol. Pharm. Bull. 2021, 44, 266–270. [CrossRef] [PubMed] 33. Pilon-Thomas, S.; Kodumudi, K.N.; El-Kenawi, A.E.; Russell, S.; Weber, A.M.; Luddy, K.; Damaghi, M.; Wojtkowiak, J.W.; Mulé, J.J.; Ibrahim-Hashim, A.; et al. Neutralization of Tumor Acidity Improves Antitumor Responses to Immunotherapy. Cancer Res. 2016, 76, 1381–1390. [CrossRef] 34. Faes, S.; Duval, A.P.; Planche, A.; Uldry, E.; Santoro, T.; Pythoud, C.; Stehle, J.-C.; Horlbeck, J.; Letovanec, I.; Riggi, N.; et al. Acidic Tumor Microenvironment Abrogates the Efﬁcacy of MTORC1 Inhibitors. Mol. Cancer 2016, 15, 78. [CrossRef] [PubMed] 35 Liu G Y; Sabatini D M MTOR at the Nexus of Nutrition Growth Ageing and Disease Nat Rev Mol Cell Biol 2020 21 183 203 34. Faes, S.; Duval, A.P.; Planche, A.; Uldry, E.; Santoro, T.; Pythoud, C.; Stehle, J.-C.; Horlbeck, J.; Letova Tumor Microenvironment Abrogates the Efﬁcacy of MTORC1 Inhibitors. Mol. Cancer 2016, 15, 78. 34. Faes, S.; Duval, A.P.; Planche, A.; Uldry, E.; Santoro, T.; Pythoud, C.; Stehle, J.-C.; Horlbeck, J.; Letovanec, I.; Riggi, N.; et al. Acidic Tumor Microenvironment Abrogates the Efﬁcacy of MTORC1 Inhibitors. Mol. Cancer 2016, 15, 78. [CrossRef] [PubMed] 35. Liu, G.Y.; Sabatini, D.M. MTOR at the Nexus of Nutrition, Growth, Ageing and Disease. Nat. Rev. Mol. Cell Biol. 2020, 21, 183–203. [CrossRef] [PubMed] g y 35. Liu, G.Y.; Sabatini, D.M. MTOR at the Nexus of Nutrition, Growth, Ageing and Disease. Nat. Rev. Mol. Cell Biol. 2020, 21, 183–203. [CrossRef] [PubMed] y, N. Is Akt the “Warburg Kinase”?-Akt-Energy Metabolism Interactions and Oncogenesis. Semin. Cancer B CrossRef] [PubMed] 36. Robey, R.B.; Hay, N. Is Akt the “Warburg Kinase”?-Akt-Energy Metabolism Interactions and Onc 2009, 19, 25–31. [CrossRef] [PubMed] 37. Chae, Y.C.; Vaira, V.; Caino, M.C.; Tang, H.-Y.Y.; Seo, J.H.; Kossenkov, A.V.; Ottobrini, L.; Martelli, C.; Lucignani, G.; Bertolini, I.; et al. Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming. Cancer Cell 2016, 30, 257–272. [CrossRef] g yp p g g 38. References Developmental Biol 2021 475 205 221 [CrossRef] [PubMed] Persistent YAP/TAZ Activation: New Hallmarks in Head and Neck Cancer. Cells 2022, 11, 1370. [CrossRef] [PubMed] 24. Szulzewsky, F.; Holland, E.C.; Vasioukhin, V. YAP1 and Its Fusion Proteins in Cancer Initiation, Progression and Therapeutic Resistance. Persistent YAP/TAZ Activation: New Hallmarks in Head and Neck Cancer. Cells 2022, 11, 1370. [CrossRef] [PubMed] 24. Szulzewsky, F.; Holland, E.C.; Vasioukhin, V. YAP1 and Its Fusion Proteins in Cancer Initiation, Progression and Therapeutic Resistance. Developmental. Biol. 2021, 475, 205–221. [CrossRef] [PubMed] p 25. Mohamed, A.; Sun, C.; De Mello, V.; Selfe, J.; Missiaglia, E.; Shipley, J.; Murray, G.I.; Zammit, P.S.; Wackerhage, H. The Hippo Effector TAZ (WWTR1) Transforms Myoblasts and TAZ Abundance Is Associated with Reduced Survival in Embryonal Rhabdomyosarcoma. J. Pathol. 2016, 240, 3–14. [CrossRef] [PubMed] y y 26. Lee, J.-H.; Liu, R.; Li, J.; Zhang, C.; Wang, Y.; Cai, Q.; Qian, X.; Xia, Y.; Zheng, Y.; Piao, Y.; et al. Stabiliz 1 Platelet Isoform by AKT Promotes Tumorigenesis. Nat. Commun. 2017, 8, 949. [CrossRef] 27. Koppenol, W.H.; Bounds, P.L.; Dang, C.V. Otto Warburg’s Contributions to Current Concepts of Cancer Metabolism. Nat. Rev. Cancer 2011, 11, 325–337. [CrossRef] 28. Icard, P.; Shulman, S.; Farhat, D.; Steyaert, J.-M.; Alifano, M.; Lincet, H. How the Warburg Effect Supports Aggressiveness and Drug Resistance of Cancer Cells? Drug Resist. Updates 2018, 38, 1–11. [CrossRef] g g p 29. Ren, J.-G.; Seth, P.; Ye, H.; Guo, K.; Hanai, J.; Husain, Z.; Sukhatme, V.P. Citrate Suppresses Tumor Growth in Multiple Models through Inhibition of Glycolysis, the Tricarboxylic Acid Cycle and the IGF-1R Pathway. Sci. Rep. 2017, 7, 4537. [CrossRef] g p Ye, H.; Guo, K.; Hanai, J.; Husain, Z.; Sukhatme, V.P. Citrate Suppresses Tumor Growth in Multiple Models of Glycolysis, the Tricarboxylic Acid Cycle and the IGF-1R Pathway. Sci. Rep. 2017, 7, 4537. [CrossRef] th, P.; Ye, H.; Guo, K.; Hanai, J.; Husain, Z.; Sukhatme, V.P. Citrate Suppresses Tumor Growth in Multiple M bition of Glycolysis, the Tricarboxylic Acid Cycle and the IGF-1R Pathway. Sci. Rep. 2017, 7, 4537. [CrossRe 30. Icard, P.; Coquerel, A.; Wu, Z.; Gligorov, J.; Fuks, D.; Fournel, L.; Lincet, H.; Simula, L. Understanding the Central Role of Citrate in the Metabolism of Cancer Cells and Tumors: An Update. Int. J. Mol. Sci. 2021, 22, 6587. [CrossRef] 31. Abe, K.; Yamamoto, N.; Hayashi, K.; Takeuchi, A.; Tsuchiya, H. References Class I Phosphatidylinositol 3-Kinase Inhibitors for Cancer Therapy. Acta Ph [CrossRef] [PubMed] 13. Gibault, F.; Sturbaut, M.; Bailly, F.; Melnyk, P.; Cotelle, P. Targeting Transcriptional Enhanced Associate Domains (TEADs). J. Med. Chem. 2018, 61, 5057–5072. [CrossRef] 14. García-Escudero, R.; Segrelles, C.; Dueñas, M.; Pombo, M.; Ballestín, C.; Alonso-Riaño, M.; Nenclares, P.; Álvarez-Rodríguez, R.; Sánchez-Aniceto, G.; Ruíz-Alonso, A.; et al. Overexpression of PIK3CA in Head and Neck Squamous Cell Carcinoma Is Associated with Poor Outcome and Activation of the YAP Pathway. Oral. Oncol. 2018, 79, 55–63. [CrossRef] [PubMed] y 15. Britschgi, A.; Andraos, R.; Brinkhaus, H.; Klebba, I.; Romanet, V.; Müller, U.; Murakami, M.; Radimerski, T.; Bentires-Alj, M. JAK2/STAT5 Inhibition Circumvents Resistance to PI3K/MTOR Blockade: A Rationale for Cotargeting These Pathways in Metastatic Breast Cancer. Cancer Cell 2012, 22, 796–811. [CrossRef] 16. Jeong, S.-H.; Kim, H.-B.; Kim, M.-C.; Lee, J.-M.; Lee, J.H.; Kim, J.-H.; Kim, J.-W.; Park, W.-Y.; Kim, S.-Y.; Kim, J.B.; et al. Hippo-Mediated Suppression of IRS2/AKT Signaling Prevents Hepatic Steatosis and Liver Cancer. J. Clin. Investig. 2018, 128, 1010–1025. [CrossRef] [PubMed] 10 of 13 10 of 13 Cancers 2022, 14, 2478 17. Rahmani, M.; Aust, M.M.; Attkisson, E.; Williams, D.C.; Ferreira-Gonzalez, A.; Grant, S. Dual Inhibition of Bcl-2 and Bcl-XL Strikingly Enhances PI3K Inhibition-Induced Apoptosis in Human Myeloid Leukemia Cells through a GSK3- and Bim-Dependent Mechanism. Cancer Res. 2013, 73, 1340–1351. [CrossRef] [ ] 18. Serra, V.; Scaltriti, M.; Prudkin, L.; Eichhorn, P.J.A.; Ibrahim, Y.H.; Chandarlapaty, S.; Markman, B.; Rodriguez, O.; Guzman, M.; Rodriguez, S.; et al. PI3K Inhibition Results in Enhanced HER Signaling and Acquired ERK Dependency in HER2-Overexpressing Breast Cancer. Oncogene 2011, 30, 2547–2557. [CrossRef] 19. Prior, I.A.; Lewis, P.D.; Mattos, C. A Comprehensive Survey of Ras Mutations in Cancer. Cancer Res. 2012, 72, 2457–2467. [CrossRef] 20. Yin, Y.; Shen, W.H. PTEN: A New Guardian of the Genome. Oncogene 2008, 27, 5443–5453. [CrossRef] 20. Yin, Y.; Shen, W.H. PTEN: A New Guardian of the Genome. Oncogene 2008, 27, 5443–5453. [CrossRef] 21. Peng, W.; Chen, J.Q.; Liu, C.; Malu, S.; Creasy, C.; Tetzlaff, M.T.; Xu, C.; McKenzie, J.A.; Zhang, C.; Liang, X.; et al. Loss of PTEN g Peng, W.; Chen, J.Q.; Liu, C.; Malu, S.; Creasy, C.; Tetzlaff, M.T.; Xu, C.; McKenzie, J.A.; Zhang, C.; Liang, Promotes Resistance to T Cell Mediated Immunotherapy Cancer Discov 2016 6 202 216 [CrossRef] [Pu 21. References Peng, W.; Chen, J.Q.; Liu, C.; Malu, S.; Creasy, C.; Tetzlaff, M.T.; Xu, C.; McKenzie, J.A.; Zhang, C.; Liang, X.; et al. Loss of PTEN Promotes Resistance to T Cell-Mediated Immunotherapy. Cancer Discov. 2016, 6, 202–216. [CrossRef] [PubMed] Peng, W.; Chen, J.Q.; Liu, C.; Malu, S.; Creasy, C.; Tetzlaff, M.T.; Xu, C.; McKenzie, J.A.; Zhang, C.; Liang, X Promotes Resistance to T Cell-Mediated Immunotherapy. Cancer Discov. 2016, 6, 202–216. [CrossRef] [Pub Resistance to T Cell-Mediated Immunotherapy. Cancer Discov. 2016, 6, 202–216. [CrossRef] [PubMed] Resistance to T Cell Mediated Immunotherapy. Cancer Discov. 2016, 6, 202 216. [CrossRef] [PubMed] abnis, A.J.; Chan, E.; Olivas, V.; Cade, L.; Pazarentzos, E.; Asthana, S.; Neel, D.; Yan, J.J.; Lu, X.; et al. The Hip 22. Lin, L.; Sabnis, A.J.; Chan, E.; Olivas, V.; Cade, L.; Pazarentzos, E.; Asthana, S.; Neel, D.; Yan, J.J.; Lu, X.; et al. The Hippo Effector YAP Promotes Resistance to RAF- and MEK-Targeted Cancer Therapies. Nat. Genet. 2015, 47, 250–256. [CrossRef] [PubMed] 22. Lin, L.; Sabnis, A.J.; Chan, E.; Olivas, V.; Cade, L.; Pazarentzos, E.; Asthana, S.; Neel, D.; Yan, J.J.; Lu, X.; et al. The Hippo Effector YAP Promotes Resistance to RAF- and MEK-Targeted Cancer Therapies. Nat. Genet. 2015, 47, 250–256. [CrossRef] [PubMed] 23 Faraji F ; Ramirez S I ; Anguiano Quiroz PY; Mendez-Molina A N ; Gutkind J S Genomic Hippo Pathway Alterations and , ; S s, J ; C , ; O s, ; C e, ; e os, ; s , S ; Nee , ; , J J ; , ; e e ppo ec o YAP Promotes Resistance to RAF- and MEK-Targeted Cancer Therapies. Nat. Genet. 2015, 47, 250–256. [CrossRef] [PubMed] 23 F ji F R i S I A i Q i PY M d M li A N G ki d J S G i Hi P h Al i d pp YAP Promotes Resistance to RAF- and MEK-Targeted Cancer Therapies. Nat. Genet. 2015, 47, 250–256. [CrossRef] [PubMed] 23. Faraji, F.; Ramirez, S.I.; Anguiano Quiroz, P.Y.; Mendez-Molina, A.N.; Gutkind, J.S. Genomic Hippo Pathway Alterations and g p 23. Faraji, F.; Ramirez, S.I.; Anguiano Quiroz, P.Y.; Mendez-Molina, A.N.; Gutkind, J.S. Genomic Hippo Pathway Alterations and Persistent YAP/TAZ Activation: New Hallmarks in Head and Neck Cancer. Cells 2022, 11, 1370. [CrossRef] [PubMed] 24. Szulzewsky, F.; Holland, E.C.; Vasioukhin, V. YAP1 and Its Fusion Proteins in Cancer Initiation, Progression and Therapeutic Resistance. References YAP/TAZ Functions and Their Regulation at a Glance. J. Cell Sci. 2020, 133 f] 48. Pocaterra, A.; Romani, P.; Dupont, S. YAP/TAZ Functions and Their Regulation at a Glance. J. Cell Sci. 2020, 133, jcs230425. [CrossRef] 49. Tumaneng, K.; Schlegelmilch, K.; Russell, R.C.; Yimlamai, D.; Basnet, H.; Mahadevan, N.; Fitamant, J.; Bardeesy, N.; Camargo, F.D.; Guan, K.-L. YAP Mediates Crosstalk between the Hippo and PI(3)K–TOR Pathways by Suppressing PTEN via MiR-29. Nat. Cell Biol. 2012, 14, 1322–1329. [CrossRef] 50. Qian, X.; He, L.; Hao, M.; Li, Y.; Li, X.; Liu, Y.; Jiang, H.; Xu, L.; Li, C.; Wu, W.; et al. YAP Mediates the Interaction between the Hippo and PI3K/Akt Pathways in Mesangial Cell Proliferation in Diabetic Nephropathy. Acta Diabetol. 2021, 58, 47–62. [CrossRef] 51. Zanconato, F.; Cordenonsi, M.; Piccolo, S. YAP/TAZ at the Roots of Cancer. Cancer Cell 2016, 29, 783–803. [CrossRef] [PubMed] 52. Rosenbluh, J.; Nijhawan, D.; Cox, A.G.; Li, X.; Neal, J.T.; Schafer, E.J.; Zack, T.I.; Wang, X.; Tsherniak, A.; Schinzel, A.C.; et al. β-Catenin-Driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis. Cell 2012, 151, 1457–1473. 50. Qian, X.; He, L.; Hao, M.; Li, Y.; Li, X.; Liu, Y.; Jiang, H.; Xu, L.; Li, C.; Wu, W.; et al. YAP Mediates the Interaction between the Hippo and PI3K/Akt Pathways in Mesangial Cell Proliferation in Diabetic Nephropathy. Acta Diabetol. 2021, 58, 47–62. [CrossRef] 51. Zanconato, F.; Cordenonsi, M.; Piccolo, S. YAP/TAZ at the Roots of Cancer. Cancer Cell 2016, 29, 783–803. [CrossRef] [PubMed] pp / y g p p y , , [ ] 51. Zanconato, F.; Cordenonsi, M.; Piccolo, S. YAP/TAZ at the Roots of Cancer. Cancer Cell 2016, 29, 783–803. [CrossRef] [PubMed] 52. Rosenbluh, J.; Nijhawan, D.; Cox, A.G.; Li, X.; Neal, J.T.; Schafer, E.J.; Zack, T.I.; Wang, X.; Tsherniak, A.; Schinzel, A.C.; et al. β-Catenin-Driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis. Cell 2012, 151, 1457–1473. [CrossRef] [PubMed] pp y g p p y 51. Zanconato, F.; Cordenonsi, M.; Piccolo, S. YAP/TAZ at the Roots of Cancer. Cancer Cell 2016, 29, 78 52. Rosenbluh, J.; Nijhawan, D.; Cox, A.G.; Li, X.; Neal, J.T.; Schafer, E.J.; Zack, T.I.; Wang, X.; Tsherniak, A.; Schinzel, A.C.; et al. β-Catenin-Driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis. Cell 2012, 151, 1457–1473. [CrossRef] [PubMed] 53. References Zanconato, F.; Forcato, M.; Battilana, G.; Azzolin, L.; Quaranta, E.; Bodega, B.; Rosato, A.; Bicciato, S.; Cordenonsi, M.; Piccolo, S. Genome-Wide Association between YAP/TAZ/TEAD and AP-1 at Enhancers Drives Oncogenic Growth. Nat. Cell Biol. 2015, 17, 1218–1227. [CrossRef] [PubMed] 54. Cordenonsi, M.; Zanconato, F.; Azzolin, L.; Forcato, M.; Rosato, A.; Frasson, C.; Inui, M.; Montagner, M.; Parenti, A.R.; Poletti, A.; et al. The Hippo Transducer TAZ Confers Cancer Stem Cell-Related Traits on Breast Cancer Cells. Cell 2011, 147, 759–772. [CrossRef] [ ] 55. Janse van Rensburg, H.J.; Azad, T.; Ling, M.; Hao, Y.; Snetsinger, B.; Khanal, P.; Minassian, L.M.; Graham, C.H.; Rauh, M.J.; Yang, X. The Hippo Pathway Component TAZ Promotes Immune Evasion in Human Cancer through PD-L1. Cancer Res. 2018, 78, 1457–1470. [CrossRef] 56. Lee, J.H.; Liu, R.; Li, J.; Wang, Y.; Tan, L.; Li, X.J.; Qian, X.; Zhang, C.; Xia, Y.; Xu, D.; et al. EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation. Molecular. Cell 2018, 70, 197–210.e7. [CrossRef] 57. Shen, J.; Jin, Z.; Lv, H.; Jin, K.; Jonas, K.; Zhu, C.; Chen, B. PFKP Is Highly Expressed in Lung Cancer and Regulates Glucose Metabolism. Cell Oncol. 2020, 43, 617–629. [CrossRef] 58. Enzo, E.; Santinon, G.; Pocaterra, A.; Aragona, M.; Bresolin, S.; Forcato, M.; Grifoni, D.; Pession, A.; Z Aerobic Glycolysis Tunes YAP/ TAZ Transcriptional Activity. EMBO J. 2015, 34, 1349–1370. [Cross nzo, E.; Santinon, G.; Pocaterra, A.; Aragona, M.; Bresolin, S.; Forcato, M.; Grifoni, D.; Pession, A.; Zanconato, robic Glycolysis Tunes YAP/ TAZ Transcriptional Activity. EMBO J. 2015, 34, 1349–1370. [CrossRef] 59. Lim, S.-O.; Li, C.-W.; Xia, W.; Lee, H.-H.; Chang, S.-S.; Shen, J.; Hsu, J.L.; Raftery, D.; Djukovic, D.; Gu, H.; et al. EGFR Signaling Enhances Aerobic Glycolysis in Triple-Negative Breast Cancer Cells to Promote Tumor Growth and Immune Escape. Cancer Res. 2016, 76, 1284–1296. [CrossRef] 60. Yang, W.; Xia, Y.; Ji, H.; Zheng, Y.; Liang, J.; Huang, W.; Gao, X.; Aldape, K.; Lu, Z. Nuclear PKM Transactivation upon EGFR Activation. Nature 2011, 480, 118–122. [CrossRef] 61. Vallée, A.; Lecarpentier, Y.; Vallée, J.-N. The Key Role of the WNT/β-Catenin Pathway in Metabolic Reprogramming in Cancers under Normoxic Conditions. Cancers 2021, 13, 5557. [CrossRef] 62. Konsavage, W.M.; Kyler, S.L.; Rennoll, S.A.; Jin, G.; Yochum, G.S. Wnt/β-Catenin Signaling Regulates Yes-Associated Protein (YAP) Gene Expression in Colorectal Carcinoma Cells. J. Biol. Chem. 2012, 287, 11730–11739. [CrossRef] 63. Demaria, M.; Poli, V. References Husain, Z.; Huang, Y.; Seth, P.; Sukhatme, V.P. Tumor-Derived Lactate Modiﬁes Antitumor Immune Response: Effect on Myeloid-Derived Suppressor Cells and NK Cells. J. Immunol. 2013, 191, 1486–1495. [CrossRef] y pp J 39. Taylor, S.; Spugnini, E.P.; Assaraf, Y.G.; Azzarito, T.; Rauch, C.; Fais, S. Microenvironment Acidity as a Major Determinant of Tumor Chemoresistance: Proton Pump Inhibitors (PPIs) as a Novel Therapeutic Approach. Drug Resist. Updat 2015, 23, 69–78. [CrossRef] pp ; Spugnini, E.P.; Assaraf, Y.G.; Azzarito, T.; Rauch, C.; Fais, S. Microenvironment Acidity as a Major Dete 39. Taylor, S.; Spugnini, E.P.; Assaraf, Y.G.; Azzarito, T.; Rauch, C.; Fais, S. Microenvironment Acidit Tumor Chemoresistance: Proton Pump Inhibitors (PPIs) as a Novel Therapeutic Approach. Drug [CrossRef] 40. Molina, J.R.; Adjei, A.A. The Ras/Raf/MAPK Pathway. J. Thorac. Oncol. 2006, 1, 7–9. [CrossRef] 40. Molina, J.R.; Adjei, A.A. The Ras/Raf/MAPK Pathway. J. Thorac. Oncol. 2006, 1, 7–9. [CrossRef] 41. Jancík, S.; Drábek, J.; Radzioch, D.; Hajdúch, M. Clinical Relevance of KRAS in Human Cancers. J. Biomed. Biotechnol. 2010, 2010, 150960. [CrossRef] [PubMed] j 2010, 150960. [CrossRef] [PubMed] 42. Peeters, K.; Van Leemputte, F.; Fischer, B.; Bonini, B.M.; Quezada, H.; Tsytlonok, M.; Haesen, D.; Vanthienen, W.; Bernardes, N.; Gonzalez-Blas, C.B.; et al. Fructose-1,6-Bisphosphate Couples Glycolytic Flux to Activation of Ras. Nat. Commun. 2017, 8, 922. [CrossRef] [PubMed] 42. Peeters, K.; Van Leemputte, F.; Fischer, B.; Bonini, B.M.; Quezada, H.; Tsytlonok, M.; Haesen, D.; Vanthienen, W.; Bernardes, N.; Gonzalez-Blas, C.B.; et al. Fructose-1,6-Bisphosphate Couples Glycolytic Flux to Activation of Ras. Nat. Commun. 2017, 8, 922. [CrossRef] [PubMed] 11 of 13 11 of 13 Cancers 2022, 14, 2478 44. Borreguero-Muñoz, N.; Fletcher, G.C.; Aguilar-Aragon, M.; Elbediwy, A.; Vincent-Mistiaen, Z.I.; Thompson, B.J. The Hippo Pathway Integrates PI3K-Akt Signals with Mechanical and Polarity Cues to Control Tissue Growth. PLoS Biol. 2019, 17, e3000509. [CrossRef] ] rvine, K.D. Integration of Hippo-YAP Signaling with Metabolism. Dev. Cell 2020, 54, 256–267. [CrossRef] [ ] 45. Ibar, C.; Irvine, K.D. Integration of Hippo-YAP Signaling with Metabolism. Dev. Cell 2020, 54, 256– g pp g g 46. Gomez, M.; Gomez, V.; Hergovich, A. The Hippo Pathway in Disease and Therapy: Cancer and Beyond. Clin. Transl. Med. 2014, 3, 22. [CrossRef] 47. Fan, R.; Kim, N.-G.; Gumbiner, B.M. Regulation of Hippo Pathway by Mitogenic Growth Factors via Phosphoinositide 3-Kinase and Phosphoinositide-Dependent Kinase-1. Proc. Natl. Acad. Sci. USA 2013, 110, 2569–2574. [CrossRef] p p a, A.; Romani, P.; Dupont, S. References Cancer 2021, 149, 778–789. [CrossRef] 83. Li, X.; Liu, J.; Qian, L.; Ke, H.; Yao, C.; Tian, W.; Liu, Y.; Zhang, J. Expression of PFKFB3 and Ki67 in Lung Adenocarcinomas and Targeting PFKFB3 as a Therapeutic Strategy. Mol. Cell Biochem. 2018, 445, 123–134. [CrossRef] 84. Gupta, S.; Ramjaun, A.R.; Haiko, P.; Wang, Y.; Warne, P.H.; Nicke, B.; Nye, E.; Stamp, G.; Alitalo, K.; Dow to Phosphoinositide 3-Kinase P110alpha Is Required for Ras-Driven Tumorigenesis in Mice. Cell 2007, 84. Gupta, S.; Ramjaun, A.R.; Haiko, P.; Wang, Y.; Warne, P.H.; Nicke, B.; Nye, E.; Stamp, G.; Alitalo, K.; Downward, J. Binding of Ras to Phosphoinositide 3-Kinase P110alpha Is Required for Ras-Driven Tumorigenesis in Mice. Cell 2007, 129, 957–968. [CrossRef] 85. Fernández-Medarde, A.; Santos, E. Ras in Cancer and Developmental Diseases. Genes Cancer 2011, 2, 344–358. [CrossRef] to Phosphoinositide 3-Kinase P110alpha Is Required for Ras-Driven Tumorigenesis in Mice. Cell 2007, 129, 957–968. [CrossRef] 85. Fernández-Medarde, A.; Santos, E. Ras in Cancer and Developmental Diseases. Genes Cancer 2011, 2, 344–358. [CrossRef] p p q g , , [ ] 85. Fernández-Medarde, A.; Santos, E. Ras in Cancer and Developmental Diseases. Genes Cancer 2011, 2, 344–358. [CrossRef] p 86. Mondal, S.; Roy, D.; Sarkar Bhattacharya, S.; Jin, L.; Jung, D.; Zhang, S.; Kalogera, E.; Staub, J.; Wang, Y.; Xuyang, W.; et al. Therapeutic Targeting of PFKFB3 with a Novel Glycolytic Inhibitor PFK158 Promotes Lipophagy and Chemosensitivity in Gynecologic Cancers. Int. J. Cancer 2019, 144, 178–189. [CrossRef] y g 87. Lypova, N.; Dougherty, S.M.; Lanceta, L.; Chesney, J.; Imbert-Fernandez, Y. PFKFB3 Inhibition Impairs Erlotinib-Induced Autophagy in NSCLCs. Cells 2021, 10, 1679. [CrossRef] 88. Nissler, K.; Petermann, H.; Wenz, I.; Brox, D. Fructose 2,6-Bisphosphate Metabolism in Ehrlich Ascites Tumour Cells. J. Cancer Res. Clin. Oncol. 1995, 121, 739–745. [CrossRef] x, M.M. Lehninger Principles of Biochemistry, 8th ed.; W.H. Freeman: New York, NY, USA, 2021. 89. Nelson, D.L.; Cox, M.M. Lehninger Principles of Biochemistry, 8th ed.; W.H. Freeman: New York, NY, 89. Nelson, D.L.; Cox, M.M. Lehninger Principles g p f y 90. Mosaoa, R.; Kasprzyk-Pawelec, A.; Fernandez, H.R.; Avantaggiati, M.L. The Mitochondrial Citrate Carrier SLC25A1/CIC and the Fundamental Role of Citrate in Cancer, Inﬂammation and Beyond. Biomolecules 2021, 11, 141. [CrossRef] 91. Icard, P.; Fournel, L.; Coquerel, A.; Gligorov, J.; Alifano, M.; Lincet, H. Citrate Targets FBPase and Constitutes an Emerging Novel Approach for Cancer Therapy. Cancer Cell Int. 2018, 18, 175. [CrossRef] 92. References Shu, Z.; Gao, Y.; Zhang, G.; Zhou, Y.; Cao, J.; Wan, D.; Zhu, X.; Xiong, W. A Functional Interaction between Hippo-YAP Signalling and SREBPs Mediates Hepatic Steatosis in Diabetic Mice. J. Cell Mol. Med. 2019, 23, 3616–3628. [CrossRef] 74. Zhang, X.; Li, Y.; Ma, Y.; Yang, L.; Wang, T.; Meng, X.; Zong, Z.; Sun, X.; Hua, X.; Li, H. Yes-Associated Protein (YAP) Binds to HIF-1α and Sustains HIF-1α Protein Stability to Promote Hepatocellular Carcinoma Cell Glycolysis under Hypoxic Stress. J. Exp. Clin. Cancer Res. 2018, 37, 216. [CrossRef] 75. Kilic-Eren, M.; Boylu, T.; Tabor, V. Targeting PI3K/Akt Represses Hypoxia Inducible Factor-1α Activation and Sensitizes Rhabdomyosarcoma and Ewing’s Sarcoma Cells for Apoptosis. Cancer Cell Int. 2013, 13, 36. [CrossRef] y g p p 76. Zheng, X.; Han, H.; Liu, G.-P.; Ma, Y.-X.; Pan, R.-L.; Sang, L.-J.; Li, R.-H.; Yang, L.-J.; Marks, J.R.; Wa up Hippo and Hedgehog Signaling to Reprogramme Glucose Metabolism. EMBO J. 2017, 36, 3325– up Hippo and Hedgehog Signaling to Reprogramme Glucose Metabolism. EMBO J. 2017, 36, 3325–3335. [CrossRef] 77. Wang, W.; Xiao, Z.-D.; Li, X.; Aziz, K.E.; Gan, B.; Johnson, R.L.; Chen, J. AMPK Modulates Hippo Pathway Activity to Regulate Energy Homeostasis. Nat. Cell Biol. 2015, 17, 490–499. [CrossRef] p pp g g g g p g 77. Wang, W.; Xiao, Z.-D.; Li, X.; Aziz, K.E.; Gan, B.; Johnson, R.L.; Chen, J. AMPK Modulates Hippo P Energy Homeostasis. Nat. Cell Biol. 2015, 17, 490–499. [CrossRef] 78. Cox, A.G.; Tsomides, A.; Yimlamai, D.; Hwang, K.L.; Miesfeld, J.; Galli, G.G.; Fowl, B.H.; Fort, M.; Ma, K.Y.; Sullivan, M.R.; et al. Yap Regulates Glucose Utilization and Sustains Nucleotide Synthesis to Enable Organ Growth. EMBO J. 2018, 37, e100294. [CrossRef] 79. Actis Dato, V.; Sánchez, M.C.; Chiabrando, G.A. LRP1 Mediates the IGF-1-Induced GLUT1 Expression on the Cell Surface and Glucose Uptake in Müller Glial Cells. Sci. Rep. 2021, 11, 4742. [CrossRef] p p 80. Fang, J.; Zhou, S.-H.; Fan, J.; Yan, S.-X. Roles of Glucose Transporter-1 and the Phosphatidylinositol 3-kinase/Protein Kinase B Pathway in Cancer Radioresistance. Mol. Med. Rep. 2015, 11, 1573–1581. [CrossRef] y p 81. Wang, F.; Li, L.; Zhang, Z. Platelet Isoform of Phosphofructokinase Promotes Aerobic Glycolysis and the Progression of Non-small Cell Lung Cancer. Mol. Med. Rep. 2021, 23, 74. [CrossRef] 82. Ferlay, J.; Colombet, M.; Soerjomataram, I.; Parkin, D.M.; Piñeros, M.; Znaor, A.; Bray, F. Cancer Statistics for the Year 2020: An Overview. Int. J. References PKM2, STAT3 and HIF-1α: The Warburg’s Vicious Circle. JAK-STAT 2012, 1, 194– Poli, V. PKM2, STAT3 and HIF-1α: The Warburg’s Vicious Circle. JAK-STAT 2012, 1, 194–196. [CrossRef] , ; , , g J , , [ ] 64. Marín-Hernández, A.; Gallardo-Pérez, J.C.; Ralph, S.J.; Rodríguez-Enríquez, S.; Moreno-Sánchez, R. HIF-1alpha Modulates Energy Metabolism in Cancer Cells by Inducing over-Expression of Speciﬁc Glycolytic Isoforms. Mini Rev. Med. Chem. 2009, 9, 1084–1101. [CrossRef] 65. Park, H.W.; Kim, Y.C.; Yu, B.; Moroishi, T.; Mo, J.-S.; Plouffe, S.W.; Meng, Z.; Lin, K.C.; Yu, F.-X.; Alexander, C.M.; et al. Alternative Wnt Signaling Activates YAP/TAZ. Cell 2015, 162, 780–794. [CrossRef] 66. Hanai, J.I.; Doro, N.; Sasaki, A.T.; Kobayashi, S.; Cantley, L.C.; Seth, P.; Sukhatme, V.P. Inhibition of Lung Cancer Growth: ATP Citrate Lyase Knockdown and Statin Treatment Leads to Dual Blockade of Mitogen-Activated Protein Kinase (MAPK) and Phosphatidylinositol-3-Kinase (PI3K)/AKT Pathways. J. Cell Physiol. 2012, 227, 1709–1720. [CrossRef] 67. Potapova, I.A.; El-Maghrabi, M.R.; Doronin, S.V.; Benjamin, W.B. Phosphorylation of Recombinant Human ATP:Citrate Lyase by CAMP-Dependent Protein Kinase Abolishes Homotropic Allosteric Regulation of the Enzyme by Citrate and Increases the Enzyme Activity. Allosteric Activation of ATP:Citrate Lyase by Phosphorylated Sugars. Biochemistry 2000, 39, 1169–1179. [CrossRef] 68. Han, Q.; Chen, C.-A.; Yang, W.; Liang, D.; Lv, H.-W.; Lv, G.-S.; Zong, Q.-N.; Wang, H.-Y. ATP-Citrate Lyase Regulates Stemness and Metastasis in Hepatocellular Carcinoma via the Wnt/β-Catenin Signaling Pathway. Hepatobiliary Pancreat. Dis. Int. 2021, 20, 251–261. [CrossRef] 12 of 13 12 of 13 Cancers 2022, 14, 2478 69. Sorrentino, G.; Ruggeri, N.; Specchia, V.; Cordenonsi, M.; Mano, M.; Dupont, S.; Manfrin, A.; Ingallina, E.; Sommaggio, R.; Piazza, S.; et al. Metabolic Control of YAP and TAZ by the Mevalonate Pathway. Nat. Cell Biol. 2014, 16, 357–366. [CrossRef] y y [ ] 70. Zhou, Y.; Tao, J.; Calvisi, D.F.; Chen, X. Role of Lipogenesis Rewiring in Hepatocellular Carcinoma. Semin. Liver Dis. 2022, 42, 77–86. [CrossRef] 71. Liu, L.; Yan, H.; Ruan, M.; Yang, H.; Wang, L.; Lei, B.; Sun, X.; Chang, C.; Huang, G.; Xie, W. An AKT/PRMT5/SREBP1 Axis in Lung Adenocarcinoma Regulates de Novo Lipogenesis and Tumor Growth. Cancer Sci. 2021, 112, 3083–3098. [CrossRef] 72. Sato, R.; Okamoto, A.; Inoue, J.; Miyamoto, W.; Sakai, Y.; Emoto, N.; Shimano, H.; Maeda, M. Transcriptional Regulation of the ATP Citrate-Lyase Gene by Sterol Regulatory Element-Binding Proteins. J. Biol. Chem. 2000, 275, 12497–12502. [CrossRef] 73. 69. Sorrentino, G.; Ruggeri, N.; Specchia, V.; Cordenonsi, M.; Mano, M.; Dupont, S.; Manfrin, A.; Ingallina, E.; Sommaggio, R.; Piazza, S.; et al. Metabolic Control of YAP and TAZ by the Mevalonate Pathway. Nat. Cell Biol. 2014, 16, 357–366. [CrossRef] References Díaz-Ruiz, R.; Avéret, N.; Araiza, D.; Pinson, B.; Uribe-Carvajal, S.; Devin, A.; Rigoulet, M. Mitochondrial Oxidative Phosphoryla- tion Is Regulated by Fructose 1,6-Bisphosphate. A Possible Role in Crabtree Effect Induction? J. Biol. Chem. 2008, 283, 26948–26955. [CrossRef] [PubMed] 93. Zhou, X.; Chen, R.; Yu, Z.; Li, R.; Li, J.; Zhao, X.; Song, S.; Liu, J.; Huang, G. Dichloroacetate Restores Drug Sensitivity in Paclitaxel-Resistant Cells by Inducing Citric Acid Accumulation. Mol. Cancer 2015, 14, 63. [CrossRef] [PubMed] y g 94. Wang, T.-A.; Zhang, X.-D.; Guo, X.-Y.; Xian, S.-L.; Lu, Y.-F. 3-Bromopyruvate and Sodium Citrate Target Glycolysis, Suppress Survivin, and Induce Mitochondrial-Mediated Apoptosis in Gastric Cancer Cells and Inhibit Gastric Orthotopic Transplantation Tumor Growth. Oncol. Rep. 2016, 35, 1287–1296. [CrossRef] [PubMed] p 95. Fan, X.; Zhou, J.; Yan, X.; Bi, X.; Liang, J.; Lu, S.; Luo, L.; Zhou, D.; Yin, Z. Citrate Activates Autophagic Death of Prostate Cancer Cells via Downregulation CaMKII/AKT/MTOR Pathway. Life Sci. 2021, 275, 119355. [CrossRef] [PubMed] 96. Zhang, X.; Varin, E.; Allouche, S.; Lu, Y.; Poulain, L.; Icard, P. Effect of Citrate on Malignant Pleural Mesothelioma Cells: A Synergistic Effect with Cisplatin. Anticancer. Res. 2009, 29, 1249–1254. 13 of 13 13 of 13 Cancers 2022, 14, 2478 97. Lincet, H.; Kafara, P.; Giffard, F.; Abeilard-Lemoisson, E.; Duval, M.; Louis, M.-H.; Poulain, L.; Icard, P. Inhibition of Mcl-1 Expression by Citrate Enhances the Effect of Bcl-XL Inhibitors on Human Ovarian Carcinoma Cells. J. Ovarian Res. 2013, 6, 72. [CrossRef] 98. Guo, X.; Zhang, X.; Wang, T.; Xian, S.; Lu, Y. 3-Bromopyruvate and Sodium Citrate Induce Apoptosis in Human Gastric Cancer Cell Line MGC-803 by Inhibiting Glycolysis and Promoting Mitochondria-Regulated Apoptosis Pathway. Biochem. Biophys. Res. Commun. 2016, 475, 37–43. [CrossRef] 99. Zhou, S.; Sakamoto, K. Citric Acid Promoted Melanin Synthesis in B16F10 Mouse Melanoma Cells, but Inhibited It in Human Epidermal Melanocytes and HMV-II Melanoma Cells via the GSK3β/β-Catenin Signaling Pathway. PLoS ONE 2020, 15, e0243565. [CrossRef] 100. Xu, X.; LI, B.; Huang, P.; Wan, X.; QIN, Y.; Zhou, L.; Liu, H.; BAI, H.; GAO, Y.; Wang, C.; et al. Citrate Induces Apoptosis of the Acute Monocytic Leukemia U937 Cell Line through Regulation of HIF-1α Signaling. Mol. Med. Rep. 2013, 8, 1379–1384. [CrossRef] [ ] 101. Hung, K.-C.; Wang, S.-G.; Lin, M.-L.; Chen, S.-S. Citrate-Induced P85α–PTEN Complex Formation Causes G2/M Phase Arrest in Human Pharyngeal Squamous Carcinoma Cell Lines. Int. J. Mol. Sci. 2019, 20, 2105. 103. Wei, X.; Shi, J.; Lin, Q.; Ma, X.; Pang, Y.; Mao, H.; Li, R.; Lu, W.; Wang, Y.; Liu, P. Corrigendum: Targeting ACLY Attenuates Tumor Growth and Acquired Cisplatin Resistance in Ovarian Cancer by Inhibiting the PI3K-AKT Pathway and Activating the AMPK-ROS Pathway. Front. Oncol. 2021, 11, 742374. [CrossRef] [PubMed] References [CrossRef] y g q 102. Hanai, J.I.; Doro, N.; Seth, P.; Sukhatme, V.P. ATP Citrate Lyase Knockdown Impacts Cancer Stem Cells in Vitro. Cell Death Dis. 2013, 4, e696. [CrossRef] [PubMed] 103. Wei, X.; Shi, J.; Lin, Q.; Ma, X.; Pang, Y.; Mao, H.; Li, R.; Lu, W.; Wang, Y.; Liu, P. Corrigendum: Targeting ACLY Attenuates Tumor Growth and Acquired Cisplatin Resistance in Ovarian Cancer by Inhibiting the PI3K-AKT Pathway and Activating the AMPK-ROS Pathway. Front. Oncol. 2021, 11, 742374. [CrossRef] [PubMed]
https://openalex.org/W4392500315	http://www.ati.ac.cn/en/article/pdf/preview/10.61977/ati2024010.pdf	Latin	null	Solar Image Reconstruction Method Under Strong Turbulence at Fuxian Lake Solar Observatory	Deleted Journal	2,024	cc-by	9,140	Astronomical Techniques and Instruments, Vol. 1, March 2024, 128–139 Astronomical Techniques and Instruments, Vol. 1, March 2024, 128–139 Open Access Article Sizhong Zou1,3 , Zhenyu Jin1,2, Kaifan Ji1,2 , Jun Xu1,2, Lei Yang1,2* Sizhong Zou1,3 , Zhenyu Jin1,2, Kaifan Ji1,2 , Jun Xu1,2, Lei Yang1,2* 1Yunnan Observatories, Chinese Academy of Sciences, Kunming 650216, China 2Yunnan Key Laboratory of Solar Physics and Space Science, Kunming 650216, China 3University of Chinese Academy of Sciences, Beijing 101408, China 1Yunnan Observatories, Chinese Academy of Sciences, Kunming 650216, China 2Yunnan Key Laboratory of Solar Physics and Space Science, Kunming 650216, China 3University of Chinese Academy of Sciences, Beijing 101408, China Correspondence: yanglei@ynao.ac.cn Received: November 30, 2023; Accepted: December 26, 2023; Published Online: March 6, 2024; https://doi.org/10.61977/ati 2024010 © 2024 Editorial Office of Astronomical Techniques and Instruments, Yunnan Observatories, Chinese Academy of Sciences. This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/) Citation: Zou, S. Z., Jin, Z. Y., Ji, K. F., et al. 2024. Solar image reconstruction method under atmospheric turbulence at Fuxian Lake Solar Observatory. Astronomical Techniques and Instruments, 1(2): 128−139. https://doi.org/10.61977/ati 2024010. Abstract: Strong atmospheric turbulence reduces astronomical seeing, causing speckle images acquired by ground- based solar telescopes to become blurred and distorted. Severe distortion in speckle images impedes image phase deviation in the speckle masking reconstruction method, leading to the appearance of spurious imaging artifacts. Relying only on linear image degradation principles to reconstruct solar images is insufficient. To solve this problem, we propose the multiframe blind deconvolution combined with non-rigid alignment (MFBD-CNRA) method for solar image reconstruction. We consider image distortion caused by atmospheric turbulence and use non-rigid alignment to correct pixel-level distortion, thereby achieving nonlinear constraints to complement image intensity changes. After creating the corrected speckle image, we use the linear method to solve the wavefront phase, obtaining the target image. We verify the effectiveness of our method results, compared with others, using solar observation data from the 1 m new vacuum solar telescope (NVST). This new method successfully reconstructs high-resolution images of solar observations with a Fried parameter r0 of approximately 10 cm, and enhances images at high frequency. When r0 is approximately 5 cm, the new method is even more effective. It reconstructs the edges of solar graining and sunspots, and is greatly enhanced at mid and high frequency compared with other methods. Comparisons confirm the effectiveness of this method, with respect to both nonlinear and linear constraints in solar image reconstruction. This provides a suitable solution for image reconstruction in ground-based solar observations under strong atmospheric turbulence. Sizhong Zou1,3 , Zhenyu Jin1,2, Kaifan Ji1,2 , Jun Xu1,2, Lei Yang1,2 Keywords: Astronomical seeing; Solar telescopes; Solar observatories; Astronomy image processing; Phase error; Deconvolution 1. INTRODUCTION When strong turbulence occurs in the free atmosphere, r0 reduces to 4−6 cm, causing short-expo- sure images acquired by ground-based solar telescopes to suffer from severe distortion and degradation. The flaws of the inverse convolution method are also obvious. It relies only on the degraded image as an input value, and uses an optimization algorithm to find the wave- front phase and target image close to the real solution. The deconvolution method ignores the existence of severe aberration and random displacements when r0 decreases. Therefore, it cannot accurately describe the nonlinear influ- ence of atmospheric turbulence on short-exposure images, and the accuracy of the restored wavefront phase is also greatly reduced. Presently, the NVST uses the speckle masking method to reconstruct high-resolution solar images[4,5]. Rep- resentative of statistical reconstruction methods, speckle interferometry[6] and speckle masking[7] have become main- stream solar image reconstruction methods. Speckle mask- ing reconstructs the modes and phases of target images based on the statistical properties of short-exposure speckle images of each order. The statistical method used in speckle masking to describe the influence of atmo- spheric turbulence on images is of physical significance. In recent years, Liu et al. proposed Non-rigid Align- ment-based Solar Image Reconstruction (NASIR), which is an image reconstruction method suitable for fast search- ing and filtering data[12]. This method uses the novel appli- cation of a computational optical flow image method to obtain image phase information by using pixel-by-pixel non-rigid alignment of each group image based on a refer- ence frame. Non-rigid alignment is the correction between pixels according to a small area in the image, which is dif- ferent to the overall size image displacement of the speckle images. NASIR can reconstruct the whole field at once without splitting the image into sub-blocks, substan- tially reducing the required reconstruction time. However, this method only considers nonlinear correction of aligned image aberration, and ignores the use of linear con- straints to reconstruct the phase from further image infor- mation. Because the speckle masking method is based on the triply correlated speckle images, the phase of the target image is estimated recursively from the low-frequency phase to the high-frequency phase. The recursive process will inevitably be affected by noise, such as photon noise, which accumulates in the high-frequency component, poten- tially causing noticeable distortion of the fine structure of the object. 1. INTRODUCTION C2 N(h) where γ is the zenith angle of the observed object, λ is the wavelength of the observing band, h is the height above ground, is the refractive index structure con- stant of the atmosphere and H is the total height of the tur- bulent atmosphere[1]. Atmospheric turbulence has always been the main fac- tor limiting the resolution of ground-based solar tele- scope imaging systems, causing acquired images to be fuzzy, distorted and randomly displaced. To describe atmo- spheric turbulence with respect to optics, Fried postulated that the physical definition of seeing is the coherence diam- eter of the turbulent atmosphere, expressed by parameter r0 in cm. The better the seeing, the higher the r0 value. The Fried parameter can be expressed as Liu et al. and Lou et al. have conducted long-term on-site atmospheric visualization tests at the Fuxian Lake Solar Observatory (FLSO)[2,3]. Table 1 lists the annual mean seeing data at FLSO monitored by the solar differ- ence image motion monitor from 1999 to 2002. In Table 1, r0 is uncalibrated; r0* is calibrated to the zenith and can be categorized into boundary layer seeing r0B and free atmosphere seeing r0F. From these data, the distribution of seeing at FLSO is not ideal. FLSO has more sunny days in winter and spring than in summer and fall, but the monthly mean seeing is generally lower than annual r0 = 0.185cos 3 5 (γ)  λ2 ∫H 0 C2 N (h)dh  3 5 , (1) (1) Table 1. Statistical information of the monthly average atmospheric seeing at Fuxian Lake Solar Observatory from 1999 to 2002. Values are in cm. r0: the monthly average atmospheric seeing. r0: the monthly average atmospheric seeing calibrated to the zenith. r0B: the monthly average boundary layer seeing. r0F: the monthly average free atmosphere seeing [1] May. Jun. Jul. Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. r0 10.42 12.91 14.29 13.44 11.07 9.34 9.26 7.84 6.85 6.74 7.67 8.42 r0 10.82 14.52 15.03 14.46 12.63 11.25 12.07 10.40 9.62 7.76 9.05 9.32 r0B 13.70 18.25 17.16 15.60 13.06 11.35 12.30 10.73 9.74 8.25 11.39 12.13 r0F 21.18 28.94 39.71 52.03 73.16 139.09 98.56 62.27 98.14 31.21 18.00 17.33 phase diversity method can be regarded as a special MFBD technology with stronger constraints[11]. mean seeing. Astronomical Techniques and Instruments, 1(2): 128–139, 2024　129 2.1. Non-rigid Alignment Technique 2.1. Non-rigid Alignment Technique For ground-based solar telescopes, the short-expo- sure image has the problems of distortion and random dis- placement caused by atmospheric turbulence. The tradi- tional way to solve this problem is to shift the whole image using the inter-correlation method, but this rigid alignment (i.e., with the same amount of displacement of pixels at different positions in the image) only over- comes the displacement problem, and cannot eliminate the aberration of different regions in the short-exposure image. 1. INTRODUCTION (4) Anisotropic filters are used to effectively smooth and suppress noise, and restore image details. between the image intensity change and displacement field for target detection in moving images, to obtain the displacement of each pixel of the target between consecu- tive image frames, and finally to get the motion trajec- tory of the spatial target. Non-rigid alignment draws on the principle of the optical flow method to establish the relationship model between pixel displacement and inten- sity change for solar speckle images. However, unlike calcu- lating the motion consistency of adjacent frames in the field of visual processing, the adjacent frame in the field of solar astronomy has random displacement and distor- tion. Consequently, it is impossible to accurately calcu- late the displacement field of short-exposure sequence images by relying on the adjacent frame images. Here, we present the specific principles and methods in Section 2. Section 3 presents observational NVST data and experimental results. Section 4 discusses the advan- tages and disadvantages of MFBD-CNRA and the role of non-rigid alignment. Finally, we summarize the results of our work and future plans. 1. INTRODUCTION Consequently, in the winter and spring when free atmo- spheric turbulence activity is strong, the resolution of FLSO images reconstructed by speckle masking will be sig- nificantly reduced. Full image quality cannot be accu- rately restored, and imaging artifacts will appear. This can be verified through image data from January to April in each year at the website, http://fso.ynao.ac.cn. Another class of solar image reconstruction tech- niques uses the inverse convolution method, which mainly includes the blind deconvolution method and phase diver- sity. Blind deconvolution refers to the technique of estimat- ing the target image directly from a small number of short-exposure images without prior knowledge of the point spread function (PSF). The multiframe blind deconvo- lution (MFBD) algorithm[8] estimates the target image based on the principle of maximum likelihood, by employ- ing a sequence of short-exposure solar speckle images. The phase diversity technique is a further refinement of blind deconvolution, introducing a known aberration (e.g. defocus) into the imaging system in addition to the target focal plane image[9,10]. Then, images are obtained, and the uncertainty of the phase recovery is eliminated by using two (or more) images. Löfdahl & Scharmer unified the problems of phase diversity and MFBD into the problem of solving the wavefront phase coefficients under the lin- ear constraints of maximum likelihood theory, and the This paper proposes the MFBD-CNRA solar image reconstruction technique. This algorithm yields improved image alignment and phase recovery. Primary considera- tions are as follows. (1) Effects of distortion and random displacement on short-exposure images, caused by strong atmospheric turbulence. We rationalize the geometrical form and structure of a sequence of speckle images using the non-rigid alignment technique, and the accurate align- ment of images between frames is realized. (2) In poor see- ing conditions, short-exposure images contain broken struc- tures. It is necessary to use an improved frame selection method to select lucky images with sufficient high-fre- quency information and stable structure to restrict the gen- eral structural shape of the target, preventing the image from reconstructing the wrong deviation structure. (3) Using the non-rigid aligned average frame to supplement the a priori knowledge of the target image, we obtain the initial values of wavefront Zernike aberration coefficients in the optimization process. Searching from reliable ini- 129 tial values effectively limits the confidence interval of opti- mization and avoids falling into local optima prematurely. 2. TECHNIQUES AND METHODS The MFBD-CNRA image reconstruction technique is still essentially a wavefront phase estimation and target recovery method based on image intensity. However, the process of image reconstruction considers the nonlinear degradation process of the solar speckle pattern, which com- pensates for the lack of linearity. When MFBD-CNRA adopts the optical flow method, it is necessary to select an appropriate reference frame. For each frame of the sequence of speckle images, the opti- cal flow vector between the reference image and the speckle image is calculated to recover the different displace- ments on each image area. To correct the distortion and displacement of speckle images caused by atmospheric turbulence, the non-rigid alignment technology uses the principle of optical flow (explained in Section 2.1) to align each frame of short-expo- sure images with the reference image (RI) pixel by pixel, completing the inter-frame image alignment and yielding the corrected short-exposure image sequence. On this basis, image sub-blocks are segmented, and the lucky frame selection strategy (explained in Section 2.2) based on spectral ratio and structural similarity are adopted to select the images with more high-frequency information and stable structure as input images for MFBD. The aver- aged frame image of the post-alignment speckle image sub-blocks is taken as the prior target image. The initial value of the phase iteration of the cost generalization func- tion is derived to effectively narrow the confidence inter- val of the optimization search. An anisotropic filter is intro- duced to smooth and suppress noise while restoring the image. Finally, the recovered image is stitched to recon- struct the large field-of-view image. This pixel-by-pixel "non-rigid" displacement is differ- ent from the overall rigid movement of the whole image, and it can reasonably and efficiently correct the target's aberration displacement in the spatial domain. At the same time, major alignment between the frames is accom- plished based on the same reference image. 130　www.ati.ac.cn Selection of the initial reference image Accurate estimation of the displacement field needs an image with a high signal-to-noise ratio as a reference frame. When constructing a reference frame for the first time, the phase of the average frame image is obtained by superimposing short-exposure sequence images filtered by the band-pass filter, and the mode is obtained according to the statistical reconstruction of speckle interferometry. After combining the mode and the phase, a reference frame image with a stable structure and high-frequency information is obtained. The initial reference frame image mode is recon- structed as The above principle involves key steps such as non- rigid alignment technique, lucky frame selection method, estimation of wavefront phase Zernike coefficients, and denoising. \|RI (u)\|2 = ⟨ \|I (u)\|2 · Ot f (u) S it f (u)+S noise ⟩ , (2) (2) where u is the frequency domain coordinates, I(u) is the speckle image, Snoise is the estimated noise power spec- trum in the speckle images, Otf(u) is the ground-based solar telescope theoretical optical transfer function, which is related to the shape of the telescope pupil and the aper- ture, and Sitf(u) is the theoretical speckle interferometry transfer function, which is estimated by the seeing[13]. The angle brackets denote taking the average value. 2.1.3. Reference image update with iterative alignment 2.1.3. Reference image update with iterative alignment d = −1 2 A−1 1 (b2 −b1). (7) (7) After the initial reference frame is set, each pixel in the set area of each speckle image frame finds the corre- sponding pixel in the reference frame, and obtains the dis- placement field corresponding to the two pixels. Then, the distortion displacement of the speckle image can be recov- ered pixel by pixel by inverse displacement and interpola- tion, according to the solved displacement field vector. Per- forming a single non-rigid alignment of all speckle images in the sequence is referred to as a “round”. The ref- erence frame of each subsequent round is set to be the aver- age frame of the speckle image sequence after the comple- tion of the previous round, to continuously improve the sig- nal-to-noise ratio of the reference image, which is con- ducive to a more accurate restoration of distortion. After the preset number of correction rounds is reached, the dis- tortion displacement field of each frame is calculated, and every original short-exposure image is moved pixel by pixel according to the finally calculated displacement field for complete alignment with the reference frame, cor- recting the geometry and structure of the image sequences. Therefore, we can develop a calculation model for the image pixel intensities of translation transformations. When we assume that the displacement field is only slowly varying so that the information can be integrated within the neighborhood of each pixel, we try to find the amount of translation d in Equation (7) in the neighbor- hood of the image region, given by Therefore, we can develop a calculation model for the image pixel intensities of translation transformations. When we assume that the displacement field is only slowly varying so that the information can be integrated within the neighborhood of each pixel, we try to find the amount of translation d in Equation (7) in the neighbor- hood of the image region, given by d =  ∑ n wnAT n An  −1 ∑ n ( wnAT n∆bn ) , (8) (8) where n is a pixel point within the neighborhood, wn is a way of calculating the weight of the pixel points in the neighborhood, and ∆bn is the difference between bn and the reference frame vector b. 2.1.2. Calculate the displacement field (5), the coeffi- cients are equal and it follows that A2 = A1 b2 = b1 −2A1d c2 = dT A1d −bT 1 d +c1, (6) (6) which leads to the relation 2.1.3. Reference image update with iterative alignment To describe the projection of the distortion on the image more accurately, a more reasonable calculation model of the displacement field can be obtained through optimization strategies such as the affine transformation of the displacement field and the weighting of neighbor- hood pixels[12]. For the projection of general 3D motion on the image plane, the displacement field is represented by a 2D 8-parameter model as 2.2. Lucky Frame Selection Method 2.1.2. Calculate the displacement field The pixel intensity distribution in the localized region of the solar speckle image can be expressed as In computational vision processing, the optical flow method is often used to establish the relationship model f (x) ≈xTAx+ bTx+c, (3) (3) which can be written as a matrix, which can be written as a matrix, where x is the two-dimensional coordinates of the speckle image region, f(x) is the image pixel intensity at the loca- tion, A is a symmetric matrix variable, b is a vector, and c is a scalar. d = SP, (11) d = SP, (11) (11) d = SP, S =  1 x y 0 0 0 x2 xy 0 0 0 1 x y xy y2 , (12) We assume that the approximate expression for the pixel intensity distribution of the reference frame is (12) f1 (x) ≈xTA1x+ bT 1 x+c1, (4) (4) P = ( a1 a2 a3 a4 a5 a6 a7 a8 )T . (13) P = ( a1 a2 a3 a4 a5 a6 a7 a8 )T . (13) (13) when the speckle image has an ideal translation transforma- tion with this region of the reference image, the expres- sion for the relationship between pixel intensity and posi- tion in this region of the speckle image and the reference frame image is as follows: So, we get P =  ∑ n wnST n AT n AnSn  −1 ∑ n ( wnST n AT n∆bn ) . (14) (14) f2 (x) = f1(x−d) = (x−d)T A1 (x−d)+ bT 1 (x−d)+c1 = xT A1x+(b1 −2A1d)T x+ dTA1d −bT 1 d +c1 = xT A2x+ bT 2 x+c2. When the displacement field is slowly changing, the unknown quantity An and ∆bn of all pixel points in the neighborhood are the same. The least squares method is used to solve the equation P relating the intensity of multi- ple pixels in the neighborhood to the translation vector. Then, according to Equation (11), the translation vector d of the pixel points of the speckle images in the local region, with respect to the pixel points of the reference image, can be derived. This is extended to the whole image area, and the displacement field is established between the speckle images and the reference image. (5) When the displacement d satisfies Eq. (5), the coeffi- cients are equal and it follows that When the displacement d satisfies Eq. Astronomical Techniques and Instruments, 1(2): 128–139, 2024　131 2.2. Lucky Frame Selection Method Here, δ represents the integral of ε(u, v) in the fre- quency domain, expressed as E(O,am) = J ∑ j I j ( u)−O(u)· Hj(u)\|2, (20) (20) δ = ∫2π 0 dθ ∫ρ2(r0) ρ1(r0) ρε(ρcosθ, ρsinθ)dρ, (16) (16) where am denotes the wavefront phase Zernike coeffi- cient corresponding to the mth term, and j denotes the jth frame of the speckle image in a set of input images. where ρ represents the frequency magnitude of the radial, ρ1(r0) and ρ2(r0) represent the integral ring inner diame- ter outer diameter, and r0 is the average seeing of the image. Löfdahl adds Tikhonov regular terms about the tar- get and phase to the error cost function, and the function is expressed as[16] However, this method does not consider the problem of image structure fragmentation and distortion caused by rapidly moving atmospheric turbulence. Consequently, it is not sensitive to image structure stability, and performs poorly at rejecting images containing broken and dis- torted structures. After the non-rigid geometric correction, it is necessary to select the lucky images with more high- frequency information and stable structure to constrain the overall structure of the target to prevent the deviation of image reconstruction in the process of MFBD. Structural similarity (SSIM)[15] is a measure of the similarity between two images. Given a single image and the mean frame image, it is given by E(O,a) = J ∑ j I j ( u)−O(u)·H j(u)\|2+γ\|O(u)\|2+αJ (ϕ), (21) J (ϕ) = 1 2 M ∑ m 1 λm J ∑ j \|ajm\|2, (22) (22) where γ is the Tikhonov regular term parameter for tar- get estimation, α is the Tikhonov regular term parameter for wavefront phase information estimation, and λm is the covariance matrix corresponding to the mth term of the orthogonal polynomials. S S IM(X,Y) = (2µxµy +c1)(2σxy +c2) (µ2x +µ2y +c1)(σ2x +σ2y +c2), (17) (17) Therefore, the problem of how to estimate the wave- front information and the target image from the multi- frame speckle image can be transformed into a problem of solving the cost generalized function with respect to the minimal values of the variable O and the phase informa- tion a, when the target image and the system PSF are unknown. We can apply linear optimization to the cost gen- eralized function, but it is inefficient to solve two unknowns directly and the calculation easily converges pre- maturely. 2.2. Lucky Frame Selection Method Each group of collected solar data contains 200 speckle images. During the observation process, there are likely to be errors in some images due to telescope point- ing errors and weather conditions. It is wise to select images that are less affected by atmospheric turbulence, which will retain more high-frequency information and be closer to the true target image. dx(x,y) = a1 +a2x+a3y+a7x2 +a8xy, (9) dy(x,y) = a4 +a5x+a6y+a7xy+a8y2, (10) dx(x,y) = a1 +a2x+a3y+a7x2 +a8xy, (9) dy(x,y) = a4 +a5x+a6y+a7xy+a8y2, (10) dx(x,y) = a1 +a2x+a3y+a7x2 +a8xy, (9) (10) dy(x,y) = a4 +a5x+a6y+a7xy+a8y2, Astronomical Techniques and Instruments, 1(2): 128–139, 2024　131 The lucky frame selection technology[14] used by NVST is a frame selection method based on speckle inter- ferometry. The less the image is affected by atmospheric turbulence, the larger the value of its transfer function will be in each frequency band. We divide the power spec- trum of the single-frame short-exposure image by the aver- age of the speckle image power spectrum to normalize and obtain ε(u, v) which is related to the telescope and the atmosphere. O(u, v) is the frequency domain representa- tion of the target image, and H(u, v) is the frequency domain representation of the PSF. cation of the target image and the PSF of the system in the frequency domain. For the convenience of formula expression and to avoid misunderstanding, in the follow- ing formula, the two-dimensional coordinates (u, v) of the image are expressed as a single coordinate (u), given by I(u) = O(u)· H (u). (19) (19) MFBD can recover the target and estimate the instanta- neous PSF or wavefront phase information of the system by establishing the error cost function and finding the mini- mum values for the aberration coefficient and target estima- tion. To determine the degree of approximation between the intensity of the reconstructed target image and the inten- sity of the actual acquired speckle image, we can define an error cost generalization function based on the princi- ple of maximum likelihood estimation, expressed as ϵ (u,v) = \|I (u,v)\|2 ⟨\|I (u,v)\|2⟩ = \|O(u,v)\|2 ⟨\|O(u,v)\|2⟩ \|H (u,v)\|2 ⟨\|H (u,v)\|2⟩ = \|H (u,v)\|2 ⟨\|H (u,v)\|2⟩ (15) We can evaluate image quality using circular integra- tion. 2.2. Lucky Frame Selection Method where µx is the mean of X; µy is the mean of Y; σx is the variance of X; σy is the variance of Y; and σxy is the covari- ance between X and Y; c1 = (k1L), c2 = (k2L) are con- stants used to maintain stability, L is the range level of pixel values, k1 = 0.03, k2 = 0.03; and the range of SSIM is −1 to 1. When the two images are identical, SSIM is equal to 1. The frame selection evaluation criterion used in this paper is given by LuckIndex = δ·S S IM . (18) (18) In this paper, we take an approach that complements the prior knowledge of the target image. Consequently, we choose to use the non-rigid corrected mean short-expo- sure image On as the initial priori estimate of the target. 2.3. Estimation of Wavefront Phase Zernike Coefficients 2.3. Estimation of Wavefront Phase Zernike Coefficients The short-exposure image can be viewed as a multipli- This leads to the solution, the expression is 132　www.ati.ac.cn www.ati.ac.cn caused by the energy decay of the PSF at high frequen- cies. When the regularization parameter is not selected prop- erly, it will produce a serious ringing effect, causing unclear edges in the recovered image. E(On,a) = J ∑I j ( u)−O(u)· Hj(u)\|2 +γ\|On(u)\|2 +αJ (ϕ), (23) To better deal with noise and image details, we con- struct a filter in the form[11] after replacing the target estimate O with On, solving for the first-order partial derivative of the cost generalized func- tion E(O, a) with respect to the unknown quantity a, and thus solving for a set of aberration coefficients am−initial, such that E(On, a) is minimized under the condition that the initial image On is known. Filter = 1− ⟨J ∑ j Noise j ⟩⟨ J ∑ j \|Hj\|2 \| J ∑ j I j · H∗ j\|2 ⟩ , (26) Filter = 1− ⟨J ∑ j Noise j ⟩⟨ J ∑ j \|Hj\|2 \| J ∑ j I j · H∗ j\|2 ⟩ , (26) (26) Considering the reduction of the number of unknowns to eliminate the objective image O, Löfdahl sim- plifies E(O, a) as[16] where Noisej is the estimated noise power spectrum out- side the selected cutoff frequency, and the angle brackets represent taking the mean average value. We then set the values of Filter to 0.2−1, and all values less than 0.2 and greater than 1 to 0. All isolated peaks in the filter that can- not be connected to the zero-frequency peak are removed to obtain a smooth filter. This constructs a filter that satis- fies anisotropy by calculating the ratio of the noise to the signal at different locations, effectively smoothing and sup- pressing the noise. E (a) =  J ∑ j \|Ij\|2 − J ∑ j I∗ j · Hj 2 J ∑ j \|Hj\|2 +γ  + α 2 M ∑ m 1 λm J ∑ j \|a jm\|2. (24) The initial iteration value of am−initial is used as the ini- tial iteration value of the E(a) optimization solving pro- cess. Equation (24) is solved iteratively from the initial value, and the iteration is stopped when the maximum num- ber of iterations is reached or the resultant value meets the set error value. 2.4. Construction of the Image Filter At present, the channels of the high-resolution imag- ing system are mainly the chromosphere H-alpha (center wavelength 656.26 nm, channel bandwidth 0.025 nm), and the photosphere TiO (center wavelength 705.8 nm, channel bandwidth 1 nm). The TiO channel is a common optical channel for observing the solar photosphere, and the reconstruction of TiO data is representative of solar observations in all bands. In actual observation, speckle images usually have addi- tional noise. The key to estimating the clear image from the blurred image is to make an appropriate strategy between obtaining the clear target image and restraining the noise. The solar short-exposure image with additional noise can be expressed as (25) I (u) = O(u)· H (u)+ Noise(u) . (25) The experimental data in Table 2 are all Level 0 obser- vations in the TiO band from 2020 to 2022. Each batch consists of 200 short-exposure images, each with an expo- sure time of approximately 0.15 s. The atmospheric Fried parameters r0 of the experimental data are calculated by the spectral ratio method[18]. Due to the existence of noise and the influence of the PSF in suppressing high-frequency information, the sig- nal-to-noise ratio of the image is insufficient, and the direct deconvolution method will lead to serious noise and ringing in the target image. 3. RESULTS The main scientific goal of NVST is to carry out high-resolution imaging and spectral observation of the sun in the 0.3−2.5 micron band, including measuring the fine structure and evolution process of the solar magnetic field with high temporal and spatial resolution. The tele- scope has a vacuum sealing window with a diameter of 1 200 mm, an effective telescope aperture of 980 mm, an effec- tive field of view of more than 3', and a focal length of 45 m at F3. By setting the initial iteration value, the search inter- val of the final solution is greatly constrained, and some local optimal values are skipped, thereby converging to more accurate aberration coefficients. By using the phase aberration coefficients and Zernike’s formula, we recon- struct the PSF of the telescope. 2.3. Estimation of Wavefront Phase Zernike Coefficients Eventually, the final resultant value of aberration coefficients am is calculated. Astronomical Techniques and Instruments, 1(2): 128–139, 2024　133 3.1. With Seeing of Approximately 1.5" Wiener filtering is a linear filtering method based on the minimum mean square error criterion[17], but the power spectra of the noisy and clear images are difficult to obtain accurately in practical problems. An approxi- mate ratio is commonly used to estimate in specific applica- tions, which still leaves a lot of noise in images with low signal-to-noise ratios. The Tikhonov regularization denois- ing method[17], in the form of frequency domain trunca- tion, can suppress the amplification of noise energy In summer and autumn, the seeing at FLSO is usu- ally approximately 1.5" and the Fried parameter r0 is above 10 cm. The speckle masking reconstruction effect is quite good. Fig. 1 and Fig. 2 show the reconstruction results of data sets 1 and 2 using speckle masking, MFBD, NASIR, and MFBD-CNRA. As can be seen, the result images of all algorithms clearly show details such as sunspot, solar graining and Astronomical Techniques and Instruments, 1(2): 128–139, 2024　133 Table 2. Solar speckle image reconstruction test data set from the NVST. Information includes experimental data number, observation band, observation time, observation area, r0 Data No. Band Time Area r0 /cm 1 TiO 2021-09-30 UT 05:38:42 12 880 11.70 2 TiO 2020-12-08 UT 03:57:16 12 790 10.06 3 TiO 2020-10-06 UT 01:27:41 12 882 9.31 4 TiO 2021-11-29 UT 04:02:58 12 898 7.35 5 TiO 2023-03-06 UT 06:31:57 13 242 5.32 6 TiO 2023-01-20 UT 03:50:04 12 297 4.90 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 (″) (″) (″) (″) 5 10 15 20 25 (″) (″) (″) (″) A B C D Fig. 1. Reconstruction results of NVST TiO 2021-09-30 UT 05:38:42, observation active area 12880, from (A) Speckle masking, (B) MFBD, (C) NASIR, (D) MFBD-CNRA. Table 2. Solar speckle image reconstruction test data set from the NVST. www.ati.ac.cn 3.1. With Seeing of Approximately 1.5" Information includes experimental data number, observation band, observation time, observation area, r0 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) B 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) A 0 0 5 10 15 20 25 (″) 5 10 15 20 25 (″) D 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) C Fig. 1. Reconstruction results of NVST TiO 2021-09-30 UT 05:38:42, observation active area 12880, from (A) Speckle masking, (B) MFBD, (C) NASIR, (D) MFBD-CNRA. solar magnetic highlights. Visually, the four algorithms are close in reconstruction quality. To intuitively com- pare the quality in the frequency domain, we convert the 2D power spectrum image to polar coordinates, then aver- age along the polar angle to obtain a 1D power spectrum curve with the length of the polar diameter as the fre- quency and the average intensity as the energy. In Fig. 3, we plot the power spectrum curves of the reconstructed image of Fig. 2. The power spectrum curve of MFBD- CNRA image is higher than the other curves in the mid and high frequency region, which is closer to the limit- ing resolution of the telescope. Fig. 4 shows the intensity profile at the red line pixels and supports the correctness of the MFBD-CNRA intensity distribution. accurately, indicators such as SSIM and the coefficient of variation of the intensity profile (CVoIP) are used to ana- lyze each result. The SSIM index can be used to evalu- ate differences between images by combining three differ- ent factors: brightness, contrast and structure. CVoIP, which is the ratio of the standard deviation of the inten- sity profile to its mean, can measure the difference in image contrast. The results of quantitative comparison between speckle masking and the other three methods are given in Table 3, which shows that the reconstructed images of speckle masking have high correlation and SSIM with those of NASIR and MFBD-CNRA. 3.1. With Seeing of Approximately 1.5" The quantitative analysis indices show that the reconstructed images of To describe the quality of image reconstruction more 134 www.ati.ac.cn 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 (″) 25 5 10 15 20 25 0 0 5 10 15 20 25 (″) (″) (″) 5 10 15 20 25 (″) (″) (″) (″) A B C D Fig. 2. Reconstruction results of NVST TiO 2020-12-08 UT 03:57:16, observation active area 12790, from (A) Speckle masking, (B) MFBD, (C) NASIR, (D) MFBD-CNRA. 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) B 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) A 0 0 5 10 15 20 25 (″) 5 10 15 20 25 (″) D 0 0 5 10 15 20 (″) 25 5 10 15 20 25 (″) C Fig. 2. Reconstruction results of NVST TiO 2020-12-08 UT 03:57:16, observation active area 12790, from (A) (B) MFBD, (C) NASIR, (D) MFBD-CNRA. tion results of NVST TiO 2020-12-08 UT 03:57:16, observation active area 12790, from (A) Speckle masking, ASIR, (D) MFBD-CNRA. Speckle masking MFBD-CNRA 0 0.6 25 50 75 100 125 150 175 200 0.8 1.0 1.2 1.4 1.6 Pixel Intensity of pixel Intensity profile (Normalized by divide mean) Fig. 4. Comparison of the intensity profiles between the reconstruction results (the red line region in Fig. 2 A) of speckle masking and MBFD-CNRA. Speckle masking MFBD-CNRA 0 0.6 25 50 75 100 125 150 175 200 0.8 1.0 1.2 1.4 1.6 Pixel Intensity of pixel Intensity profile (Normalized by divide mean) 1/spatial frequency (″) Log (power spectrum) 1.0 0 0.5 0.25 0.1 0.08 5 10 20 Power spectrum of Speckle masking Power spectrum of MFBD Power spectrum of MASIR Power spectrum of MFBD-CNRA Fig. 3. Power spectrum curves of reconstruction results of NVST TiO 2020-12-08 UT 03:57:16, observation active area 12790. 1/spatial frequency (″) Log (power spectrum) 1.0 0 0.5 0.25 0.1 0.08 5 10 20 Power spectrum of Speckle masking Power spectrum of MFBD Power spectrum of MASIR Power spectrum of MFBD-CNRA Fig. 3. Power spectrum curves of reconstruction results of NVST TiO 2020-12-08 UT 03:57:16, observation active area 12790. Fig. 4. 3.2. With Seeing of Approximately 3 Arcseconds In FLSO short-exposure image data, the seeing reaches the lowest from January to March every year, usu- ally approximately 3". And r0 range is usually 4 cm to 5 cm. As seeing declines, the high-frequency information of the speckle image is severely impaired after atmospheric effects, and the signal-to-noise ratio also begins to decrease. Fig. 5 illustrates the reconstruction results for data set 5. Fig. 6 shows reconstruction results of data set 6 for comparison. These two sets of data are representa- tive of low seeing data in actual observation. With the weakening of seeing, the phase recursion error gradually accumulates, and the reconstruction results of the speckle- mask method show spurious structures, which have a great impact on the reconstructed images. The multi- frame blind deconvolution algorithm can reconstruct solar graining in the local area, but detailed structures such as sunspot contours, penumbral filaments, and edges of the solar graining cannot be revealed. In Fig. 7, we draw the power spectrum outline of the reconstructed image of data set 5. The MFBD power spec- tral profile decreases significantly, and the speckle mask- ing power spectral profile also decreases rapidly in the high-frequency region. The power spectrum curves for the NASIR and MFBD-CNRA images are very close in the mid and high frequency regions, and they are both higher than the other methods. Fig. 8 shows the intensity curve of the red line in Fig. 5, showing that the quality of the reconstructed image using MFBD-CNRA gradually improves. 3.1. With Seeing of Approximately 1.5" Comparison of the intensity profiles between the reconstruction results (the red line region in Fig. 2 A) of speckle masking and MBFD-CNRA. Table 3. Quantitative comparison of the reconstruction results from four solar image reconstruction algorithms of test data in Table 1 Observation Time r0 SSIM CVoIP Masking MFBD NASIR MFBD-CNRA Masking MFBD NASIR MFBD-CNRA 2021-09-30 UT 05:38:42 11.70 1 0.971 03 0.975 12 0.975 22 0.317 12 0.309 67 0.314 04 0.317 77 2020-12-08 UT 03:57:16 10.06 1 0.843 35 0.873 72 0.907 97 0.241 95 0.238 16 0.264 22 0.265 48 2020-10-06 UT 01:27:41 9.31 1 0.931 70 0.965 54 0.949 84 0.161 91 0.135 52 0.164 08 0.170 23 2021-11-29 UT 04:02:58 7.35 1 0.786 35 0.830 14 0.848 13 0.273 82 0.238 48 0.272 24 0.258 25 2023-03-06 UT 06:31:57 5.32 0.324 34 0.847 51 0.831 51 1 0.165 79 0.196 04 0.245 11 0.256 33 2023-01-20 UT 03:50:04 4.90 0.524 34 0.883 35 0.907 97 1 0.201 95 0.226 14 0.245 22 0.273 64 Astronomical Techniques and Instruments, 1(2): 128–139, 2024　135 Table 3. Quantitative comparison of the reconstruction results from four solar image reconstruction algorith Table 1 comparison of the reconstruction results from four solar image reconstruction algorithms of test data in NASIR and MFBD-CNRA are close to speckle masking when seeing is good, and even exceed speckle masking in image contrast. NASIR and MFBD-CNRA are close to speckle masking when seeing is good, and even exceed speckle masking in image contrast. NASIR and MFBD-CNRA are close to speckle masking when seeing is good, and even exceed speckle masking in image contrast. as the contours of the solar graining. However, in the NASIR algorithm, there is an error in the reconstruction for the penumbra portion: The penumbra portion has no real contrast. The high-frequency structures of the MFBD- CNRA algorithm are better than the results of the other reconstruction algorithms, retaining a realistic contrast. 3.2. With Seeing of Approximately 3 Arcseconds 136　www.ati.ac.cn illustrates the power spectrum curves of Fig. 9A and F. illustrates the power spectrum curves of Fig. 9A and F. Geometrically, non-rigid alignment eliminates the influ- ence of random image displacement caused by atmo- spheric turbulence. The pixel alignment between the cor- rected image sequences is improved, which is more con- ducive to overcoming strong turbulence. Astronomical Techniques and Instruments, 1(2): 128–139, 2024　137 4. DISCUSSION The reconstruction of observational data by compar- ing MFBD and MFBD-CNRA verify the necessity of non-rigid alignment, which is a nonlinear constraint, to solve the problem of solar image degradation. Meanwhile, The NASIR algorithm image does not contain spuri- ous structures and shows low-frequency information such 0 0 5 10 15 20 5 10 15 20 25 0 0 5 10 15 20 25 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 (″) (″) (″) (″) 5 10 15 20 25 (″) (″) (″) (″) A B C D Fig. 5. Reconstruction results of NVST TiO 2023-03-06 UT 06:31:57 at observation active area 13242, from (A) Speckle masking, (B) MFBD, (C) NASIR, (D) MFBD-CNRA. 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) B 0 0 5 10 15 20 5 10 15 20 25 25 (″) (″) A 0 0 5 10 15 20 25 (″) 5 10 15 20 25 (″) D 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) C Fig. 5. Reconstruction results of NVST TiO 2023-03-06 UT 06:31:57 at observation active area 13242, from (A) Speckle masking, (B) MFBD, (C) NASIR, (D) MFBD-CNRA. 136　www.ati.ac.cn 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 (″) (″) (″) (″) 5 10 15 20 25 (″) (″) (″) (″) A B C D Fig. 6. Reconstruction results of NVST TiO 2023-01-20 UT 03:50:04, observation active area 12297, from (A) Speckle masking, (B) MFBD, (C) NASIR, (D) MFBD-CNRA. 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) B 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) A 0 0 0 0 5 10 15 20 25 (″) 5 10 15 20 25 (″) D 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) C Fig. 6. Reconstruction results of NVST TiO 2023-01-20 UT 03:50:04, observation active area 12297, from (A) (B) MFBD, (C) NASIR, (D) MFBD-CNRA. 4. DISCUSSION tion results of NVST TiO 2023-01-20 UT 03:50:04, observation active area 12297, from (A) Speckle masking, ASIR, (D) MFBD-CNRA. g. 6. Reconstruction results of NVST TiO 2023-01-20 UT 03:50:04, observation active area 12297, from (A) Speckle masking ) MFBD, (C) NASIR, (D) MFBD-CNRA. Speckle masking MFBD-CNRA Intensity profile (Normalized by divide mean) 0 0.7 25 50 75 100 125 150 175 200 0.8 0.9 1.0 1.1 1.2 Pixel Intensity of pixel Fig. 8. Comparison of the intensity profile between the reconstruction results (the red line region in Fig. 5A) of speckle masking and MBFD-CNRA. Speckle masking MFBD-CNRA Intensity profile (Normalized by divide mean) 0 0.7 25 50 75 100 125 150 175 200 0.8 0.9 1.0 1.1 1.2 Pixel Intensity of pixel Log (power spectrum) 1.0 0 0.5 0.25 0.1 0.08 5 10 20 1/Spatial frequency (″) Power spectrum of Speckle masking Power spectrum of MFBD Power spectrum of MFBD-CNRA Power spectrum of NASIR Fig. 7. Power spectrum curves of reconstruction results of NVST TiO 2023-03-06 UT 06:31:57, observation active area 13242. Log (power spectrum) 1.0 0 0.5 0.25 0.1 0.08 5 10 20 1/Spatial frequency (″) Power spectrum of Speckle masking Power spectrum of MFBD Power spectrum of MFBD-CNRA Power spectrum of NASIR Fig. 7. Power spectrum curves of reconstruction results of NVST TiO 2023-03-06 UT 06:31:57, observation active area 13242. Fig. 8. Comparison of the intensity profile between the reconstruction results (the red line region in Fig. 5A) of speckle masking and MBFD-CNRA. comparing the results of NASIR and MFBD-CNRA, we find that the high-resolution images cannot be recon- structed perfectly without optimizing the solving phase by linear constraint either. 4.1. The Role of Non-rigid Alignment To solve the problem of solar image reconstruction, we must consider the effect of the atmosphere on the image. Fig. 9 shows the average frames of the initial speckle frame sequence and the average frames of the sequence from the first through fifth alignments. Fig. 10 From the power spectrum curve, the reconstructed image after correction has a good enhancement effect in the mid and high frequency area. This is helpful for the reconstructed image resolution to be closer to the diffrac- tion limit of the telescope. Astronomical Techniques and Instruments, 1(2): 128–139, 2024　137 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 5 10 15 20 25 0 0 5 10 15 20 25 (″) (″) (″) (″) (″) (″) 5 10 15 20 25 (″) (″) (″) (″) (″) (″) A B C D E F Fig. 9. Average frame images of NVST TiO 2023-01-20 UT 03:50:04, observation active area 12297. (A) The average frame image before alignment. (B) to (F)：The average frame images after 1st to 5th non-rigid alignment. 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) A B 0 0 5 10 15 20 25 5 0 5 0 5 (″) ) C 0 0 5 10 15 20 25 5 10 15 20 25 (″) ″) 0 0 0 0 0 0 0 0 0 0 5 10 15 20 25 5 10 15 20 25 (″) (″) D E 0 0 5 10 15 20 25 (″) 5 10 15 20 25 (″) 0 0 5 10 15 20 25 5 0 5 0 5 (″) ) F 0 0 20 Fig. 9. Average frame images of NVST TiO 2023-01-20 UT 03:50:04, observation active area 12297. (A) The average frame image before alignment. (B) to (F)：The average frame images after 1st to 5th non-rigid alignment. Fig. 9. Average frame images of NVST TiO 2023-01-20 UT 03:50:04, observation active area 12297. (A) The av before alignment. (B) to (F)：The average frame images after 1st to 5th non-rigid alignment. 4.2. 4.1. The Role of Non-rigid Alignment Necessary Linear Constraint Log (power spectrum) 1.0 0 0.5 0.25 0.1 0.08 5 10 20 1/Spatial frequency (″) Power spectrum of the average frame before alignment Power spectrum of the average frame after 5th non-rigid alignment Fig. 10. Comparison of power spectral curves. Log (power spectrum) 1.0 0 0.5 0.25 0.1 0.08 5 10 20 1/Spatial frequency (″) Power spectrum of the average frame before alignment Power spectrum of the average frame after 5th non-rigid alignment on speckle imaging requires a reference image. It should be noted that the number of iterations for the reference image here is roughly five, and it is not the case that more corrections are more effective. The next step of the optimization strategy should be to build a reasonable model and choose the number of frame iterations accord- ing to the value of r0. (2) Time efficiency. The MFBD-CNRA reconstruc- tion time efficiency is slower than NASIR and does not improve significantly over the time efficiency of the speckle masking method. The reason is that it still needs to perform the processes of sub-block splitting, reconstruc- tion, and stitching together the final image. Further work is needed to optimize the algorithm for high-performance parallel computing to improve the reconstruction effi- ciency. Fig. 10. Comparison of power spectral curves. 4.2. Necessary Linear Constraint Fig. 10. Comparison of power spectral curves. 4.2. Necessary Linear Constraint NASIR shows some improvement in the power spec- trum but still lacks in the image phase, because it only merges the mean frame phase and the interferometry speckle mode into a rebuilt image. The disadvantage of this method is that the calculated Sitf is the same for each value of r0 without considering the variability of the actual image. MFBD-CNRA solves the phase close to the true value through the linear constraint method. For differ- ent groups of input images, it is possible to compute the corresponding PSF. Depending on the corrected image and the calculated PSF, the reconstructed image can be solved more satisfactorily. (3) Phase optimization. Phase reconstruction is a diffi- cult problem, and this work currently still reconstructs the phase by employing the same solution method used to solve the Zernike coefficients. Traditional linear optimiza- tion methods are effective but may not be ideal. Utilizing deep learning to search for phase coefficients is also a promising method to consider in future work. 138　www.ati.ac.cn REFERENCES REFERENCES MFBD methods is poor. Combining the advantages of the non-rigid alignment method and phase reconstruction, we propose a new solar image reconstruction method, termed the MFBD-CNRA method, which successfully obtains high-resolution images under strong turbulence. The recon- structed image results from NVST observational data show the robustness and applicability of the MFBD- CNRA algorithm in cases where the r0 parameter is less than 6 cm. Chen, L. H., Liu, Z., Chen, D. 2019. Climatological analysis of the seeing at Fuxian Solar Observatory. Research in Astronomy and Astrophysics, 19(1): 015. [1] y y Liu, Z., Lou, K., Zhang, R. L., et al. 2000. The day-time seeing monitor at Fuxian lake and some primary results. Publication of Yunnan Observatory, 4(4): 95−100. (in Chinese) [2] Lou, K., Liu, Z., Zhang, R. L., et al. 2001. The site testing day of daytime seeing and vapor from Fuxian lake. Progress In Astronomy, 19(2): 147−150. (in Chinese) [3] Non-rigid alignment is a nonlinear method that can overcome atmospheric turbulence's effect. The main effect is pixel-by-pixel alignment of images and correction of aberrations, thereby allowing the target image to retain more high-frequency information. Considering the differ- ences in actual images, linear constraints are essential in the accurate calculation of PSF. Xiang, Y. Y., Liu, Z., Jin, Z.Y., et al. 2016. High resolution solar image reconstruction. Progress In Astronomy, 34(1): 94−110. (in Chinese) [4] ( ) Xiang, Y. Y., Liu, Z., Jin, Z.Y. 2016. High resolution reconstruction of solar prominence images observed by the New Vacuum Solar Telescope. New Astronomy, 49: 8−12. [5] Xiang, Y. Y., Jin, Z. Y. 2011. Simulation and analysis of signal-to-noise ratios for statistics of speckle interferometry. Astronomical Research and Technology, 8(3): 293−297, 305. (in Chinese) [6] For day-to-day solar telescope observations, MFBD- CNRA can effectively process data, allowing continuous data collection in the event of sudden unfavorable changes in weather conditions. The MFBD-CNRA algo- rithm can compensate for algorithm-level defects in data reconstruction in the case of strong atmospheric turbu- lence. Liu, Z., Xu, J., Gu, B. Z.,et al. 2014. New vacuum solar telescope and observations with high resolution. Research in Astronomy and Astrophysics, 14(6): 705−718. [7] Van Noort, M.,Van Der Voort, L. R., Löfdahl, M. G. 2005. Solar image restoration by use of multi-frame blind deconvolution with multiple objects and phase diversity. Solar physics, 228(1/2): 191−215. AUTHOR CONTRIBUTIONS Sizhong Zou and Lei Yang conceived the ideas, Sizhong Zou implemented the study and wrote the paper. Lei Yang and Jun Xu collected the data, Zhenyu Jin and Kaifan Ji improved the algorithm and innovation. All authors read and approved the final manuscript. Wang, Z., Bovik, A. C., Sheikh, H. R., et al. 2004. Image quality assessment: from error visibility to structural similarity. IEEE Transactions on Image Processing, 13(4): 600−612. [15] Löfdahl, M. G. 2002. Multi-frame blind deconvolution with linear equality constraints. In Society of Photo-Optical Instrumentation Engineers (SPIE) Conference Series. DOI:10.1117/12.451791. [16] ACKNOWLEDGMENTS This work is sponsored by the National Natural Sci- ence Foundation of China (NSFC) under the grant num- bers (11773073, 11873027, U2031140, 11833010), Yun- nan Key Laboratory of Solar Physics and Space Science under the number 202205AG070009, Yunnan Provincial Science and Technology Department (202103AD50013, 202105AB160001, 202305AH340002), the GHfund A202302013242 and CAS “Light of West China” Pro- gram 202305AS350029. Löfdahl, M. G., Scharmer, G. B. 1994. Wavefront sensing and image restoration from focused and focused solar images. Astronomy and Astrophysics, Supplement Series, 107(2): 243−264. [11] Liu, H., Jin, Z. Y., Xiang, Y. Y., et al. 2022. High resolution solar image reconstruction based on non-rigid alignment. Research in Astronomy and Astrophysics, 22(9): 095005 [12] Li, M. L., Xiang, Y. Y., Jin, Z. Y., et al. 2018. The analysis of anisoplanatism’s influence in different econstruction sub- block size of NVST. Astronomical Research and Technology, 15(1): 87−94. (in Chinese) [13] Liu, C. Y., Xiang, Y. Y., Jin, Z. Y. 2014. Image processing with frame selection for Hα solar chromosphere images. Astronomical Research and Technology, 11(2): 140−144. (in Chinese) [14] REFERENCES [8] Future work will involve parallelization of the MFBD-CNRA algorithm to further improve reconstruc- tion efficiency. This study can provide an effective refer- ence for data reconstruction pipelines in other ground- based solar telescopes. p y , ( ) Yu, X. G., Liu, Z., Jin, Z. Y., et al. 2010. Design of a phase diversity wavefront sensor. Astronomical Research and Technology, 7(1): 55−59. (in Chinese) [9] Yang L., Liu, Z., Jin, Z. Y., et al. 2009. The effect of uncertainty in defocus distanceon the wave-front reconstruction with the phase diversity algorithm. Astronomical Research and Technology, 6(1): 43−50. (in Chinese) [10] 5. CONCLUSION While achieving successful experiments, MFBD- CNRA deserves further optimization and improvement in the following areas: Strong atmospheric turbulence in winter and spring leads to a significant decrease in the seeing at FLSO, and the reconstruction of solar images using the masking and (1) Number of iterations. Non-rigid alignment based Astronomical Techniques and Instruments, 1(2): 128–139, 2024　139 DECLARATION OF INTERESTS Hang, Y. 2022. Survey of non-blind image restoration. Chinese Optics, 15(5): 954−972. (in Chinese) [17] Zhenyu Jin is an associate editor-in-chief number and Kaifan Ji is an editorial board member for Astronomical Techniques and Instruments. They are not involved in the editorial review or the decision to publish this article. The authors declare no competing interests. Hu, G., Xiang, Y. Y., Liu, Z., et al. 2021. Seeing estimation accuracy study based on a sequence of speckle images. Astronomical Research and Technology, 18(2): 213−225. (in Chinese) [18] Astronomical Techniques and Instruments, 1(2): 128–139, 2024　139
https://openalex.org/W2976325607	https://eprints.gla.ac.uk/243876/1/243876.pdf	English	null	The association between intimate partner violence against women and newborn telomere length	Translational psychiatry	2,019	cc-by	6,596	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/. A R T I C L E O p e n A c c e s s Abstract Abstract Intimate partner violence (IPV) against women negatively impacts infant health. However, its impact on infant’s biology, in particular on telomere length (TL) is unknown. The aim of this study was to examine the association between IPV against women before childbirth and cord blood TL in their newborn. A total of 774 pregnant women in the 20th–24th week of gestation were recruited at a public hospital in Hong Kong. The mothers’ exposure to IPV before childbirth, demographic characteristics, obstetric outcomes, health and mental health were measured at the time of recruitment and 4 weeks after childbirth. Umbilical cord blood was collected by midwives at the time of delivery. The newborn TL was quantiﬁed using quantitative PCR method and expressed in T/S ratio (the ratio of telomere repeat copy numbers to single-copy gene numbers). After adjusting for a number of confounding variables, the mothers’ exposure to any IPV before childbirth (β = −0.08, 95% CI = −0.14, −0.01) was associated with shorter TL. Speciﬁcally, psychological abuse against women before childbirth (β = −0.08, 95% CI = −0.15, −0.02) and sexual abuse against women before childbirth (β = −0.22, 95% CI = −0.43 to −0.01) were signiﬁcantly associated with reduced newborn TL. This study is the ﬁrst to provide evidence of an association between IPV against women before childbirth and TL shortening in their newborns. Through TL- dependent transcription and epigenetic mechanisms, our ﬁnding suggests maternal exposure to IPV may exert a life-long impact on the offspring’s health. Chan et al. Translational Psychiatry (2019) 9:239 https://doi.org/10.1038/s41398-019-0575-6 Chan et al. Translational Psychiatry (2019) 9:239 https://doi.org/10.1038/s41398-019-0575-6 Translational Psychiatry Translational Psychiatry Correspondence: Camilla K.M. Lo (camilla.lo@polyu.edu.hk) or Patrick Ip (patricip@hku.hk) 1Department of Applied Social Sciences, The Hong Kong Polytechnic University, Hong Kong, China 2Institute of Health and Wellbeing, University of Glasgow, Glasgow, United Kingdom Full list of author information is available at the end of the article. Methods Participants before adulthood. Whilst TL shortening occurs natural as part of aging, heritability, and early life environment are the major determinants of TL9. One important advance- ment is the appreciation that TL erosion may result from childhood exposure to adversities, including childhood abuse and neglect10. However, these studies were pre- dominantly conducted in adults, whose recalls of expo- sure to violence years after the events happened could be subject to bias. In addition, the TL obtained in adult subjects is likely also inﬂuenced by other confounding factors, such as lifestyle and engagement in health- compromising behaviors11,12. Participants were pregnant women recruited from the antenatal clinic of Kwong Wah Hospital, a public hospital managed by the Hospital Authority in Hong Kong. Of note, public hospitals in Hong Kong are highly subsidized and provide ~80% of in-patient services in the region. Kwong Wah Hospital has one of the city’s major obste- trics and gynaecology departments, providing services to ~5000 child births annually. During the study period (~3 months in 2017), we recruited 774 pregnant women who met the study’s inclusion criteria: (1) were age 18 years or above, (2) gave their informed consent, and (3) were in the 20th–24th week of gestation at the time of recruitment. At the time of recruitment, the mothers’ report of any history of exposure to IPV, demographic characteristics, health-related quality of life, engagement in risk behaviors, and mental health symptoms such as depression, stress, and anxiety were collected. Umbilical cord blood samples from the newborns were subsequently collected by midwives during delivery. The cord blood samples were used for assessing newborn TL. The mothers were contacted 4 weeks after delivery. The mothers reported again about their history of exposure to IPV at any time in their lives up to childbirth and their obstetric outcomes. The development and the health of children are inter- woven with the well-being of their family. Children’s exposure to violence could be particularly detrimental to their long-term health given that the early development of multiple biological systems is highly sensitive to early life stressors and cumulative effects of these stressors13. A few studies have reported the association between children’s TL shortening and family problems, including family violence and marital conﬂict14,15. However, these studies have been challenged on methodological grounds, such as small sample size and reliance on a cross-sectional design. Thus far, Shalev et al. Introduction sensitive to aging and may mediate some of the long-term adverse health outcomes in abused women3. However, the precise impacts of IPV against women on children’s TL has yet been unequivocally ascertained. Intimate partner violence (IPV) against women has substantial impacts on the women’s physical, mental, and reproductive health1. The negative impacts of IPV against women on infants health include preterm delivery, low birth weight, and a range of social and emotional pro- blems2. Recently, the biological mechanisms related to transgenerational health risk as a result of IPV against women have attracted increasing attention. One line of investigation into the possible impacts of IPV at the cel- lular level has focused on telomere function, in particular through the examination of telomere length (TL). TL is a useful marker for telomere integrity, which is known to be Telomeres are nucleoprotein complexes comprising repetitive DNA sequences -TTAGGG- and are located at the ends of the eukaryotic chromosomes, which serve to protect genomic integrity from damages caused by repli- cation ﬂaws4. Shorter TL has been linked to a higher risk of developing chronic diseases, such as type 2 diabetes, cancers, and cardiovascular diseases in adults5,6. Growing studies have pointed to the particular importance of TL dynamics in early stage of life than in adult life. A study has shown that the ranking of TL of most of the study participants did not change across their adult life7. Another study has shown a wide variation of TL across newborns but not within newborns8. These ﬁndings suggest that TL attrition is faster during early stage of life than in adulthood and that TL is largely determined Page 2 of 8 Chan et al. Translational Psychiatry (2019) 9:239 Page 2 of 8 Chan et al. Translational Psychiatry (2019) 9:239 Methods Participants have provided the only longitudinal study involving 236 children. Those who experienced multiple types of violence, including domestic violence, child physical abuse, and bullying victimization, had sig- niﬁcantly shorter TL in buccal DNA sampled from the age of 5–10 compared with children exposed to less or no violence16. However, still critically missing from the existing literature is an assessment of children’s TL at birth. Covariates M l Maternal anxiety and stress symptoms These were assessed using the anxiety and stress subscales of the Chinese version of the Depression Anxiety Stress Scale (DASS)25. The DASS has been widely used in research and clinical settings. The anxiety and stress subscales each consist of seven items that assess the severity (identiﬁed as normal, mild, moderate, and severe) of respective core symptoms. The DASS has been validated in a Chinese population26. The symptom scores were adjusted in the regression models as continuous variable. Data analysis Descriptive statistics of the participants’ characteristics and exposure to IPV before childbirth were ﬁrst computed. To examine the association between IPV against the women before childbirth and TL in their newborns, four regression models were generated with increasing number of covariates to examine the inﬂuence of adjustment covariates on the association. This approach is to illustrate the robustness of the association regardless of adjustment schemes, rather than for variable selection as in traditional stepwise approach. The statistical signiﬁ- cance of the adjustment variables is not of primary interest in this study and serve as a reference only. The ﬁrst model was of crude associations between different types of IPV against the women before childbirth and their newborns’ TL. In the second model, the associations were examined with adjustments for demographic variables, obstetric problems, and newborn characteristics. The third model additionally included the mothers’ health-related quality of life as the adjusted variables. The fourth model additionally included the mothers’ mental health variables as the adjusted variables. These adjustment variables were selected due to previous reports on their simultaneous association with the exposure (IPV) and outcome (TL). Due to a highly positive skewness of the distribution (2.34), a logarithmic transformation of the TL was performed before the analysis. Data analyses were performed using R Statistical Software. All tests were two-tailed, and P-values below 0.05 were regarded as statistically signiﬁcant. Multicollinearity was examined using variance inﬂation factor (VIF) where variables with VIF >10 were removed as a sensitivity analysis. Maternal depressive symptoms Maternal depressive symptoms were assessed using the 10-item Edinburgh Postnatal Depression Scale (EPDS)27. The EPDS assesses the presence of maternal depressive symptoms during the perinatal period. The mothers were asked to report the presence of depressive symptoms. The Chinese version of EPDS has been validated in a previous study28. The symptom scores were adjusted in the regression models as a continuous variable. Predictor Maternal risk behaviors Maternal risk behaviors, including gambling, cigarette smoking, alcohol drinking, and drug use were assessed using four items. The mothers were asked to report how often they engaged in each of those risk behaviors, by rating them as 1 = never; 2 = rarely; 3 = sometimes; and 4 = always. Because these risk behaviors were not common in this sample, the variables were grouped as binary variables (never vs. ever engaged in the behavior) in the analysis. IPV against women by current partners before child- birth The mothers reported on their exposure to violence by their current partners in the form of psychological, physical, and sexual abuse at any time in their lives up to childbirth, using the ﬁve-item Abuse Assessment Screen (AAS)23. The AAS has been commonly used in different healthcare settings, and the validated Chinese AAS demonstrated satisfactory measurement accuracy for identifying IPV against women when it was used to evaluate Chinese women in a previous study24. The mothers who reported any history of exposure to IPV at any time in their lives up to the time of childbirth were categorized into “abused group” and those who did not have any history of exposure to IPV were categorized into “non-abused group.” Demographic characteristics and obstetric factors Demographic information about the mothers, such as maternal age, educational level, marital status, family income, self-reported obstetric problems (hyperemesis gravidarum, hypertension, diabetes mellitus, multiple pregnancy, and vaginal bleeding), gestation age, and birth weight were collected and considered in our analyses. Maternal age, family income, gestation age, and birth weight were adjusted as continuous variables, while education level, marital status, and obstetric problems were adjusted as categorical variables. Outcome measure Newborn telomere lengths Samples of the newborns’ DNA were extracted from the umbilical cord blood collected by midwives during each baby’s delivery. Genomic DNA samples were isolated and extracted from the collected samples using the QIAamp DNA Mini kit (Qiagen), according to the manufacturer’s instructions. Isolated DNA samples were eluted to the buffer solution (10 mM Tris-HCl and 1 mM ethylenediamine-tetraacetic acid, pH 8.0) for quality checking and quantiﬁcation by spectrophotometry (NanoDrop 2000c, Thermo Scienti- ﬁc). Upon conﬁrming that the DNA quality and quantity were in the acceptable range for the determination of telomere length, each sample was processed following the procedures described in previous literature22. Each sample was handled in triplicate for the telomere length assay by quantitative polymerase chain reaction (qPCR) using a 7900HT Thermocycler (Applied Biosystems). After the assay, the telomere length was presented by a relative ratio of the telomere repeat copy number (T) to single-copy gene 36B4 copy number (S) using the formula of T/S = 2(−ΔCt), where ΔCt is the mean difference between the threshold cycle (Ct) value of the 36B4 gene and telomere repeats obtained from the qPCR performed. Recently, Enlow et al.17 were the ﬁrst to report that maternal exposure to childhood sexual abuse before age 11 was related to TL shortening already at birth, parti- cularly among boys. Their study suggested that variations of newborn TL may be associated with childhood abuse of the mother that may have exerted an effect in the intrauterine environment. In addition, observations link- ing newborn TL and other maternal factors, such as maternal demographic characteristics (e.g., maternal age and educational level18,19) and health and mental health factors (e.g., depression and stress) have been found20,21. Despite the indication of the link between newborn TL and maternal risk factors, maternal childhood abuse, and family problems, no study has speciﬁcally investigated the association between IPV against women and their off- spring’s TL at birth. Hence, this study aimed to provide a more accurate estimation of the association between IPV against women and newborn TL by using a larger sample size, assessing child TL at birth using cord blood samples, and taking into consideration potential confounding fac- tors in the analysis. Page 3 of 8 Chan et al. Translational Psychiatry (2019) 9:239 Covariates M l Maternal health-related quality of life The Chinese version of the Short-Form Health Survey, version 2 (SF- 12)29 was used to assess the mothers’ maternal health- related quality of life (HRQoL). The SF-12 consists of 12 items that measure eight health domains, including physical functioning, role limitations due to physical health, bodily pain, general health, vitality, social functioning, role limitations due to emotional health, and mental health. The SF-12 generates two composite scores, the physical component summary (PCS) and the mental component summary (MCS), which were adjusted in the regression models as continuous variables. Page 4 of 8 Chan et al. Translational Psychiatry (2019) 9:239 Ethical approval The research protocol was approved by the Institutional Review Board of the Hospital Authority Kowloon West Cluster Research Ethics Committee (Reference number: KW/FR-16-042(97-01)(1)). Ethical approval The research protocol was approved by the Institutional Review Board of the Hospital Authority Kowloon West Cluster Research Ethics Committee (Reference number: KW/FR-16-042(97-01)(1)). (92.1%) were married, and 7.9% were single or divorced. More than half (63.3%) of them had obtained a post- secondary education or above. The mean monthly family income was equivalent to USD 4827 (SD = 3188). The mothers in the abused group had higher levels of depressive, anxiety, and stress symptoms, compared to the mothers in the non-abused group (p-values < 0.0001). The mothers in the non-abused group were more likely than the mothers in the abused group to have hypertension Numbers presented are n (%) unless otherwise speciﬁed. Tests for difference between non-abused and any abuse groups were chi-square test/Fisher exact test for independence (categorical variables) and independent t-test (continuous variables) Numbers presented are n (%) unless otherwise speciﬁed. Tests for difference between non-abused and any abuse groups were chi-sq independence (categorical variables) and independent t-test (continuous variables) Discussion Main ﬁndings Despite a wealth of research showing that early life adversities such as violence can negatively affect health and morbidity13, little is known about the potential bio- logical impacts of such adversities at the cellular level. This study provided the ﬁrst evidence that IPV against women by current partners before childbirth was asso- ciated with TL shortening in their children that could already be detected at birth. This ﬁnding extends the concept of biological embedding31 by showing that the mother’s exposure to violence is important in under- standing the newborn’s telomere biology. (Fisher’s exact test = 4.88, p = 0.02) and ever drunk alcohol (Fisher’s exact test = 7.65, p = 0.006). In regard to the infants, the mean age of gestation was 38.73 weeks (SD = 1.43). The mean birth weight was 3074.28 grams (SD = 553.08). The mean telomere length (T/S) was 15.12 (SD = 6.34) and it was signiﬁcantly shorter in the abused group (Mean = 14.26, SD = 5.54) than the non- abused group (Mean = 15.39, SD = 6.54), t(772) = 4.44, p < 0.04. The mean T/S ratio measured in this study is similar to that reported by a previous study in children (with a median T/S ratio of 10.87 ± 6.4)30. Table 2 shows the percentages of the women who were exposed to different types of IPV by their current partners. Nearly one-quarter (23.5%) of the mothers in this study reported exposure to IPV by their current partners before child- birth. Psychological abuse was the most common type of abuse (23.3%), followed by physical abuse (3.5%) and sexual abuse (1.8%). Among the women in this study, 18.9% were exposed to one type of IPV, 4.3% were exposed to two types of IPV, and 0.4% were exposed to three types of IPV. In this study, approximately a quarter of women (23.5%) were exposed to IPV by their current partners before childbirth, with psychological abuse being the most common type of abuse (23.3%), followed by phy- sical abuse (3.5%) and sexual abuse (1.8%). The pattern is comparable to previous ﬁndings on IPV in Chinese couples32. Results Translational Psychiatry (2019) 9:239 Page 5 of 8 Table 2 Participants’ exposure to IPV before childbirth Type of IPV before childbirth All (N = 774) n (%) Psychological abuse 180 (23.3) Physical abuse 27 (3.5) Sexual abuse 14 (1.8) Any abuse 182 (23.5) Number of types of abuse 0 592 (76.5) 1 146 (18.9) 2 33 (4.3) 3 3 (0.4) Table 2 Participants’ exposure to IPV before childbirth Table 2 Participants’ exposure to IPV before childbirth characteristics, and maternal health and mental health factors, any IPV by current partners before childbirth (β = −0.08, 95% CI = −0.14, −0.01, p = 0.02) was associated with the logarithm of the TL in the newborns. Speciﬁcally, psychological abuse (β = −0.08, 95% CI = −0.15, −0.02, p = 0.01) and sexual abuse (β = −0.22, 95% CI = −0.43 to −0.01, p = 0.04) before childbirth were signiﬁcantly associated with the newborns’ TL, which was shorter by 21.4% and 61.5% of standard deviation of the log- transformed TL, respectively. Mental health-related vari- ables showed strong inter-correlation (VIFs ranged 2.25–2.75) but there was no evidence for severe multi- collinearity (all VIFs ≤2.75). Results As Table 1 shows, the mean age of the mothers in this study was 31.05 years (SD = 4.5). The majority of them Table 1 Characteristics of study participants All (N = 774) Non-abused (N = 592) Any abuse (N = 182) Chi-squared-/t-statistic (df) P-value Mean (SD) maternal age at recruitment 31.05 (4.50) 31.24 (4.40) 30.44 (4.78) 4.40 (772) 0.04 Marital status 2.76 (1) 0.10 Single/divorced 60 (7.9) 40 (6.9) 20 (11.1) Married 698 (92.1) 538 (93.1) 160 (88.9) Maternal educational level 8.78 (4) 0.07 Junior secondary school or below 61 (7.9) 44 (7.4) 17 (9.3) Senior secondary school 223 (28.8) 176 (29.7) 47 (25.8) Post-secondary 91 (11.8) 77 (13.0) 14 (7.7) Bachelor’s degree 151 (19.5) 105 (17.7) 46 (25.3) Postgraduate degree 248 (32.0) 190 (32.1) 58 (31.9) Mean (SD) family monthly income (USD) 4827.35 (3188.22) 4792.08 (3136.56) 4939.51 (3353.99) 0.29 (738) 0.59 Mean (SD) gestation age (weeks) 38.73 (1.43) 38.71 (1.39) 38.79 (1.54) 0.30 (457) 0.58 Mean (SD) birth weight (g) 3074.28 (553.08) 3074.07 (535.60) 3074.88 (601.69) 0.00 (454) 0.99 Mean (SD) telomere length (T/S) 15.12 (6.34) 15.39 (6.54) 14.26 (5.54) 4.44 (772) 0.04 Mean (SD) SF-12 PCS 46.19 (6.82) 46.30 (6.87) 45.81 (6.64) 0.72 (772) 0.40 Mean (SD) SF-12 MCS 48.42 (8.67) 49.39 (8.27) 45.24 (9.19) 33.26 (772) <0.0001 Obstetric problems Hyperemesis gravidarum 17 (2.2) 15 (2.5) 2 (1.1) 0.75 (1) 0.39 Hypertension 19 (2.5) 10 (1.7) 9 (4.9) 4.88 (1) 0.02 Diabetes mellitus 56 (7.2) 40 (6.8) 16 (8.8) 0.58 (1) 0.45 Multiple pregnancy 5 (0.6) 4 (0.7) 1 (0.5) 0.00 (1) 1.00 Vaginal bleeding 27 (3.5) 21 (3.5) 6 (3.3) 0.00 (1) 1.00 Ever gambled 255 (32.9) 190 (32.1) 65 (35.7) 0.67 (1) 0.41 Ever smoked 90 (11.6) 66 (11.1) 24 (13.2) 0.38 (1) 0.54 Ever drunk alcohol 232 (30.0) 162 (27.4) 70 (38.5) 7.65 (1) 0.006 Ever use drugs 2 (0.3) 2 (0.3) 0 (0.0) – – Mean (SD) depressive symptom score 7.15 (4.63) 6.68 (4.44) 8.67 (4.92) 26.44 (772) <0.0001 Mean (SD) anxiety symptom score 5.63 (5.15) 5.21 (4.89) 7.00 (5.71) 17.25 (772) <0.0001 Mean (SD) stress symptom score 7.43 (6.77) 6.64 (6.44) 10.01 (7.18) 36.16 (772) <0.0001 Numbers presented are n (%) unless otherwise speciﬁed. Tests for difference between non-abused and any abuse groups were chi-square test/Fisher exact test for independence (categorical variables) and independent t-test (continuous variables) Table 1 Characteristics of study participants Chan et al. Discussion Main ﬁndings Translational Psychiatry (2019) 9:239 Page 6 of 8 Table 3 Associations between IPV against women by their current partners before childbirth and their newborns’ TL Model 1: Crude associations Model 2: IPV, Controlled for maternal demographics, obstetric problems, and newborn characteristics Model 3: IPV, Controlled for demographics, health variables Model 4: IPV, Controlled for demographics, health variables, and mental health variables β (95% CI) P β (95% CI) P β (95% CI) P β (95% CI) P IPV before childbirth Psychological abuse −0.08 (−0.14, −0.02) 0.01 −0.08 (−0.14, −0.02) 0.01 −0.09 (−0.15, −0.02) 0.009 −0.08 (−0.15, −0.02) 0.01 Physical abuse 0.00 (−0.14, 0.14) 0.95 −0.06 (−0.20, 0.09) 0.45 −0.06 (−0.21, 0.09) 0.40 −0.06 (−0.21, 0.09) 0.42 Sexual abuse −0.19 (−0.39, 0.00) 0.048 −0.22 (−0.42, −0.02) 0.03 −0.24 (−0.44, −0.03) 0.02 −0.22 (−0.43, −0.01) 0.04 Any abuse −0.08 (−0.14, −0.02) 0.01 −0.08 (−0.14, −0.01) 0.02 −0.08 (−0.15, −0.02) 0.01 −0.08 (−0.14, −0.01) 0.02 Covariates Gestation age 0.00 (0.00, 0.00) 0.27 Birth weight −0.01 (−0.03, 0.02) 0.69 Hyperemesis gravidarum −0.19 (−0.38, −0.01) 0.04 Hypertension −0.01 (−0.19, 0.17) 0.9 Diabetes mellitus 0.02 (−0.09, 0.12) 0.78 Multiple pregnancy 0.66 (0.29, 1.04) 0.0006 Vaginal bleeding 0.05 (−0.10, 0.19) 0.51 Maternal age 0.01 (0.00, 0.01) 0.11 Married −0.06 (−0.16, 0.05) 0.28 Family income −0.02 (−0.05, 0.01) 0.16 Education level Junior secondary −0.03 (−0.17, 0.10) 0.62 Senior secondary −0.02 (−0.13, 0.09) 0.73 Post-secondary −0.08 (−0.20, 0.03) 0.16 Bachelor’s degree 0.00 (−0.11, 0.11) 0.99 Postgraduate degree 0 (Reference) SF-12 PCS −0.02 (−0.05, 0.01) 0.21 SF-12 MCS 0.00 (−0.03, 0.03) 0.95 Ever gamble −0.03 (−0.09, 0.03) 0.38 Ever smoke 0.04 (−0.05, 0.13) 0.41 Ever drink alcohol 0.00 (−0.07, 0.06) 0.91 Ever use drugs 0.00 (−0.51, 0.52) 0.99 Depressive symptoms 0.01 (−0.03, 0.05) 0.54 Anxiety symptoms −0.01 (−0.05, 0.03) 0.61 Stress symptoms 0 03 ( 0 08 0 01) 0 13 Table 3 Associations between IPV against women by their current partners before childbirth and their newborns’ TL Table 3 Associations between IPV against women by their current partners before childbirth and their newborns’ TL IPV against women by their current partners before childbirth and their newborns’ TL The association between IPV against women and TL in their newborns in this study raises the question of what possible biological mechanisms could explain such an association. The developing fetus is particularly sensitive to intrauterine inﬂuences35. Discussion Main ﬁndings After controlling for a number of con- founding factors, including maternal age, educational level, family income, obstetric problems, obstetric outcomes, health-related quality of life, engagement in risky behaviors (e.g., gambling, smoking, alcohol drinking, and substance use), depression, anxiety, and stress symptoms, IPV against women by current part- ners before childbirth was a signiﬁcant predictor of the TL of the women’s newborns. Speciﬁcally, psychologi- cal abuse and sexual abuse perpetrated by a partner on a woman before childbirth were signiﬁcantly associated with newborn TL shortening. However, this study did not ﬁnd a signiﬁcant association between physical abuse to the mothers and their newborns’ TL, suggesting psychological and sexual abuse may be more important than physical abuse in explaining newborn TL short- ening. This explanation is consistent with previous studies that found robust and unique effects of psy- chological IPV on mental health, after controlling for the effect of physical IPV33, and a stronger effect of sexual IPV than physical IPV on depression34. y Table 3 shows the results of regression analyses on the associations between IPV against women by their current partners before childbirth and their newborns’ TL. The ﬁrst regression analysis model showed the crude asso- ciations between different types of IPV against women by their current partners before childbirth and newborn TL. Any abuse (β = −0.08, 95% CI = −0.14 to −0.02, p = 0.01), psychological abuse (β = −0.08, 95% CI = −0.14 to −0.02, p = 0.01) and sexual abuse (β = −0.19, 95% CI = −0.39 to 0.00, p = 0.048) of the mothers were signiﬁcantly associated with the logarithm of their newborns’ TL. Physical abuse was not signiﬁcantly associated with newborn TL. In the ﬁnal model, after adjusting for a number of confounding variables, such as gestation age, birth weight, maternal age, educational level, family income, marital status, obstetrics problems, newborn Chan et al. Strengths g One of the methodological strengths of this study is the use of a relatively large sample (N = 774) of mother-infant dyads, particularly for a study of cord blood TL. In addition, the use of cord blood TL permitted us to assess the impact of pre-birth factors on TL by minimizing the postnatal environmental confounding factors. As mater- nal factors may also inﬂuence newborn TL, this study statistically adjusted for a number of confounding vari- ables related to maternal demographic characteristics, newborn characteristics, obstetric problems, maternal health and mental health. Discussion Main ﬁndings One of the possible mechanisms to explain the ﬁndings of this study is the maternal-placental-fetal neuroendocrine processes36. Exposing mothers to a violent experience heightens their stress levels, and that added stress may in turn transmit from the mothers to the fetus through the release of maternal and placental hormone including cortisol and placental corticotropin-releasing hormone that may induce changes in placental physiology, which may in turn inﬂuence TL of the offspring. However, the contribution of maternal stress as a potential mediator of the reported associations here warrant further investigation. Limitations The study sample consisted solely of pregnant women recruited at a single antenatal clinic of a public hospital in Page 7 of 8 Chan et al. Translational Psychiatry (2019) 9:239 Hong Kong. This sampling method limited the external generalizability of the ﬁndings to the rest of the popula- tion. The use of self-report measurements of IPV against women was subject to several limitations, such as parti- cipants’ social desirability in responding, and their recall bias, which might result in underestimation of the vio- lence. Also, this study did not examine the father’s char- acteristics and lacked data on infant sex, which may have an inﬂuence on the newborn TL. Another limitation is that this study only assessed TL in venous cord blood and was unable to adjust for cell types in the analyses. Although TL in different tissues such as white blood cell, foreskin and umbilical artery within the newborn are highly correlated8, further study to include TL in different cell types is needed to validate whether cell type is important in understanding the impact of IPV against women on newborn TL. effects of IPV on child health, as positive parenting has been found to potentially decrease the rate of postnatal TL shortening in children42. Acknowledgements g We would like to thank the midwives of the Department of Obstetrics & Gynaecology of Kwong Wah Hospital, Hong Kong for their contribution to this study. This study was funded by Health and Medical Research Fund, the Government of the Hong Kong Special Administrative Region (Project Number: 15161101). Conﬂict of interest The authors declare that they have no conﬂict of interest. The authors declare that they have no conﬂict of interest. Author details 1 f 1Department of Applied Social Sciences, The Hong Kong Polytechnic University, Hong Kong, China. 2Institute of Health and Wellbeing, University of Glasgow, Glasgow, United Kingdom. 3Department of Obstetrics & Gynaecology, Kwong Wah Hospital, Hong Kong, China. 4Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hong Kong, China. 5Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China Human TL is largely determined during the ﬁrst two decades of life and to a large degree at birth7. This study advanced our knowledge that IPV against women is a relevant prenatal predictor of newborn TL shortening. Although existing evidence suggests that TL is a biological marker for various chronic diseases in adults5,6, the link between TL shortening and pediatric health has been less consisent37,38. Also, the causal roles of TL in diseases have yet to be established. Longer-term longitudinal investi- gations to track children’s TL from birth through early childhood, using multiple measurement points and including a comprehensive family proﬁle of exposure to violence, would shed more light on the complexity of exposure to violence and any subsequent changes in the TL of children during their developmental years. Author contributions K.L.C. conceptualized and designed the study, collected the data, interpreted the data, critically reviewed the paper, and approved the ﬁnal paper as submitted. C.K.L. interpreted the data, drafted the paper, and approved the ﬁnal paper as submitted. F.K.H. analyzed and interpreted the data, and approved the ﬁnal paper as submitted. WCL assisted in study conceptualization, interpreted the data, critically reviewed the paper, and approved the ﬁnal paper as submitted. B.K.Y. interpreted the data, critically reviewed and revised the paper, and approved the ﬁnal paper as submitted. P. I. assisted in study conceptualization, critically reviewed the paper, and approved the ﬁnal paper as submitted. In addition, a previous study showed that IPV against pregnant women is associated with subsequent risk of child maltreatment39 and such intergenerational abuse will further jeopardize children’s health and well-being. Moreover, consistent evidence has shown a dose-response relationship between exposure to IPV and various out- comes in victims and their children40,41. Prolonged and severe violence may moderate intergenerational trans- mission of abuse and the effect of IPV against women on newborn TL. Hence, health professionals should routinely screen for IPV against women to safeguard the overall well-being of children and women. In particular, preg- nancy is a period that offers an opportunity for health professionals to screen for pregnant women’s exposure to IPV. Although IPV in form of psychological abuse does not leave visible wounds on the victims, its impact on abused women’s newborns is signiﬁcant. More attention should be given to this form of IPV. Providing psycho- logical services and parenting support for mothers exposed to IPV may be helpful in buffering the long-term Received: 16 May 2019 Revised: 4 July 2019 Accepted: 30 July 2019 Received: 16 May 2019 Revised: 4 July 2019 Accepted: 30 July 2019 Received: 16 May 2019 Revised: 4 July 2019 Accepted: 30 July 2019 Conclusions This study demonstrates an association between IPV against women by their current partners before childbirth and their newborns’ biology, in the form of TL shortening, even after controlling for a number of confounding factors. This ﬁnding extends the concept of biological embedding by showing that the mother’s exposure to violence is a relevant predictor of newborn’s telomere biology. 1. Campbell, J. C. Health consequences of intimate partner violence. Lancet 359, 1331–1336 (2002). 1. Campbell, J. C. Health consequences of intimate partner violence. Lancet 359, 1331–1336 (2002). 2. Hill, A., Pallitto, C., McCleary-Sills, J. & Garcia-Moreno, C. A systematic review and meta-analysis of intimate partner violence during pregnancy and selected birth outcomes. Int J. Gynaecol. Obstet. 133, 269–276 (2016). 3. Humphreys, J. et al. Telomere shortening in formerly abused and never abused women. Biol. Res Nurs. 14, 115–123 (2012). 4. Cawthon, R. M. Telomere measurement by quantitative PCR. Nucleic Acids Res. 30, e47 (2002). 5. Zhu, H., Belcher, M. & van der Harst, P. Healthy aging and disease: role for telomere biology? Clin. Sci. (Lond.). 120, 427–440 (2011). 5. Zhu, H., Belcher, M. & van der Harst, P. Healthy aging and disease: role for telomere biology? Clin. Sci. (Lond.). 120, 427–440 (2011). 4. Cawthon, R. M. Telomere measurement by quantitative PCR. Nucleic Acids Res. 30, e47 (2002). 2. Hill, A., Pallitto, C., McCleary-Sills, J. & Garcia-Moreno, C. A systematic review and meta-analysis of intimate partner violence during pregnancy and selected birth outcomes. Int J. Gynaecol. Obstet. 133, 269–276 (2016). y 3. Humphreys, J. et al. Telomere shortening in formerly abused and never abused women. Biol. Res Nurs. 14, 115–123 (2012). Publisher’s note S i N Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. References Telomere length and early severe social deprivation: linking early adversity and cellular aging. Mol. Psychiatry 17, 719–727 (2012). y y g g y y 31. Hertzman, C. Putting the concept of biological embedding in historical per- spective. PNAS 109, 17160–17167 (2012). 13. Shonkoff, J. P., Boyce, W. & McEwen, B. S. Neuroscience, molecular biology, and the childhood roots of health disparities: building a new framework for health promotion and disease prevention. JAMA 301, 2252–2259 (2009). 32. Tiwari, A. et al. The impact of psychological abuse by an intimate partner on the mental health of pregnant women. BJOG 115, 377–384 (2008). 14. Drury, S. S. et al. The association of telomere length with family violence and disruption. Pediatrics 134, e128–e137 (2014). 14. Drury, S. S. et al. The association of telomere length with family violence and disruption. Pediatrics 134, e128–e137 (2014). 15. Robles, T. F. et al. Emotions and family interactions in childhood: associations with leukocyte telomere length. Psychoneuroendocrinology 63, 343–350 (2016). 33. Mechanic, M. B., Weaver, T. L. & Resick, P. A. Mental health consequences of intimate partner abuse: a multidimensional assessment of four different forms of abuse. Violence Women 14, 634–654 (2008). 15. Robles, T. F. et al. Emotions and family interactions in childhood: associations with leukocyte telomere length. Psychoneuroendocrinology 63, 343–350 (2016). 16. Shalev, I. et al. Exposure to violence during childhood is associated with telomere erosion from 5 to 10 years of age: a longitudinal study. Mol. Psy- chiatry 18, 576 (2013). 34. Bonomi, A. E., Anderson, M. L., Rivara, F. P. & Thompson, R. S. Health outcomes in women with physical and sexual intimate partner violence exposure. J. Women’s. Health 16, 987–997 (2007). 17. Enlow, M. B. et al. Sex differences in effects of maternal risk and protective factors in childhood and pregnancy on newborn telomere length. Psycho- neuroendocrinology 95, 74–85 (2018). 35. Entringer, S. et al. Fetal programming of body composition, obesity, and metabolic function: the role of intrauterine stress and stress biology. J. Nutr. Metab. 2012, 632548 (2012). 18. Wojcicki, J. M. et al. Early exclusive breastfeeding is associated with longer telomeres in Latino preschool children. Am. J. Clin. Nutr. 104, 397–405 (2016). 36. Wadhwa, P. D. Psychoneuroendocrine processes in human pregnancy inﬂu- ence fetal development and health. Psychoneuroendocrinology 30, 724–743 (2005). 19. Broer, L. et al. References 1. Campbell, J. C. Health consequences of intimate partner violence. Lancet 359, 1331–1336 (2002). 2. Hill, A., Pallitto, C., McCleary-Sills, J. & Garcia-Moreno, C. A systematic review and meta-analysis of intimate partner violence during pregnancy and selected birth outcomes. Int J. Gynaecol. Obstet. 133, 269–276 (2016). 5. Zhu, H., Belcher, M. & van der Harst, P. Healthy aging and disease: role for telomere biology? Clin. Sci. (Lond.). 120, 427–440 (2011). Chan et al. Translational Psychiatry (2019) 9:239 Page 8 of 8 Page 8 of 8 6. D’Mello, M. J. et al. Association between shortened leukocyte telomere length and cardiometabolic outcomes: systematic review and meta-analysis. Circ. Genom. Precis Med. 8, 82–90 (2015). 25. Lovibond, S. & Lovibond, P. Manual for the Depression Anxiety Stress Scales. (Psychology Foundation, Sydney, 1995). 26. Chan, R. C. K. et al. Extending the utility of the depression anxiety stress scale by examining its psychometric properties in Chinese settings. Psychiatry Res. 200, 879–883 (2012). 7. Benetos, A. et al. Tracking and ﬁxed ranking of leukocyte telomere length across the adult life course. Aging Cell. 12, 615–621 (2013). 7. Benetos, A. et al. Tracking and ﬁxed ranking of leukocyte te across the adult life course. Aging Cell. 12, 615–621 27. Cox, J. L., Holden, J. M. & Sagovsky, R. Detection of postnatal depression: development of the 10-item Edinburgh Postnatal Depression Scale. Br. J. Psychiatry 150, 782–786 (1987). 8. Okuda, K. et al. Telomere length in the newborn. Pediatr. Res. 52, 377–381 (2002). 9. Hjelmborg, J. B. et al. The heritability of leucocyte telomere length dynamics. J. Med Genet. 52, 297–302 (2015). 28. Lee D. T. et al. Detecting postnatal depression in Chinese women: validation of the Chinese version of the Edinburgh Postnatal Depression Scale. Br. J. Psy- chiatry 172, 433–437 (1998) 10. Ridout, K. et al. Early life adversity and telomere length: a meta-analysis. Mol. Psychiatry 23, 858 (2018). 11. Mundstock, E. et al. Effects of physical activity in telomere length: systematic review and meta-analysis. Ageing Res Rev. 22, 72–80 (2015). 29. Lam, C. L., Eileen, Y. & Gandek, B. Is the standard SF-12 health survey valid and equivalent for a Chinese population? Qual. Life Res. 14, 539–547 (2005). 12. Astuti, Y., Wardhana, A., Watkins, J. & Wulaningsih, W. Cigarette smoking and telomere length: A systematic review of 84 studies and meta-analysis. Environ. Res. 158, 480–489 (2017). 30. Drury, S. S. et al. Chan et al. Translational Psychiatry (2019) 9:239 References Meta-analysis of telomere length in 19 713 subjects reveals high heritability, stronger maternal inheritance and a paternal age effect. Eur. J. Hum. Genet. 21, 1163 (2013). 37. Suh, D. I. et al. Leukocyte telomere length reﬂects prenatal stress exposure, but does not predict atopic dermatitis development at 1 year. Allergy Asthma Immun. 11, 357–366 (2019). 20. Send, T. S. et al. Telomere length in newborns is related to maternal stress during pregnancy. Neuropsychopharmacology 42, 2407 (2017). Send, T. S. et al. Telomere length in newborns is related to materna 38. Chen, X. et al. Associations between early life parent-child separation and shortened telomere length and psychopathological outcomes during ado- lescence. Psychoneuroendocrinology 103, 195–202 (2019). 21. Wojcicki, J. et al. Telomere length is associated with oppositional deﬁant behavior and maternal clinical depression in Latino preschool children. Transl. Psychiatry 5, e581 (2015). 39. Chan, K. L. et al. Violence against pregnant women can increase the risk of child abuse: A longitudinal study. Child Abus. Negl. 36, 275–284 (2012). 22. Ip, P. et al. Prenatal tobacco exposure shortens telomere length in children. Nicotine Tob. Res. 19, 111–118 (2016). 40. Basile, K. C., Arias, I., Desai, S. & Thompson, M. P. The differential association of intimate partner physical, sexual, psychological, and stalking violence and posttraumatic stress symptoms in a nationally representative sample of women. J. Trauma Stress 17, 413–421 (2004). 23. Soeken K. L., McFarlane J., Parker B., Lominack M. C. The abuse assessment screen: a clinical instrument to measure frequency, severity, and perpetrator of abused against women. in Empowering survivors of abuse: Health care for battered women and their children (ed Campbell J.). (Sage Publications, Thousand Oaks, 1998) 41. Wood, S. L. & Sommers, M. S. Consequences of intimate partner violence on child witnesses: A systematic review of the literature. J. child Adolesc. Psychiatr. Nurs. 24, 223–236 (2011). 42. Asok, A., Bernard, K., Roth, T., Rosen, J. & Dozier, M. Parental responsiveness moderates the association between early-life stress and reduced telomere length. Dev. Psychopathol. 25, 577–585 (2013). 24. Tiwari, A. et al. Identifying intimate partner violence: comparing the Chinese abuse assessment screen with the Chinese revised conﬂict tactics scales. BJOG 114, 1065–1071 (2007).
https://openalex.org/W2913251300	https://www.iiste.org/Journals/index.php/JAAS/article/download/45988/47484	English	null	Physical Quality Characteristics of Potato (Solanum tuberosum L.) Tubers as Influenced by Cultivar and Plant Spacing in Eastern Ethiopia	International Journal of African and Asian Studies	2,019	cc-by	4,245	INTRODUCTION Potato (Solanum tuberosum L.) is a crop of major economic importance worldwide. On a global scale, potato is the fourth most cultivated food crop after wheat, rice, and maize (FAOSTAT, 2012). The relatively high carbohydrate and low fat content of potato makes it an excellent energy source for human consumption (Dean, 1994). Physical characteristics of agricultural products are the most important parameter in the design of grading, handling, processing and packaging systems. Among these physical characteristics, mass, volume, projected area, and centre of gravity are the most important ones in the handling systems (Peleg, 1985). Other important parameters are width, length, and thickness (Peleg, 1985). Knowledge of length, width, volume, surface area and centre location of mass may be applied in the designing of sorting machinery, in predicting surface needed when applying chemicals, shape factor (sphericity), and yield in the peeling operation (surface area) (Wright et al., 1986). Other characteristics worth of concentration are width, length, and thickness (Mohsenin, 1970; Peleg, 1985). Moreover, the appearance of fresh agricultural products is a primary criterion in making purchasing decisions (Kays, 1991). ( y , ) When tubers are marketed for industrial processing, the portions of certain size-grades and the tuber shape play an important role (Haase et al., 2007). Therefore, the industry processing potatoes demands tubers grade > 50 mm for French fries and 40 to 65 mm for crisps. In this connection, the choice of cultivar for industry processing may also be an efficient agronomic measure to increase financial returns when high portions of larger tubers are required Zehra (2011). Tuber shape is an important characteristic in influencing peeling and trimming efficiency during processing (George et al., 2010). Potato tubers that are round (spherical) in shape have been shown to be suitable for crisps processing for most processors because they easily make the required crisp diameters (Kulkarni and Govinden, 1994; Kabira and Lemaga, 2006). The long and oval tubers, however, lend themselves easily for processing of French fries (Kabira and Lemaga, 2006; Abong’ et al., 2009). Factors that influence potato yield and quality include cultivar, soil type, weather conditions, water management, plant population, seed piece size, pests and diseases (Khalafalla, 2001). Plant spacing should depend on type of variety, fertility status of soil, plant architecture or growth habit etc. Potato varieties also differ on growth habit and quality attributes. www.iiste.org www.iiste.org International Journal of African and Asian Studies ISSN 2409-6938 An International Peer-reviewed Journal Vol.52, 2019 DOI: 10.7176/JAAS Physical Quality Characteristics of Potato (Solanum tuberosum L.) Tubers as Influenced by Cultivar and Plant Spacing in Eastern Ethiopia Birhanu Tsegaye1* Nigussie Dechassa2 Wassu Mohammed2 1. Wolkite University, College of Agriculture and Natural Resource, P O Box 07, Wolkite, Ethiopia 2. Haramaya University, School of Plant Sciences, P O Box 138, Dire Dawa, Ethiopia Abstract A field experiment was conducted at Haramaya and Hirna during the main cropping season of 2013 to determine the appropriate plant spacing for potato cultivars in relation to physical tuber quality characteristic. The treatments consisted of five seed tuber spacing between ridges and seed tubers (75 cm x 30 cm, 60 cm x 30 cm, 60 cm x 25 cm, 50 cm x 25 cm and 45 cm x 20 cm) and four potato varieties (Bubu, Badhassa, Zemen and Chiro). The experiment was laid out as a randomised complete block design with three replications. All physical quality attributes of potato responded significantly (P < 0.01) for the main effect of variety and spacing. At Haramaya, Bubu had the higher geometric mean diameter (46.76 mm3) and surface area (6958 mm2) of tuber than the other varieties. Tuber sphericity was higher for Bubu (85.28%), Badhasa (86.54%) and Chiro (82.26%). At both locations, wider spacing of 75 cm x 30 cm, 60 cm x 30 cm and 60 cm x 25 cm gave the highest geometric mean diameter and tuber surface area. However, narrow spacing of 45 cm x 20 cm and 50 cm x 25 cm resulted in the higher sphericity of tuber. g p y Keywords: Inter and intra row spacing, Solanum tuberosum L., variety, geometric diameter, sphericity, surface area DOI: 10.7176/JAAS/52-06 INTRODUCTION Therefore, using the same spacing for all varieties may not lead to optimum tuber quality. Thus, this experiment was conducted with the objective of determining some physical quality attributes of potato cultivars under different spacing and growing conditions. MATERIALS AND METHODS Management of the Experiment The experimental fields were cultivated by a tractor and then levelled after which ridges were made by hand. Well-sprouted medium sized seed tubers were planted according to the specified treatments. Cultivation, weeding and harvesting were done at the appropriate time. Untifengicidal chemical (Mancozeb 80% WP) was applied on 15 days interval at the rate of 1.5 kg ha-1 diluted at the rate of 40 g per 20 liter to control late blight disease. Phosphorus fertilizer was applied at the rate of 92 kg P2O5 ha-1 was done by banding the granules of DAP (diammonium phosphate) (18% N, 46% P2O5) at the depth of 10 cm below and around the seed tuber at planting. Nitrogen fertilizer was applied at the rate of 111 kg N ha-1 (Anonymous, 2004). Treatments and Experimental Design The experiment consisted of four improved potato varieties (Bubu, Badhasa, Zemen and Chiro) and five seed tuber spacing between rows (ridges) and between plants (75 cm x 30 cm, 60 cm x 30 cm, 60 cm x 25 cm, 50 cm x 25 cm and 45 cm x 20 cm). The treatments were laid out as a randomized complete block design (RCBD) in a factorial arrangement and replicated three times per treatment. Gross plot size was 3.6 m x 4.0 m (14.4 m2). The spacing between adjacent plots was 1.0 m and the spacing between adjacent blocks was 1.5 m. MATERIALS AND METHODS MATERIALS AND METHODS Description of Experimental Sites p p The study was conducted under rain-fed condition during the 2013 main cropping season at Haramaya and Hirna 63 International Journal of African and Asian Studies www.iiste.org ISSN 2409-6938 An International Peer-reviewed Journal DOI: 10.7176/JAAS Vol.52, 2019 districts, in eastern and western Hararghe zones of the Oromia Regional State in Ethiopia, respectively. Table 1. Description of the experimental sites Characteristics /features Haramaya Site Hirna Site Latitude Longitude Altitude Mean annual rainfall Soil type Organic carbon content Total nitrogen content Available Phosphorus content Exchangeable potassium Soil pH Sand content Silt content Clay content 9o 26' North latitude 42o 3' East longitude 2015 masl 760 mm well-drained deep alluvial 1.15% 0.11% 18.2 mg kg soil-1 0.65cmolc kg soil-1 8.0 63% 20% 17% 9o 12' North latitude 41o 4' East longitude 1870 masl 990 to 1010 mm vertisol 1.75% 0.18% 32 mg kg soil-1 0.68 cmolc kg soil-1 7.09 27% 28% 45% Source; Belay et al., 1998, Tamire, 1973, Simret, 2010, HURC, 1996, Nebret, 2011 DOI: 10.7176/JAAS cts, in eastern and western Hararghe zones of the Oromia Regional State in Ethiopia, respectively. 1. Description of the experimental sites Source; Belay et al., 1998, Tamire, 1973, Simret, 2010, HURC, 1996, Nebret, 2011 Description of Experimental Materials ion of Experimental Materials eriment was conducted with four improved potato varieties (Bubu, Badhasa, Zemen and Chiro) which y cultivated in eastern Ethiopia. p p The experiment was conducted with four improved potato varieties (Bubu, Badhasa, Zemen and Chiro) which are widely cultivated in eastern Ethiopia. are widely cultivated in eastern Ethiopia. Table 2. Description of the potato varieties used for the experiment No Variety Year of release Growth habit Plant height (cm) Area of adaptation Altitude (metres above sea level) Rainfall (mm) 1 Bubu 2011 Erect 66.8 1650-2330 700-800 2 Badhasa 2001 Erect 50-55 1700-2000 700-800 3 Zemen 2001 Erect 55-60 1700-2000 700-800 4 Chiro 1998 Semi-erect 60 1600-2000 700-800 Source: MoARD (2012). y p Table 2. Description of the potato varieties used for the experiment Data Collection and Measurements Geometric mean diameter (Dg) (mm): The size of ten randomly selected tubers from each plot were measured as length, width and thickness using a digital caliper with an accuracy of 0.01 mm. The geometric mean diameter (Dg) was calculated by using the following equation as described by Mohsenin (1970) as cited by Shehzad et al. (2013): Dg = (LWT) 0.333 ( ) g ( ) Where, L is the length; W is the width and T is thickness of the tuber. Where, L is the length; W is the width and T is thickness of the tuber. Where, L is the length; W is the width and T is thickness of the tuber. Sphericity of the tuber (Ф) (%): Tuber sphericity was determined by the following formula as described by Ahmadi et al. (2008): Ф= (Dg/ L) ×100 Sphericity of the tuber (Ф) (%): Tuber sphericity was determined by the following formula as described by Ahmadi et al. (2008): Ф= (Dg/ L) ×100 Where, Ф is sphercity of the tuber, Dg is geometric mean diameter and L is length 2 Surface area (S) (mm2): Tubers surface area was determined according to Baryeh (2001) by the following formula: S = π Dg 2 64 International Journal of African and Asian Studies ISSN 2409-6938 An International Peer-reviewed Journal Vol.52, 2019 DOI: 10.7176/JAAS Geometric mean diameter The main effects of variety and seed tuber spacing significantly (P < 0.01) affected geometric mean diameter of the tuber at both locations. However, variety and plant spacing did not interact to influence this parameter at both locations (Appendix Table 1, 2 and 3). At Haramaya, Bubu had the highest geometric mean diameter (46.76 mm3) than the other varieties; whereas the other varieties are in statistical parity with each other. However, at Hirna, Bubu, Zemen and Chiro had higher geometric mean diameters (50.49, 49.34 and 49.11 mm3, respectively) while Badhasa had the lower (45.83 mm3). This is because of the production of large-sized tubers by Bubu, Zemen and Chiro varieties which resulted in high geometric mean diameter. Increasing plant spacing significantly increased geometric mean diameter. Thus, in general, 75 cm x 30 cm, 60 cm x 30 cm and 60 cm x 25 cm spacing resulted in higher geometric mean diameters compared to 45 cm x 20 cm and 50 cm x 25 cm spacing. This is because large-sized tubers are produced in response to wider spacing than narrower spacing, consequently resulted in higher geometric mean diameters (Table 14). Habtamu (2013) indicated that geometric mean diameter of potato was significantly influenced by variety and growing environment. Data Analysis The data were subjected to analysis of variance (ANOVA) using the General Linear Model of the SAS statistical package (SAS, 2007) version 9.1. All significant pairs of treatment means were compared using Tulkey Test at 5% level of significance. T-test was conducted to determine differences between the two locations in the performance of the potato varieties to plant spacing. F-test was computed for determining homogeneity of variance for the locations. Sphericity of tuber The main effects of variety and spacing significantly (P < 0.01) affected sphericity of tubers produced at both locations and the mean results of the locations. However, the interaction effect of variety and spacing did not influence this parameter at both locations (Appendix Tables 1, 2and 3). Mean result of the two locations showed that Bubu, Badhasa and Chiro are more spherical in shape as compared to Zemen. On the other hand, decreasing plant spacing significantly increased tuber sphericity. More spherical tubers were obtained from narrow spacing of 45 cm x 20 cm (94.09%) and 50 cm x 25 cm (89.38%) (Table 14). This is because at narrower spacing, small-sized tubers are produced which are more or less spherical in shape as compared to tubers that are produced in response to wider spacing, which are usually wide and large in size and oval in shape. p The t-test of sphericity of the overall mean of the two locations revealed a non-significant difference. This indicates that tuber sphericity did not influenced by varied environmental factors across the locations. Geometric mean diameter Geometric mean diameter Surface area Both the main effects of variety and plant spacing significantly (P < 0.01) influenced surface area of potato tubers at both locations. However, the interaction effect of variety and plant spacing did not influence this parameter at both locations (Appendix Tables 1, 2 and 3). At Haramaya, Bubu had the highest tuber surface area (6958 mm2), while the remaining varieties had the lowest surface area and were in statistical parity with each other. However, under Hirna condition, the varieties Bubu, Zemen and Chiro had higher surface area of (8055, 7676 and 7622 mm2, respectively). Consistent with the results of this study, Habtamu (2013) observed significant varietal and location differences for surface area of potato tubers. Increasing seed tuber spacing significantly increased surface area of potato tuber. Thus, at both locations, spacing of 75 cm x 30 cm, 60 cm x 30 cm and 60 cm x 25 cm resulted in the highest surface area while spacing of 45 cm x 20 cm and 50 cm x 25 cm led to the lowest surface area (Table 14). This is because at wider spacing large-sized tubers are produced which have higher surface area due to less stiffer competition for resources whereas at narrow spacing small-sized tubers are produced due to stiffer competition for growth factors. 65 International Journal of African and Asian Studies ISSN 2409-6938 An International Peer-reviewed Journal DOI: 10.7176/JAAS Vol.52, 2019 www.iiste.org DOI: 10.7176/JAAS Vol.52, 2019 Table 3. Geometric mean diameter, sphericity and Surface area of potato tubers as influenced by the main effects of variety and spacing at Haramaya and Hirna during the 2013 main cropping season. Table 3. Geometric mean diameter, sphericity and Surface area of potato tubers as influenced by the main effects of variety and spacing at Haramaya and Hirna during the 2013 main cropping season. SUMMARY AND CONCLUSION The experiment was conducted out at Haramaya and Hirna, Hararghe highlands of Eastern Ethiopia. Randomized complete black design in factorial arrangement was used with three replications which comprised of five levels of plant spacing (75 cm x 30 cm, 60 cm x 30 cm, 60 cm x 25 cm and 50 cm x 25 cm and 45cm x 20cm) and four levels potato varieties (Bubu, Badhassa, Zemen and Chiro). All physical quality attributes of potato responded significantly (P < 0.01) for the main effect of variety and spacing. At Haramaya, Bubu had the higher geometric mean diameter (46.76 mm3) and surface area (6958 mm2) of tuber than the other varieties. Spericity of tuber was higher for Bubu (85.28%), Badhasa (86.54%) and Chiro (82.26%). At both locations, the wider spacing of 75 cm x 30 cm and 60 cm x 30 cm and 60 cm x 25 cm gave the highest geometric mean diameter and tuber surface area. However, narrow spacing (high planting density) of 45 cm x 20 cm and 50 cm x 25 cm resulted in the higher sphericity of tuber. Surface area Variety Geometric mean diameter (mm3) Sphericity of tuber (%) Mean Surface area (mm2) Haramaya Hirna Haramaya Hirna Haramaya Hirna Bubu 46.76a 50.49a 85.58ab 84.99ab 85.28a 6958a 8055a Badhasa 37.64b 45.83b 86.04a 87.04a 86.54a 4588b 6663b Zemen 37.55b 49.34ab 78.39b 79.55c 78.97b 4562b 7676ab Chiro 41.14b 49.11ab 82.14ab 82.37bc 82.26ab 5367b 7622ab LSD (0.05) 3.461 2.662 5.543 3.107 3.454 839.3 770.2 F-test * CV% 11.5 7.4 9 5 5.6 21.1 13.9 Spacing 75 cm x 30 cm 44.95a 52.56a 76.27c 71.27e 73.77c 6506a 8682a 60 cm x 30 cm 43.35ab 49.23ab 77.81c 76.30d 77.05bc 6073a 7651ab 60 cm x 25 cm 41.34abc 48.91ab 81.38bc 82.65c 82.02b 5433ab 7569ab 50 cm x 25 cm 38.05bc 46.99b 87.86ab 90.90b 89.38a 4632b 6975b 45 cm x 20 cm 36.15c 45.77b 91.87a 96.31a 94.09a 4199b 6643b LSD (0.05) 3.87 2.976 6.197 3.474 3.862 938.4 861.1 F-test CV% 11.5 7.4 9 5 5.6 21.1 13.9 Overall mean Location 40.77 48.69 83.04 83.49 5369 7504 T-test NS LSD (0.05) 1.5 2.181 391.4 Means followed by the same letter within a column for the main effects of variety and plant spacing are not significantly different at 5% level of significance. = significant at 1% probability level, * = significant at 5% probability level. NS= non-significant difference. LSD = Least significant difference; CV % = Coefficient of variation and NS= non-significant difference. Table 3. Geometric mean diameter, sphericity and Surface area of potato tubers as influenced by the main effects of variety and spacing at Haramaya and Hirna during the 2013 main cropping season. G i di Means followed by the same letter within a column for the main effects of variety and plant spacing are not significantly different at 5% level of significance. ** = significant at 1% probability level, * = significant at 5% probability level. NS= non-significant difference. LSD = Least significant difference; CV % = Coefficient of variation and NS= non-significant difference. y , y p p g f g g pp Belay, S., Wortman, W., and Hoogen boom, G. 1998. Haricot bean agro-ecology in Ethiopia: definition using agro-climatic and crop growth stimulation models. African Crop Science Journal 6: pp. 9-18. Dean, B.B. 1994. Managing the Potato Production System. Food Products Press, USA, 61p. AT. 2012. Food and Agriculture Organization of the United Nations, for a world without hunger. Rome, y. http://faostat.fao.org /site/567/. REFERENCES Abong’, G.O., Okoth, M.W., Karuri, E.G., Kabira, J.N. and Mathooko, F.M. 2009. Levels of reducing sugars in eight Kenyan potato cultivars as influenced by stage of maturity and storage conditions. Journal of Animal and Plant Science 2 (2): pp. 76 - 84. Ahmadi, H., Fathollahzadeh, H., Mobli, H. 2008. Some Physical and Mechanical Properties of Apricot Fruits, Pits and Kernels (C.V Tabarzeh). American-Eurasian Journal of Agricultural and Environmental Sciences 3(5), pp. 703-707. Anonymous. 2004. Directory of Released Crop Varieties and Their Recommended Cultural Practices. Ethiopian Agricultural Research Organization, Addis Ababa, Ethiopia. Baryeh, E.A. 2001. Physical properties of Bambara groundnuts. Journal of Food Engineering 47: pp. 32 y , y p p g f g g pp Belay, S., Wortman, W., and Hoogen boom, G. 1998. Haricot bean agro-ecology in Ethiopia: definition using agro-climatic and crop growth stimulation models. African Crop Science Journal 6: pp. 9-18. g p g f p pp Dean, B.B. 1994. Managing the Potato Production System. Food Products Press, USA, 61p. FAOSTAT. 2012. Food and Agriculture Organization of the United Nations, for a world without hunge Italy. http://faostat.fao.org /site/567/. 66 International Journal of African and Asian Studies www.iiste.org DOI: 10.7176/JAAS George, O. Abong, Michael, W. Okoth, Jasper, K. Imungi and Jackson N. Kabira. 2010. Evaluation of selected Kenyan potato cultivars for processing into potato Crisps. Agriculture and biology journal of America 1(5): pp 886-893. pp Haase, T., Schüler, C., and Heb, J. 2007. The effect of different N and K sources on tuber nutrient uptake, total and graded yield of potatoes (Solanum tuberosum L.) for processing. European Journal of Agronomy, 26: pp. 187-197. Habtamu Gebreselassie. 2013. Evaluation of physicochemical and processing attributes of potato (Solanum tuberosum L.) varieties in Eastern Ethiopia. Unpublished MSc. Thesis submitted to school of plant sciences, Haramaya University, Ethiopia. y y p HURC (Haramaya University Research Centre). 1996. Proceedings of the 13th annual Research and extension review meeting. pp. 26-28. a, J.N., and Lemaga, B. 2006. Potato Processing: Quality Evaluation procedures for research and fo ndustries applications in East and Central Africa. Kenya Agricultural Research Institute, Nairobi, Kenya. pp y g , , y Kays, S. J. 1991: Postharvest physiology of perishable plant products. New York: Van Nostrand Reinholt. pp y g Kays, S. J. 1991: Postharvest physiology of perishable plant products. New York: Van Nostrand Reinho Khalafalla, A. M. 2001. REFERENCES Effect of plant density and seed size on growth and yield of Solanum potato in Khartoum state, Sudan. African Crop Science Journal Vol. 9, No. 1: pp. 77-82. Kulkarni, K.D, and Govinden, N. 1994. Crisp Quality of Two Potato Varieties: Effects of Dehydra Rehydration. J Sci. Food Agric. 64 (2): pp. 205-210. MoARD (Ministry of Agriculture and Rural Development). 2012. Animal and Plant Health R Directorate. Crop Variety Register Issue No. 12. Addis Ababa, Ethiopia. p. 180. Mohsenin, N.N. 1970. Physical properties of plant and animal material. New York. Gordon and Breach. Mohsenin, N.N. 1970. Physical properties of plant and animal material. New York. Gordon and Breach. Nebret Tadesse. 2011. The effect of Nitrogen and Sulphur on yield and yield component of common bean in E t Ethi i U bli h d M S Th i t d t th h l f d t t di f H Mohsenin, N.N. 1970. Physical properties of plant and animal material. New York. Gordon and Breach. Nebret Tadesse. 2011. The effect of Nitrogen and Sulphur on yield and yield component of common bean in Eastern Ethiopia. Unpublished M.Sc. Thesis presented to the school of graduate studies of Haramaya University. 25p. Peleg K. 1985. Produce handling, packaging, and distribution. The AVI Publishing Company. Inc. Westport, Connecticut, pp. 55-95. SAS (Statistical Analysis Software). 2007. Stat. Jahrbuch tuber Ernahrung, Landwirtschaft und Forsten In German, Landwirtschaftsverlag Munster-Hiltrup, Germany, 2008. Shehzad Hussain, Tariq Masud1, Sartaj Ali, Rahat Bano, Amjad Ali. 2013. Some physico-chemical attributes of pear (Pyrus communis L.) cultivars grown in Pakistan. International Journal of Biosciences. Vol. 3, No. 12, pp. 206-215. pp Simret Burga. 2010. Influence of inorganic nitrogen and potassium fertilizers on seed tuber yield and size distribution of potato (Solanum tuberosum L.). MSc. Thesis submitted to school of plant sciences, Haramaya University, Ethiopia. Simret Burga. 2010. Influence of inorganic nitrogen and potassium fertilizers on seed tuber yield and size distribution of potato (Solanum tuberosum L.). MSc. Thesis submitted to school of plant sciences, Haramaya University, Ethiopia. y y p Tamire Hawando. 1973. Characterization of Alemaya Soils. Soil Science Paper, Series No, 1. p. 45. Tamire Hawando. 1973. Characterization of Alemaya Soils. Soil Science Paper, Series No, 1. p. 45. Wright Malcom, E., Tappan, J. H. and Sister, F. E. 1986. The size and shape of typical sweet potatoes. Transactions of the ASAE, 29 (3), pp. 678–68. Tamire Hawando. 1973. REFERENCES Characterization of Alemaya Soils. Soil Science Paper, Series No, 1. p. 45. Wright Malcom, E., Tappan, J. H. and Sister, F. E. 1986. The size and shape of typical sweet potatoes. Transactions of the ASAE, 29 (3), pp. 678–68. Wright Malcom, E., Tappan, J. H. and Sister, F. E. 1986. The size and shape of typical sweet Transactions of the ASAE, 29 (3), pp. 678–68. ( ) pp Zehra, E. 2011. Some analytical quality characteristics for evaluating the utilization and consumption of potato (Solanum tuberosum L.) tubers. African Journal of Biotechnology Vol. 10(32), pp. 6001-6010. APPENDIX TABLE APPENDIX TABLE Appendix Table 1. Mean squares from analysis of variance (ANOVA) for some tuber quality attributes at Haramaya site Variables Replication Variety (V) Spacing (S) V x S Error Degrees of freedom 2 3 4 12 38 Geometric mean diameter 13.72 280.81 159.6 34.58ns 21.93 Sphericity of tuber 23.03 189.2* 531.58 20.63ns 56.22 Surface area 960543 18926975 11110773 2073641ns 1289236 Appendix Table 2. Mean squares from analysis of variance (ANOVA) for some tuber quality attributes at Hirna site Variables Replication Variety (V) Spacing (S) V x S Error Degrees of freedom 2 3 4 12 38 Geometric mean diameter 21.88 60.2 80.4 7.64ns 12.97 Sphericity of tuber 25.66 158.08 1263.47 21.75ns 17.66 Surface area 1772568 5268714 7309073 653607ns 1085611 APPENDIX TABLE Appendix Table 1. Mean squares from analysis of variance (ANOVA) for some tuber quality attributes at Haramaya site Variables Replication Variety (V) Spacing (S) V x S Error Degrees of freedom 2 3 4 12 38 Geometric mean diameter 13.72 280.81 159.6** 34.58ns 21.93 Sphericity of tuber 23.03 189.2* 531.58 20.63ns 56.22 Surface area 960543 18926975 11110773 2073641ns 1289236 x Table 1. Mean squares from analysis of variance (ANOVA) for some tuber quality attributes at it Appendix Table 2. Mean squares from analysis of variance (ANOVA) for some tuber quality attributes at Hirna site Variables Replication Variety (V) Spacing (S) V x S Error Degrees of freedom 2 3 4 12 38 Geometric mean diameter 21.88 60.2 80.4 7.64ns 12.97 Sphericity of tuber 25.66 158.08 1263.47 21.75ns 17.66 Surface area 1772568 5268714 7309073 653607ns 1085611 Appendix Table 2. Mean squares from analysis of variance (ANOVA) for some tuber quality attribute site x Table 2. REFERENCES Mean squares from analysis of variance (ANOVA) for some tuber quality attributes at Hirna 67 International Journal of African and Asian Studies www.iiste.org ISSN 2409-6938 An International Peer-reviewed Journal DOI: 10.7176/JAAS Vol.52, 2019 Appendix Table 3. Mean squares from analysis of variance (ANOVA) for some tuber quality attributes for the mean of the two locations Variables Replication Variety (V) Spacing (S) V x S Error Degrees of freedom 2 3 4 12 38 Geometric mean diameter 17.131 129.828 113.796 11.091ns 7.262 Sphericity of tuber 24.08 171.22 854.88 9.28ns 21.83 Surface area 1317716 9528818 8871981 653603ns 499052 International Journal of African and Asian Studies www.iiste.org ISSN 2409-6938 An International Peer-reviewed Journal DOI: 10.7176/JAAS Vol.52, 2019 www.iiste.org able 3. Mean squares from analysis of variance (ANOVA) for some tuber quality attributes for the t l ti Appendix Table 3. Mean squares from analysis of variance (ANOVA) for some tuber quality attributes for the mean of the two locations Variables Replication Variety (V) Spacing (S) V x S Error Degrees of freedom 2 3 4 12 38 Geometric mean diameter 17.131 129.828 113.796 11.091ns 7.262 Sphericity of tuber 24.08 171.22 854.88 9.28ns 21.83 Surface area 1317716 9528818 8871981** 653603ns 499052 68
https://openalex.org/W2336785207	https://letterkunde.africa/article/download/1178/1033	English	null	‘A crushing curse’: Widowhood in contemporary Anglophone Cameroon literature	Tydskrif vir letterkunde	2,016	cc-by-sa	5,732	‘A crushing curse’: Widowhood in contemporary Anglophone Cameroon literature ‘A crushing curse’: Widowhood in contemporary Anglophone Cameroon literature Eunice Ngongkum Eunice Ngongkum is Associate Professor of African Literature, Culture and Ecocriticism at the University of Yaoundé I, Cameroon, and currently a Humboldt Scholar at the Bayreuth Institute for American Studies, University of Bayreuth, Germany. Email: eunicengongkum@yahoo.com Widowhood in contemporary Anglophone Cameroon Literature Moving from the premise that widows have been at the margins of literary discourse in Cameroon, this paper examines widow hood in contemporary Anglophone Cameroon literature using John Nkemngong Nkengasong’s The Widow’s Might (2006) and Alobwed’Epie’s Patching the Broken Dream (2012) as the springboard for its discussion. It argues that the factors that influence the lives of widows, especially, the options available to them and the multiplicity of interests touching on their behavior are grounded in socio-cultural parameters that shape communal consciousness. The paper equally aims at showing how these widows attempt to or actually construct new worlds for themselves by resisting such dominant cultural scripts. The paper locates its discourse within the framework of womanist ideology as propounded by Chikwenye Okonjo Ogunyemi and Mary Modupe Kalawole. Keywords: Cameroon literature, discrimination and stigma, widowhood, womanist theory. TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 ISSN: 0041-476X E-ISSN: 2309-9070 Eunice Ngongkum Eunice Ngongkum is Associate Professor of African Literature, Culture and Ecocriticism at the University of Yaoundé I, Cameroon, and currently a Humboldt Scholar at the Bayreuth Institute for American Studies, University of Bayreuth, Germany. Email: eunicengongkum@yahoo.com Introduction The global focus on women’s issues since the 1970s has brought the activities of women within and beyond the family to the centre in the works of Anglophone Cameroon writers like Makuchi, Anne Tanyi-Tang, Margaret Afuh, Eunice Ngongkum, Bole Butake, Alobwed’Epie and John Nkemngong Nkengasong, among others. However, the space occupied by widows in a number of these texts appears to be liminal despite the increasing interest on the fate of this group of women at the local and global levels. As it were, the growing number of widows in the country due to the HIV/AIDS pan demic coupled with the efforts of international and local organizations to foreground their plight, has contributed in no small way in bringing this essential component of the Cameroon woman’s materiality to the fore. But while the widow still seems to be at the margins of literary discourse, a few Anglophone Cameroonian writers are foregrounding her plight in their works, portraying the institution of widowhood from a variety of interesting perspectives that necessitates a corresponding critical attention. While it is true that widowhood victimizes the woman, the widow’s liminal voicelessness in social discourses seems to suggest that she does very little to respond adequately to her victim status. Some of the writers like Alobwed’Epie, John Nkemngong Nkengasong and Eunice Ngongkum posit that this is far from being the norm. Writing from different ideologi TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 ISSN: 0041-476X E-ISSN: 2309-9070 2016/03/28 11:24:09 AM 09_Ngongkum.indd 138 cal positions, they foreground the subject of widowhood as a crucial paradigm in ongoing debates about woman-being in the contemporary context. In their works, essentialist perceptions of widows are interrogated and disrupted in a way that brings fascinating positions to the dialogue on the subject in the modern framework. Interestingly, the fact that these artists are both male and female, underscores the reality that “in the African woman’s quest for a positive and wholesome definition of womanhood and empowerment, the African male is not excluded” (Nwajiaku 56). John Nkemngong Nkengasong and Alobwed’Epie write against a postcolonial context that tends to assert women’s, and by extension, widows’ rights. In The Widow’s Might (2006) and Patching the Broken Dream (2012), respectively, these novelists foreground the predicament of young widows. Through setting, plot, characteriza tion, narrative perspective and varying stylistic devices, they explain, describe and interrogate widowhood. 09_Ngongkum.indd 139 Widowhood: Definition Widowhood—the state of being a widow—occurs when a wife loses her husband through death and does not remarry. According to Marjo Buitelaar, the word “widow” derives from the Latin, vidua which relates to a root meaning, “to place apart” (1). From this root word, widowhood is thus a separation, a placing apart of a wife from the husband through death. The loss of the husband through death generally represents the loss of a partner, a friend and a breadwinner. In most cases, this results in a radical change in the woman’s social status and lifestyle. Margaret Owen argues that widowhood tends to impact more traumatically on the woman, altering forever the way she is perceived and consequently affecting her self-image (8). Widowhood is a universal human phenomenon shaped by, among others, the culture within which people function. In Africa, in general, and Cameroon in par ticular, it constitutes a traumatic life event. According to a recent United Nations report on discrimination against women in Africa, widows, irrespective of their ethnic affiliations, are among the most vulnerable and destitute. This situation is fueled by cultural practices/beliefs, patriarchal domination and inheritance laws which militate against the rights of the widow. A number of perceptions of widow hood from different parts of Africa seem to confirm this. Florence Orabueze, for instance, notes that “widowhood is a word that every woman dreads to mention. In Africa, it brings to a peak all the humiliation, subordi nation, degradation and oppression which an African woman goes through in her life time. From the time of her husband’s death to the time of her own death, his family and society blame her for the passing away of the much needed male. She is indirectly asked why she should survive the man” (115). For Rose Acholonu, “widows are subjected to a whole gamut of obnoxious widowhood rites aimed at making her die within the mourning period of about one year. Most brothers-in-law are ever ready to disinherit her and […] drive her out of her marital home” (97). Teddy Kuyela, on his part, observes that, “In most of the African society, widow hood represents a ‘social death’ for women. It is not just that they have lost their husbands, the breadwinner and supporter of their children, but widowhood robs them of their status and confines them to the fringes of society where they suffer discrimination and stigma. Introduction Particularly, they lay bare not only the difficulties of its young victims but also show how the latter engage their circumstances and move on to be better or worse off, as the case may be. These works question dominant cultural practices or knowledges that widows live by, locating themselves within not only womanist discursive strategies but also within a postcolonial context that has witnessed significant changes in the situation of the woman as a whole. I argue that the factors that influence the lives of young widows, especially, the options available to them, and the multiplicity of interests touching on their behavior are grounded in socio-cultural parameters that shape communal consciousness; and I further show how these widows resist these dominant cultural scripts to carve out niches for themselves. I adopt womanism, an ideological praxis hinged on a strategy of reading that considers the woman in the African context as the theoretical framework for my analysis. Womanism’s focus is on the woman in the family and the society, especially highlighting her experience in settings chiefly defined by patriarchy. The Widow’s Might and Patching the Broken Dream portray widows in a postcolonial context that is largely patriarchal. According to Chikwenye Okonjo Ogunyemi (1996), and Mary Modupe Kolawole (1998), womanist writings criticize grossly unfair gender arrangements; arrangements drawn from “myths which recommend, prescribe or validate the society’s norms, values, code of conduct, social […] and society’s sense of identity” (Kolawole 7). Nkengasong and Alobwed’Epie interrogate the marginal and negative images of widows promoted and sustained by patriarchal cultures. They portray these women as capable of redefinition and self-evaluation even in the most tragic circumstances. In this context, men and women play vital roles for, as Charles Nnolim claims, in another context, womanism, “wants a meaningful union between black women and men and children and will see to it that men will change from their sexist stand” (46). TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 139 2016/03/28 11:24:09 AM 09_Ngongkum.indd 139 Widowhood: Definition ‘A deep dark world of anguish?’: Widowhood in The Widow’s Might ‘A deep dark world of anguish?’: Widowhood in The Widow’s Might Set principally in Bakomba Town and Ekaka Village in present day Cameroon, the novel tells the story of Akwenoh, widow of Honorable Makata Mbutuku, Member of Parliament of Bakomba Town, whose sudden death at the beginning of the nar rative sets the canvas for the novel’s discourse on the subject. Through plot, setting, characterization, narrative voice and rhetorical strategies, Nkengasong demonstrates how women’s agency in widowhood is particularly complex given the choices, inter connections, negotiated meanings and social spaces that widows must navigate in this new phase of their lives. p The plot of the novel begins on an ironical note. Honorable Mbutuku informs his wife about his imminent appointment as a government minister. The couple is ecstatic as both share how this piece of news will change their future unaware that Mbutuku will die that same night. In a series of ironical hints, the novelist builds up tension preparing the reader for Mbutuku’s demise and the eventual widowhood experience of his wife, Akwenoh. For instance, we are told that “they went to sleep with promises of greater days ahead, promises of affluence and dignity, promises of health and wealth” (11). Akwenoh’s response to the information is noteworthy: “she wished that what her husband was telling her came out to be true. That was going to mean a change in her status. A minister’s wife! She was going to rise above the ordinary woman in Bakomba and she was going to be the pride of the Global Ladies, an association of women who were married to top civil servants and promi nent businessmen and politicians in town” (10). Her dream and waking worlds are invaded by the possibility of a better life. She envisions herself in her new role as the wife who would “by law be the only person to control her husband’s property if he died” (12). These hints, at the beginning of the narrative, establish Akwenoh’s character and prepare the reader for the ironic twist to her fate as a widow. How Akwenoh will respond when she eventually discovers that she is the destitute wife of a parliamentarian constitutes the central issue in the work. The sudden demise of a husband, as in Akwenoh’s case here, is an unexpected catastrophe that thrusts the widow into a new and problematic status. Widowhood: Definition […] Widows are generally trodden upon, poor and least protected as their lives are determined by local, patriarchal interpretations of tradition, discrimination and stigma” (1). These perceptions of the widowhood landscape in Africa identify two broad defining aspects of the experience, namely, disinheritance/deprivation and manda tory observance of culturally prescribed burial rites which inflect psychologically and physically on the widow. This is the background that generally informs Nken gasong’s The Widow’s Might and Alobwed’Epie’s Patching the Broken Dream. In both TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 140 2016/03/28 11:24:09 AM 09_Ngongkum.indd 140 texts, however, this general picture is reinforced by several modern dynamic socio- cultural parameters that problematize and complicate the widowhood experience of Akwenoh and Diana, the heroines of the texts in question. 09_Ngongkum.indd 141 ‘A deep dark world of anguish?’: Widowhood in The Widow’s Might In addition to the deep physical, emotional and psychological pain that strips her of all self esteem and dignity, she must give her husband a befitting burial and perform the traditional widowhood rites demanded of her. Akwenoh had hitherto looked down on widows and once challenged her husband to marry a widow if he wanted an obedient wife, who condoned poverty. As she squats on the bare floor in mourning, she is unable to come to terms with her new position. Nkengasong’s rhetoric and diction serve TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 141 2016/03/28 11:24:09 AM 09_Ngongkum.indd 141 to delineate her emotional trauma and the subject position she must now assume as a widow. “What a cruel world she asked herself? That she will go to sleep hap pily, rise in the morning happily and out of a sudden her bright promising world is transformed into a deep dark world of anguish?” (21). “‘What will I do with my life? What will I do with my children?’ She continued weeping. ‘Now I am a widow too. […] Where do I go from here?’” (40) Phrases and sentences such as “her head reeled will pain,” “Her husband’s death had cut her heart to pieces,” “With a heavy heart” and “words cut through her like a razor,” among others, continue to highlight Akwenoh’s emotional, psychological and physical trauma as she slowly becomes aware of her vulnerability and destitu tion. All along, her husband’s real estate investments (the family house in Salaka Street and the apartments on rent at Awarawara Quarter) and political connections fuel her survival discourse. But as the plot of the novel unfolds, she learns that all of these are lost due to the late husband’s murky political and financial past. Caught in this dilemma, she must, to hide her shame, “empty her purse” to give her husband a befitting burial, as Shadrach Ambanasom rightly observes (5). And she does amidst prospects of either giving in to tradition or living in destitution. The widow’s world in this novel is a hybrid space informed by forces of tradition and modernity. These meet, clash and jostle with each other, providing a rich tap estry on which Akwenoh’s widowhood trajectory is laid bare. The setting is urban but traditional widowhood rites must be performed. She is expected to formally and openly demonstrate her grief and intense feelings. ‘A deep dark world of anguish?’: Widowhood in The Widow’s Might The cultural context being one that views death as ‘unnatural,’ she is also expected to mourn her husband in a way that will indicate that she is innocent of his death. She wails loudly and continuously while sitting on the bare floor. The corpse removal and final burial episodes serve to highlight the oppressive world of tradition. Her sister-in-law, Ma Eseke, totally insen sitive to her physical and emotional exhaustion, accuses her of not weeping enough because she is looking forward to being a ‘happy widow,’ enjoying the wealth of her late brother. Even when Akwenoh swears the mandatory oath before the villagers to prove her innocence, this sister-in-law challenges the veracity of her innocence. With the husband buried, Akwenoh’s head is shaved; she is given sackcloth to wear for a year and abandoned to hunger and loneliness in a dark room. Ma Eseke seems to embody the hard stance of tradition; however, some women, like the Global ladies, posit that “the times have changed and some old practices had to be buried” (22). They suggest that a mattress be brought to provide some comfort for the widow. But “as soon as Akwenoh was settled on the mattress, Ma Eseke rose from where she was sitting as if stung on the buttocks by a black ant. […] She walked over to Akwenoh as if to exorcise some vanity out of her. ‘Akwenoh, get up from that mattress.’ She screamed. […] ‘I have asked you to get up from that mattress and heap your buttocks on the floor like a woman who has truly lost her TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 142 2016/03/28 11:24:09 AM 09_Ngongkum.indd 142 husband’” (23). The phrase, “heap your buttocks on the floor,” denotes the widow’s subject position in the context of widowhood. Akwenoh’s refusal to comply elicits the inevitable accusations and threats from her sister-in-law. “[…] You killed your husband in order to enjoy his wealth. You have started enjoying it even when he is not a day old in the fridge. But I will see how far you will go” (23). Ma Eseke’s responses here underscore the disconcerting contradiction that African women, in their roles as mothers, sisters and daughters are the main agents for the perpetration and enforcement of patriarchy. ‘A deep dark world of anguish?’: Widowhood in The Widow’s Might a d e o ce e t o pat a c y The widow’s plight is further exacerbated by conflicts over property. Akwenoh is reminded by her brother-in-law, Chief Ekwe, that she is part of her late husband’s assets, all of which now belong to him. When she rejects this levirate posture, Ma Eseke tells her that she doesn’t have any say in the matter because tradition must be upheld. Mbutuku’s family believes that their brother, being a “big man,” left boxes of money which they expect the wife to produce so they could spend it on his funeral. In the context of widowhood in this novel, tradition enunciates a self-serving ethos, drawing from modernity for its own ends. This reinforces Allison Jaggar’s view that “The patriarchal institution is characterized by division, distinction, opposition and dualism” (36). The postcolonial environment that informs the novel is defined by such oppositions and dualisms and is paradoxically the space that equally gives room for the questioning of some of these practices hitherto taken as the norm. Through Akwenoh, Nkengasong posits that even in the most vulnerable of women’s posi tions in Africa, namely, widowhood, women are not voiceless. They speak and act even if their actions are circumscribed by choices and circumstances that somehow limit their agency Akwenoh, for instance, draws on existing traditional roles while equally playing on the intersection between the social and the economic possibilities in her setting. Her husband’s briefcase is immediately secured in a room upon his demise and she systematically refuses to give in to her in-laws’ request for money for the funeral arrangements. She reminds them of their traditional role in arranging and organizing their brother’s funeral. She roundly rejects Pa Ekwe’s marriage demand and begins to work out how she will organize her husband’s funeral and fare with her children as a widow. “Chief,” she said, “as far as marrying you is concerned I will not accept. As for my husband’s wealth, I don’t know in the first place which wealth you are talking about. The little that I shall find, if at all I find it, will be used to bring up the children” (31). She looks forward to joining the Association of Happy Widows, geared towards protecting widows’ rights, in the hope of being shielded from the greed of her in-laws. 09_Ngongkum.indd 143 ‘A deep dark world of anguish?’: Widowhood in The Widow’s Might All these indicate how Akwenoh confronts and negotiates the controlling patriar chal structures in widowhood — structures that seek to exclude and marginalize her. However, her confrontation fails to carry through given the choices made as a young TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 143 2016/03/28 11:24:09 AM 09_Ngongkum.indd 143 girl, namely, neglecting her studies for a life of promiscuity. Her self-reflection, nar rated through a flashback, elucidates her present circumstances, leading to a better understanding of her immediate predicament. She recollects that “her mother had always insisted when she was young that her education was her best husband. She ignored and went after men. If only she knew the consequences!” (133). Akwenoh can only blame “herself for the present state of affairs. It was the result of the fool ish pattern of choices she made as a young woman. […] She regretted that she did not take interest in her education” (133). Her regrets at not privileging her studies indicate the role of education in women’s empowerment, the weapon that Diana, Alobwed’Epie’s heroine in Patching the Broken Dream, uses to redefine herself within these same parameters. p Nkengasong’s exploration of Akwenoh’s limitation does not problematize his feminist/womanist stance in the novel as Blossom Fondo (2010) and Adamu Pangmeshi (2011) have argued. Rather, it can be seen to be situated in a realist and functionalist paradigm that requires an awareness of the environment and the choices that fuel or limit the heroine’s agency. ‘Hideously ugly’: Widowhood in Patching the Broken Dream y g y g Unlike his earlier novel, The Lady with a Beard, in which Alobwed’Epie deals with a widow in a purely traditional setting, Patching the Broken Dream handles the fortunes of a widow in an urban setting much like The Widow’s Might. The narrative, told from Diana’s perspective, begins with details of her married life with David. Theirs is a loving relationship in which David is not the typical “African” man. He loves and encourages his wife to pursue higher education even if it would mean she becomes more qualified than he. He takes care of the children, does laundry, brings home a salary, saves money and generally looks out for the wife’s welfare. Diana notes that he was simply “inde scribable” as a husband and as a father. While Diana is at the university, for instance, he returns home promptly every evening to see to it that their three “children were bathed, fed and sent to bed on time.” This, she says, is not to undermine her but “to remove every extra strain from” her and make her concentrate on her studies; complete university, then enter a professional school and after a few years, join him in building their dream, namely, having a successful and happy family (7). Even Diana’s mother who thinks David is “spoiling” his wife, is not happy that her son in-law is “giving a helping hand in washing the children’s dresses, even his” because for her, “domestic work was a woman’s affair” (20). However, it is noteworthy that Diana makes good use of the opportunity given to her. She works hard, completes university and by the time her third child is six months old, she “entered Ecole Normale Superieure” (8). This atmosphere of bliss, unlike that of Akwenoh’s in The Widow’s Might, prepares the reader for Diana’s eventual response to the demise of her beloved spouse; a response at odds with society’s vision of a young and working class widow. TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 144 2016/03/28 11:24:09 AM 09_Ngongkum.indd 144 David’s demise through a tragic motor accident plunges Diana into psycho logical and emotional shock. She loses touch with reality “as a million questions raced through [her] mind and [she] drifted into a world of insensitivity” (72). She attempts suicide, passes out and is hospitalized. ‘Hideously ugly’: Widowhood in Patching the Broken Dream Upon recovery, she is psychologi cally tormented at the thought of her orphaned children, “an eclipsed life without David,” his burial and the debts that will accrue from this. However, supported by family, friends and David’s employer, she buries him. It is only then that “the full torrent of what [her] new life would be buffeted [her] in the face” (82). Again, unlike Akwenoh who must perform the mandatory widowhood rites, Diana’s “tradition forbade [her] from mourning him overtly” (129). Yet she demonstrates an awareness of these traditions and out of love for her spouse, she chooses how to mourn him, “wearing black underwear, for [she] thought it was incumbent [on her] to mourn him in another way—a more liberal way” (130). This liberal vision is grounded in a postcolonial setting that has witnessed an increasing global international action in reducing women’s oppression such as The Beijing Platform of Action, Convention on the Elimination of All Forms of Discrimination Against Women (1979) and the African Charter on Human and People’s Rights (2003), among others. These human rights instruments have had significant international influence on legal and institutional reforms relative to women’s rights. The plot of the novel thus highlights how Diana defies the institutional perception of widows as vulnerable and powerless and shows how she progresses to autonomy and dignity. At home, she schools her children against extravagance while helping her mother and brother-in-law, who live with her, to “fit into the new situation” when they must relocate due to the brazen injustice of an ungrateful landlord, who takes advantage of the fact that she is a widow, to renege on his previous rental agreement with her late spouse. Rather than driving her under, the eviction notice elicits unknown reserves of determination and resilience within her. She success fully manages the leftover money from David’s funeral and other funds accruing from the sale of used material from the building project to complete the structure her husband started and moves into the house, even “without doors, windows and some sort of concrete floor” (98). When her eldest daughter passes the entrance exam into secondary school, she borrows money and sends her to one of the best schools in the country while counting on her salary arrears and David’s pension and death benefits to pay the loans. Towards the end of the novel, she turns down suitors the likes of Dr. 09_Ngongkum.indd 145 ‘Hideously ugly’: Widowhood in Patching the Broken Dream Maurice, himself a widower, and Peter, not out of what society considers as “impudence, snob bishness, impropriety” nor for enjoying life as a rich happy widow, as Peter purports. Rather, it is her love for husband and the determination to patch the broken dream they both had, namely, to “keep [their] D identity” (124) that undergird her actions. She even envisages bringing her mother-in-law to live with her so that “they shall TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 145 2016/03/28 11:24:09 AM 09_Ngongkum.indd 145 live together in their own way—the way of patching the broken dream” (124). Diana succeeds and is praised by her immediate entourage for this stance as, “She had never thought of becoming famous for mourning David” (136). Through his heroine, Alobwed’Epie foregrounds a culturally relevant and alternative vision of widow hood that is currently gaining ground on the continent. Kenda Muthoni corroborates this when she says that “families headed by widows constitute a large proportion of families in Africa; nearly 30% of adult women in Africa today are widows” (4). Further, the social networks Diana created before David’s demise continue in some form after his death to help the heroine cope better with the challenges of widow hood. This indicates that the novel is constructed on the womanist understanding that the heroine’s ability to successfully engage the oppressive world of widowhood enjoys the collaboration of a communally oriented ethos, even if at the end, the decisions concerning her life remain hers. These networks include Diana’s friends, Imelda and her husband, Roselyn and her husband, Dr. Maurice who saved her life, the neighbours, workers and her husband’s fellow club members. In negotiating the perception of widows in her context, Diana sometimes uses that view to her benefit, corroborating Muthoni’s view that in coping with their status, widows often use patriarchy to their advantage (6). For instance, she asks for and obtains help and advice from Imelda’s husband; she begs “the workers to treat [her] kindly and fairly, seeing that [she] was a widow” (97). But as time evolves, Diana is wary of depending too much on these perceptions and networks because of the stigmas accompanying young widowhood, namely, being “suspected by married and unmarried women alike, and eyed with voluptuous desires of young fellows and senile dads alike” (117). She gradually moves on to “become a husband to [herself] in thoughts, word and deed” (118). Conclusion As narratives of woman-centered experience, The Widow’s Might and Patching the Broken Dream, foreground the plight of their protagonists in an alien and oppressive context, namely, widowhood. Written against a post-independence context that tends to assert women’s rights, the works tell the stories of women who find themselves in the unwelcome state of widowhood and are constrained to seek ways to cope with it. They further underscore the fact that the transformations in the socio-cultural landscape influence the actions and perceptions of widows. It is incumbent on the women, the narratives seem to posit, to take advantage of these transformations to enrich and empower themselves. Akwenoh fails to do so and her fate is uncertain while Diana does and secures a future for herself and her family. In spite of the marked difference in response to widowhood which points to differences in ideology, two crucial conclusions regarding widowhood discourses in Anglophone Cameroon literature can be drawn from the two texts. Firstly and centrally, both investigate how the rhythms and routines of daily life for the heroines are disrupted by crisis — the sudden deaths of their husbands — and how life can and could be lived in the aftermath of such tragedy. The works portray these women making decisions about their own lives; having the liberty to live alone and managing their own affairs. The triumph of such decisions, however, is shown to be rooted in choices made before the tragic incident. Akwenoh’s vulnerability is accentuated by her lack of economic autonomy devolving from her rejection of for mal education, which in the contemporary context, liberates the woman “unleashing human energy and spirit, critical thinking, the question of authority, challenges to conventional wisdom, and adds new ways of being and doing (Saleeby 7). This is evident in Diana who, thanks to a salaried job, decides to remain unmarried while heading her household. The empowerment that education can confer on the woman in the contemporary context cannot thus be gainsaid. Secondly, both novels foreground the way in which widowhood transforms the individual woman’s relationship to the immediate cultural environment. The posi tions of the widows highlight tensions between tradition and modernity as well as between individual autonomy and group perception. While Diana is spared the scourge of traditional widowhood rites, Akwenoh undergoes them even if at the beginning, she resists. ‘Hideously ugly’: Widowhood in Patching the Broken Dream Speaking at a welcome-back home party for her daughter, Dora, the president of her husband’s club, acknowledging her resolve and success says: “you needed to take decisions that only you could take so that if they worked in either way (good or bad) you would hold yourself responsible” (133-4). Through the use of the first person narrative point of view, Alobwed’Epie allows Diana to write herself into being, thereby stressing her agency in the context of widowhood. It is important to note that of the eight chapters of this fifteen–chapter novel that foreground the widowhood experience of Diana, five are devoted to underscoring how she displays remarkable determination and courage in the face of tragedy. By closing the novel with the widowed heroine’s moral and financial inde pendence—evidence of her construction of a productive widowhood—Alobwed’Epie makes a more womanist statement than Nkengasong about postcolonial widowhood. Diana serves to demonstrate the feminine energies needed to cope in a catastrophic and complex situation. These energies are largely the product of her personality, education and the friendly/unfriendly culture around her. TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 146 2016/03/28 11:24:09 AM 09_Ngongkum.indd 146 09_Ngongkum.indd 147 Conclusion Diana, on the other hand, must confront society’s percep tion of young unmarried widows in her decision not to remarry. In some instances, both widows uphold tradition especially with regard to mourning their husbands. By this, the novelists seem to posit that this should not be considered as inherently conservative. As it were, women can employ traditional ideals and norms to their own advantage. Akwenoh calls her in-laws’ attention to the fact that by tradition, she is only a widow and they must do all to bury their brother. Diana plays on the traditional perception of widows as vulnerable to get the workers on her housing TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 147 2016/03/28 11:24:09 AM 09_Ngongkum.indd 147 project to work without cheating her. These relationships and perspectives function as a metaphor for the widowhood experience in both novels. project to work without cheating her. These relationships and perspectives function as a metaphor for the widowhood experience in both novels. 09_Ngongkum.indd 148 Works Cited Acholonu, Rose. “Women in the African Novel and the Quest for Human Rights.” Beyond the Marginal Land: Gender Perspectives in African Writing. Ed. Chioma Opara. Port Harcourt: Belpot. 1999. 97–100. Alobwed’Epie. Patching the Broken Dream. Yaoundé: Miraclaire. 2012. Ambanasom, Shadrach. “Review of John Nkemngon Nkengasong’s The Widow’s Might.” The Post 074 2006. 5. Buitelaar, Marjo.“Widows’ Worlds: Representations and Realities.” Between Poverty and the Pyre: Moments in the History of Widowhood. Ed. Jan Bremmer & Lourens von den Bosch. New York: Routledge. 1995. 1–18. Fondo, Blossom N. �� “Subtle Misogyny: The Femme Fatale Motif in the Novels of John Nkemngong Nken gasong.” Rupture et transversalité de la littérature Camerounaise. Eds. Marcelline Nnomo, Nol Alembong, Mvogo Faustin. Yaoundé: Éditions CLE. 2010. 329–41. g Jaggar, Allison. Feminist Politics and Human Nature. New York: Rowman & Little Field Publishers. 1983. Kolawole, M. Modupe. Womanism and African Consciousness. Trenton, NJ: Africa World Press. 1998. Kuyela, Teddy. “Southern Africa: Alleviating the Pangs of Widowhood.” www.allafrica.com. 14 Feb 2014. 12 Dec 2014. <http://allafrica.com/stories/200702140501.html>. p Muthoni, Kenda. Worries of the Heart. Chicago & London: The U of Chicago P. 2007. Nwajiaku, Ijioma C. “Representation of the Womanist Discourse in the Short Fiction of Akachi Ezeigbo and Chinwe Okechukwu.” New Women’s Writing in African Literature 24. Ed. Ernest Emenyonu. London: James Currey. 2004. 55–68. Nkemngong, Nkengasong J. The Widow’s Might. Yaoundé: Editions CLE. 2006. Ogunyemi, Chikwenye O. Africa Wo/Man Palava: The Nigerian Novel by Women. Chicago: U of Chicago P. 199 Orabueze, Florence. “The Feminist Crusade Against Violation of Women’s Fundamental Human Rights: Mariama Ba’s So Long A Letter and Buchi Emecheta’s Second Class Citizen.” Women in the Academy: Festschrift for Prof. Helen Chukwuma. Eds. Seiyefa Konoye and Noel C. Anyadike. Port Harcourt: Pearl Publications. 2004. 111–16. Owen, Margaret. A World of Widows. London: Zed Books. 1998. Nnolim, Charles . ”African Feminism: The Scandalous Path.” Beyond the Marginal Land: Gender Perspectives in African Writing. Ed. Chioma Opara. Port Harcourt: Belpot. 1999. 34–54. Pangmeshi, Adamu. “Failed Heroines: The Paradox of Female Liberation in John Nkemngong Nken gasong’s The Widow’s Might and Alobwed’Epie’s The Lady with a Beard.” Folia Linguistica et Litteraria: Časopis Za Nauku O Jeziku I Knijiževnosti. 2011. 213–26. Saleeby, Dennis. 2002. “Introduction. Power in the People.” The Strengths Perspective in Social Work Practic Ed. Dennis Saleeby. Boston: Allyn & Bacon. 2002. 1–22. TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 148 2016/03/28 11:24:09 AM 09_Ngongkum.indd 148 09_Ngongkum.indd 148
https://openalex.org/W2124089048	https://dash.harvard.edu/bitstream/1/12064543/1/3938467.pdf	English	null	Blockade of the Programmed Death-1 (PD1) Pathway Undermines Potent Genetic Protection from Type 1 Diabetes	PloS one	2,014	cc-by	10,679	Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12064543 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Published Version Published Version doi:10.1371/journal.pone.0089561 Citation Citation Kochupurakkal, N. M., A. J. Kruger, S. Tripathi, B. Zhu, L. T. Adams, D. B. Rainbow, A. Rossini, et al. 2014. “Blockade of the Programmed Death-1 (PD1) Pathway Undermines Potent Genetic Protection from Type 1 Diabetes.” PLoS ONE 9 (2): e89561. doi:10.1371/journal.pone.0089561. http://dx.doi.org/10.1371/journal.pone.0089561. Abstract Aims/Hypothesis: Inhibition of PD1-PDL1 signaling in NOD mice accelerates onset of type 1 diabetes implicating this pathway in suppressing the emergence of pancreatic beta cell reactive T-cells. However, the molecular mechanism by which PD1 signaling protects from type 1 diabetes is not clear. We hypothesized that differential susceptibility of Idd mouse strains to type 1 diabetes when challenged with anti PDL1 will identify genomic loci that collaborate with PD1 signaling in suppressing type 1 diabetes. Methods: Anti PDL1 was administered to NOD and various Idd mouse strains at 10 weeks of age and onset of disease was monitored by measuring blood glucose levels. Additionally, histological evaluation of the pancreas was performed to determine degree of insulitis. Statistical analysis of the data was performed using Log-Rank and Student’s t-test. Results: Blockade of PDL1 rapidly precipitated type 1 diabetes in nearly all NOD Idd congenic strains tested, despite the fact that all are moderately (Idd5, Idd3 and Idd10/18) or highly (Idd3/10/18 and Idd9) protected from spontaneous type 1 diabetes by virtue of their protective Idd genes. Only the Idd3/5 strain, which is nearly 100% protected from spontaneous disease, remained normoglycemic following PDL1 blockade. Conclusions: These results indicate that multiple Idd loci collaborate with PD1 signaling. Anti PDL1 treatment undermines a large portion of the genetic protection mediated by Idd genes in the NOD model of type 1 diabetes. Basal insulitis correlated with higher susceptibility to type 1 diabetes. These findings have important implications since the PD1 pathway is a target for immunotherapy. Citation: Kochupurakkal NM, Kruger AJ, Tripathi S, Zhu B, Adams LT, et al. (2014) Blockade of the Programmed Death-1 (PD1) Pathway Undermines Potent Genetic Protection from Type 1 Diabetes. PLoS ONE 9(2): e89561. doi:10.1371/journal.pone.0089561 Editor: Rasheed Ahmad, Dasman Diabetes Institute, Kuwait Received September 18, 2013; Accepted January 22, 2014; Published February 28, 2014 Received September 18, 2013; Accepted January 22, 2014; Published February 28, 2014 Copyright: 2014 Kochupurakkal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by JDRF Grant # 1-2007-756 (I. Guleria). LSW is supported by Wellcome Trust Grant 096388 and Juvenile Diabetes Research Foundation International Grant 9-2011-253. Blockade of the Programmed Death-1 (PD1) Pathway Undermines Potent Genetic Protection from Type 1 Diabetes Nora M. Kochupurakkal1, Annie J. Kruger2, Sudipta Tripathi1, Bing Zhu3, La Tonya Adams1, Daniel B. Rainbow4, Aldo Rossini2, Dale L. Greiner2, Mohamed H. Sayegh1, Linda S. Wicker4, Indira Guleria1* 1 Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School Renal Division, Boston, Massachusetts, United States of America, 2 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 3 Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, United States of America, 4 Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom Abstract This work was also supported by National Institute of Allergy and Infectious Disease (NIAID) grants AI 070351, P01AI039671 (LSW), and AI 46629. The Cambridge Institute for Medical Research is the recipient of a Wellcome Trust Strategic Award (100140). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Indira.Guleria@childrens.harvard.edu Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Introduction All mice were cared for in accordance with Boston Children’s Hospital and the University of Massachusetts Medical School institutional guidelines. susceptibility to autoimmune diseases, particularly in individuals harboring susceptibility alleles. To date, numerous MHC-linked and non-MHC-linked genes and genetic regions influencing the susceptibility to autoimmune diseases have been identified in humans, rats and mice. In insulin dependent type 1 diabetes, many genes implicated in the control of glycemia have also been described in the NOD Idd congenic mouse strains. Congenic NOD strains have genetic loci from diabetes resistant parental strains inserted (introgressed) into their genome (reviewed in [7]). ( g ) g ( [ ]) In recent years, NOD H2-Ag7 and H2-Enull MHC class II genes have been unequivocally identified as susceptibility genes within Idd1 [8]. Additionally, accumulated data support the existence of particular susceptibility genes within other Idd regions. Idd3 is the most well studied Idd region [9–11]. Protective alleles in Idd3 reduce type 1 diabetes frequency and Il2 and Il21 are the prime candidate genes. The protective effects of Idd3 are evident in multiple cell types including antigen-presenting cells, effector T cells and regulatory (FoxP3+) T cells which are critical for maintaining immune cell homeostasis [12,13]. In recent years, NOD H2-Ag7 and H2-Enull MHC class II genes have been unequivocally identified as susceptibility genes within Idd1 [8]. Additionally, accumulated data support the existence of particular susceptibility genes within other Idd regions. Idd3 is the most well studied Idd region [9–11]. Protective alleles in Idd3 reduce type 1 diabetes frequency and Il2 and Il21 are the prime candidate genes. The protective effects of Idd3 are evident in multiple cell types including antigen-presenting cells, effector T cells and regulatory (FoxP3+) T cells which are critical for maintaining immune cell homeostasis [12,13]. g [ 2, ] The prime gene candidate for Idd10 is Cd101 whose expression on regulatory T cells and dendritic cells is affected in NOD/B6 polymorphisms [14]. Vav3, which encodes a guanine nucleotide exchange factor important for signaling in immune cells, is the only complete gene present in the 604 kb Idd18.1 region on Chromosome 3. Gene expression evidence indicates that alter- ation of Vav3 expression is an etiological factor in the development of autoimmune beta-cell destruction in NOD mice, making it the most likely candidate [15]. The Idd5 region is composed of at least 5 sub-regions. Antibodies and Treatment Protocol Anti mouse PDL1 mAb (MIH6, rat IgG2a) was generated as previously described, [35] and was manufactured by BioXCell (West Lebanon, NH, USA). Rat IgG (Sigma-Aldrich, St.Louis, MO, USA) served as a control. Anti PDL1 was injected in PBS i.p.; 500 mg on day 0, followed by 250 mg on days 2, 4, 6, 8, and 10 unless indicated otherwise. Mice were 10 weeks of age at the start of treatment. Monitoring for Diabetes The onset of type 1 diabetes was defined as a random blood glucose reading of 250 mg/dl or greater for three consecutive days. Blood glucose levels were measured daily for the first two weeks followed by 2–3 times per week by One Touch Ultra meter and One Touch Ultra test strips (LifeScan, Milipitas, CA, USA). Introduction infectious diseases (such as HIV), since blocking the pathway results in an increased immune response against tumors and infections [1–3]. Type 1 diabetes is a multi-factorial autoimmune disease resulting from the destruction of pancreatic beta cells by autoreactive T cells. Both environmental factors and variations in multiple genetic loci have been implicated in the etiology of type 1 diabetes. The NOD mouse recapitulates many features of human type 1 diabetes and is used extensively as an experimental model. We and others have shown that PD1-PDL1 interaction is critical for the regulation of CD4 and CD8 autoreactive T cells involved in the development of type 1 diabetes [4,5]. Further, while PD1 deficiency resulted in lupus-like symptoms in C57BL6 or BALB/c mice, it led to accelerated onset and frequency of type 1 diabetes in NOD mice [6]. Programmed death-1 (PD1) and its ligand PDL1 have been shown to play an important role in regulating T cell activation and peripheral tolerance. The PD1- PDL1 pathway is being explored for developing therapies against recurrent solid tumors and In the NOD mouse model, blockade of PD1-PDL1 pathway results in accelerated onset of autoimmune diabetes, raising concern that immunotherapy by such blockade could increase February 2014 \| Volume 9 \| Issue 2 \| e89561 1 PLOS ONE \| www.plosone.org February 2014 \| Volume 9 \| Issue 2 \| e89561 PDL1 Blockade Reverses Genetic Protection from T1D 1094) [31], NOD.B10-Idd5.1 (line 2193) [31], NOD.B10-Idd5.2 (line 6146) [31], NOD.B10-Idd5.3 (line 6360) [32], NOD.B6-Idd3/ 10/18 (line 1538) [15,27], NOD.B6-Idd3 (line 1098) [12,27], NOD.B6-Idd3 B10-Idd5 (line 6109) [27], NOD.B10-Idd5.2 Idd5.3 (line 1595) [32] and NOD.B10-Idd5.2 Idd5.3 Idd3 (lines 7380 and 9245, data combined in this study)[33]. The NOD congenic strains will be referred to by their Idd numbers without adding NOD before the designated Idd region. When referring to congenic mice containing two or more Idd loci, the loci will be separated by slashes. For example, Idd9.1 Idd9.2 Idd9.3 (line 905) mice will be referred to as Idd9.1/9.2/9.3 for simplicity. Spontaneous development of diabetes in females from these strains of mice has been published (references noted above). BDC2.5 TCR Tg mice were a gift of Drs. Diane Mathis and Christophe Benoist [34]. NY8.3 mice were obtained from JDRF’s Resource Sharing Program. All mouse experiments were approved by the Institutional Animal Care and Use Committee of Children’s Hospital Boston and University of Massachusetts Medical School. Introduction Idd5 contributes to islet-specific CD8 T cell tolerance and to loss of CD4 tolerance through both lymphocytic and non-lymphocytic compartments [9,16,17]. Candidate genes for Idd5 sub regions include Ctla4 for Idd5.1 [18], Slc11a1 for Idd5.2 [19] and Acadl for Idd5.3 [20]. The Idd9 region on chromosome 4 is composed of at least three separate intervals, Idd9.1, Idd9.2, and Idd9.3 and numerous candidate genes are present. Fine mapping of type 1 diabetes regions Idd9.1 and Idd9.2 revealed further genetic complexity [21]. The Idd9.1 sub-region has been shown to influence regulatory T cells and iNKT cells [22,23]. Idd9.2 and Idd9.3 have been linked to limit the expansion of islet specific autoreactive CD8 T cells [24]. The Idd9.3 candidate gene encodes 4-1bb, which is important for CD4 and CD8 T cell activation [25]. The Idd9 locus has also been previously described to play a role in homing of islet-specific T cells [26]. Overall, Idd9 mice display profound resistance to diabetes even though nearly all develop insulitis. Adoptive transfer of BDC2.5 TCR-transgenic cells Anti-CD25 mAb (clone 7D4, ATCC, Manassas, VA, USA) and rabbit complement (Cedarlane, Burlington, NC, USA) were incubated with splenocytes of BDC2.5 TCR Tg mice at 37uC for 45 min to remove CD25+ cells (technique described in [36]. Remaining cells were labeled with 7.5 mM CFSE (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The percentage of CD4+ T cells in the splenocyte suspension was determined by flow cytometry to calculate the volume needed for injection of 0.56106 BDC2.5tg CD4+ T cells. Splenocytes were labeled with CD3, CD4 and Vb4 antibodies and analyzed by flow cytometry. Half a million CD4+ T cells were injected i.v. into the tail veins of pre-diabetic 8–10 week old female NOD and age matched Idd3/10/18 mice. Mice received 500 mg of either anti PDL1 mAb or rat IgG one day before transfer (day 0), and 250 mg Histology Pancreases were fixed in 10% neutral buffered formalin in PBS for 16 h and transferred to 70% ethanol before being embedded in paraffin. Tissue sections were stained with H&E (Dana Farber Cancer Institute’s Research Pathology Core, Boston, MA, USA) and insulitis was graded by scoring a minimum of 10 islets per mouse. Each mouse received a score from the average of graded islets. Scores were defined as: 0 -no insulitis, 1 –peri-insulitis, 2, 50% insulitis, 3.50% insulitis, 4 -100% insulitis. In this study, we made use of four loci on Chromosome 3, four on Chromosome 1, and three on Chromosome 4 to determine which Idd regions conferring resistance to type 1 diabetes remain so in the presence of anti PDL1 negative co-stimulatory blockade. We show that blockade of the PD1-PDL1 interaction results in accelerated onset of type 1 diabetes in all the NOD Idd strains except NOD Idd3/5. Additionally, basal insulitis levels correlated with higher susceptibility to type 1 diabetes induction by anti PDL1 treatment. Diabetes-resistant NOD Idd strains develop diabetes upon anti PDL1 treatment p In order to determine if blocking the PD1-PDL1 pathway would induce autoimmune diabetes in mice genetically protected from developing the disease, strains of mice protected from type 1 diabetes because they carry protective genes derived from B6 and B10 mice, were treated with anti PDL1 mAb. We tested the following 14 NOD congenic strains to examine the genetic protection due to a range of genes and gene combinations which can possibly contribute to resisting the precipitation of type 1 diabetes following PDL1 blockade: Idd3, Idd10/18, Idd3/10/18, Idd5 (which includes the subcongenic regions of Idd5.1, Idd5.2, and Idd5.3), Idd5.1, Idd5.2, Idd5.3, Idd5.2/5.3, Idd3/5, Idd3/5.2/5.3, Idd9 (which includes the sub-congenic regions of Idd9.1, Idd9.2 and Idd9.3), Idd9.1, Idd9.2 and Idd9.3. The incidence of diabetes for females from these 14 strains at 28 to 30 weeks of age are ,5% (Idd3/10/18, Idd3/5, Idd3/Idd5.2/Idd5.3 and Idd9), 15–40% (Idd3, Idd9.1 and Idd5), and 45–65% (Idd5.1, Idd5.2, Idd5.3, Idd5.2/Idd5/ 3, Idd9.1, Idd9.2, Idd9.3, and Idd10/18). Throughout the time period of defining the Idd regions using congenic strains that are resistant to type 1 diabetes (1990 to 2010) the NOD female diabetes incidence has ranged from 70–90% at 28 to 30 weeks of age. Idd9, Idd9.1, Idd9.2, Idd9.3 Idd9 mice receiving anti PDL1 treatment developed type 1 diabetes with a cumulative incidence of 46% between days 6 and 16. The sub-congenic strain Idd9.2 showed a reduced cumulative incidence of diabetes at 56% (between days 4–18), whereas the Idd9.1 and Idd9.3 strains had a much higher cumulative incidence with 95% and 90% respectively (onset from day 3 to day 22), which is quite similar to 93% type 1 diabetes in anti PDL1 treated NOD mice (between days 4–12) (Figure 1b, Table 1, 2). As the Idd9.2 strain had the lowest cumulative incidence among Idd9 subcongenic strains, we deduced that this sub-congenic strain must be associated with the protective allele in the Idd9 congenic interval. Statistical Analysis Kaplan-Meier survival analysis was performed to compare the frequency of diabetes in sub-congenic strains using the Log-Rank test. Differences in insulitis between congenic strains were analyzed by unpaired two-tailed Student’s t test. A p value of , 0.05 was considered significant. Idd3/Idd5 and Idd3/5.2/5.3 The Idd3/5 strain has protective alleles at both Idd3 and Idd5 and only 1% of mice develop spontaneous diabetes by 7–8 months of age [38]. Anti PDL1 treatment did not induce diabetes in Idd3/ 5 mice as 100% of them stayed non-diabetic over the course of 30 days PDL1 blockade (Figure 1d). The Idd3/5.2/5.3 (without protective alleles at the Idd5.1 sub-region) strain that is also almost completely protected from spontaneous diabetes shows suscepti- bility to treatment with anti PDL1, and 15% of the mice developed diabetes by day 30 (Figure 1d, Table 1, 2). Idd3, Idd10/18 and Idd3/10/18 Twenty percent of Idd3 mice spontaneously develop type 1 diabetes within 7–8 months [37]. Upon anti PDL1 administration, 50% of the mice developed type 1 diabetes between days 6 and 16 (Figure 1c). Idd10/18 mice have a 50% occurrence of spontaneous diabetes, and with anti PDL1 treatment 94% of mice developed the disease between days 4 to 27. The Idd3/10/18 strain develops diabetes with 31% incidence upon anti PDL1 treatment (days 4 to 28), which is ,6-fold greater than the spontaneous incidence at the age of 7–8 months (Figure 1c, Table 1, 2). RNA extraction and Real time PCR of pancreas tissue Pancreas tissue from Idd9 mice was stored in RNAlater solution (Ambion, Austin, TX, USA) and total RNA was extracted using the RNAeasy Mini kit (Qiagen, Gaithersburg, MD, USA). RNA was redissolved in RNAse-free water and the yield quantified by spectrophotometry. Equal amounts of RNA were used for quantitative real time PCR. First strand cDNA synthesis was performed using Superscript III (Invitrogen, Grand Island, NY, USA). All reactions were run in triplicates in an ABI Prism 7300 (Applied Biosystems, Foster City, CA, USA) and normalized to GAPDH. For a list of primers used, see Electronic Supplemental Material. Mice Female NOD mice were obtained from Taconic (Germantown, NY, USA). NOD congenics were obtained through the Taconic Emerging Models program; NOD.B10-Idd9.1/9.2/9.3 (line 905) [27], NOD.B10-Idd9.1 (line 1565) [22], NOD.B10-Idd9.2 (line 1566) [22], NOD.B10-Idd9.3 (line 1106) [22], NOD.B6-Idd10/18 (line 7754) [14,15,27–30], NOD.B10-Idd5.1 Idd5.2 Idd5.3 (line February 2014 \| Volume 9 \| Issue 2 \| e89561 PLOS ONE \| www.plosone.org 2 PDL1 Blockade Reverses Genetic Protection from T1D Idd5 on days 2 and 4. Pancreatic LN and spleens were harvested on day 6 and cells were stained for CD4, Vb4 (KT4, BD Biosciences, USA), labeled with CFSE and analyzed by flow cytometry. Untreated Idd5 mice have a 40% cumulative incidence of diabetes at 28 to 30 weeks of age [37]. With anti PDL1 treatment, 10-week old Idd5 mice started to develop the disease by day 10, and after 30 days, 62.5% had developed type 1 diabetes (Figure 1a, Table 1, 2). The sub-congenic strains Idd5.1, Idd5.2, Idd5.2/5.3 and Idd5.3 showed a faster onset of disease from day 3 to day 7. The Idd5.3 strain had the highest cumulative incidence of type 1 diabetes following anti PDL1 treatment, with 90% of the mice developing disease by day 30 (P = 0.0140 Idd5 vs Idd5.3), followed by Idd5.2 with 80% cumulative incidence (P = 0.0194 Idd5 vs Idd5.2). Idd5.1 developed diabetes with a 66% cumulative incidence. The combination of two sub-congenic strains in Idd5.2/5.3 developed type 1 diabetes with a cumulative incidence of 65% (Figure 1a, Table 1, 2). Of the control NOD mice treated with anti PDL1 93% developed type 1 diabetes by day 21. None of the control NOD mice developed type 1 diabetes during the course of the experiment (Figure 1a, Table 1, 2). Adoptive transfer of NY8.3 TCR transgenic cells Adoptive transfer of NY8.3 TCR transgenic cells Splenocytes from NY8.3-NOD TCR Tg mice were used for adoptive transfer studies. Splenocytes were enriched for CD8+ T cells using the CD8+ T cell untouched isolation kit II (Miltenyi, Auburn, CA, USA). One million CD8+ T cells were injected i.v. into the tail veins of pre-diabetic 8–10 week old female NOD and age matched Idd3/10/18 mice. The recipients received 500 mg of either anti PDL1 mAb or IgG Ab one day before transfer (day 0), and 250 mg on days 2 and 4. The pancreatic lymph node and spleen were harvested on day 6 and the cells were acquired by flow cytometry for CFSE labeling. Insulitis in anti PDL1 treated congenic strains One of the hallmarks of developing type 1 diabetes is the presence of infiltrating lymphocytes in the pancreas. Idd congenic strains have made it possible to identify checkpoints of disease progression. Ninety percent of Idd9 mice show evidence of islet insulitis, but only 3% develop diabetes spontaneously [21,37]. Analysis of insulitis scores of the Idd strains revealed that NOD congenics that were partially (Idd 9.2, Idd 3/10/18) or fully (Idd3/ PLOS ONE \| www.plosone.org February 2014 \| Volume 9 \| Issue 2 \| e89561 3 PDL1 Blockade Reverses Genetic Protection from T1D Figure 1. Incidence of diabetes in NOD congenic mouse strains undergoing anti PDL1 treatment. Treatment was started at 10 weeks of age. a): Incidence of diabetes in Idd5 and sub-congenics Idd5.1, Idd5.2, Idd5.3 and Idd5.2/5.3 until day 30 after anti PDL1 treatment. In Idd5 mice (n = 16) 62.5% developed diabetes, in Idd5.1 (n = 15) 66.6%, in Idd5.2 (n = 20) 80%, in Idd5.3 (n = 8) 87.5% and in Idd5.2/5.3 (n = 23) 66.5%. NOD mice (n = 28) had a 92.5% incidence of diabetes by day 30. All control treated mice did not develop diabetes. b): In Idd9 (n = 26) 46.15% developed diabetes, in Idd9.1 (n = 20) 95%, in Idd9.2 (n = 30) 56.6% and in Idd9.3 (n = 21) 90.5%. c): Idd3 (n = 17) developed diabetes at a rate of 50%, Idd10/18 (n = 18) at 94.1%, Idd3/10/18 (n = 26) at 30.8%. d): In Idd3/5 (n = 16) 0% of anti - PDL1 treated mice developed diabetes, in Idd3/5.2/5.3 (n = 13) 15.4%. Statistics and cumulative incidence for the strains are shown in separate Tables for Figure 1. P-values were calculated using Log-rank (Mantel-Cox test). doi:10 1371/journal pone 0089561 g001 PDL1 Blockade Reverses Genetic Protection from T1D Figure 1. Incidence of diabetes in NOD congenic mouse strains undergoing anti PDL1 treatment. Treatment was started at 10 weeks of age. a): Incidence of diabetes in Idd5 and sub-congenics Idd5.1, Idd5.2, Idd5.3 and Idd5.2/5.3 until day 30 after anti PDL1 treatment. In Idd5 mice (n = 16) 62.5% developed diabetes, in Idd5.1 (n = 15) 66.6%, in Idd5.2 (n = 20) 80%, in Idd5.3 (n = 8) 87.5% and in Idd5.2/5.3 (n = 23) 66.5%. NOD mice (n = 28) had a 92.5% incidence of diabetes by day 30. All control treated mice did not develop diabetes. Insulitis in anti PDL1 treated congenic strains b): In Idd9 (n = 26) 46.15% developed diabetes, in Idd9.1 (n = 20) 95%, in Idd9.2 (n = 30) 56.6% and in Idd9.3 (n = 21) 90.5%. c): Idd3 (n = 17) developed diabetes at a rate of 50%, Idd10/18 (n = 18) at 94.1%, Idd3/10/18 (n = 26) at 30.8%. d): In Idd3/5 (n = 16) 0% of anti - PDL1 treated mice developed diabetes, in Idd3/5.2/5.3 (n = 13) 15.4%. Statistics and cumulative incidence for the strains are shown in separate Tables for Figure 1. P-values were calculated using Log-rank (Mantel-Cox test). d i 10 1371/j l 0089561 001 ) doi:10.1371/journal.pone.0089561.g001 doi:10.1371/journal.pone.0089561.g001 Cytokine and chemokine profile in Idd9 subcongenic mice 5) protected from anti PDL1 accelerated diabetes had lower basal insulitis scores (Idd 9.2 (0.1860.11), Idd3/10/18 (0.2460.059) and Idd3/5 (0.060.0) compared to the almost unprotected Idd9.1 (1.260.3), Idd9.3 (0.8260.14) and the NOD (1.2360.24) mice. These results were statistically significant (Idd9.2 vs Idd9.1, Idd9.3, NOD p value 0.0016, 0.0031 and 0.0007, respectively; Idd3/10/ 18 vs Idd9.1, Idd9.3, NOD p value ,0.0001, 0.0002 and ,0.0001, respectively; Idd3/5 vs Idd9.1, Idd9.3, NOD p value 0.0047, 0.0014 and 0.0018 respectively). The cumulative incidence of diabetes following anti PDL1 treatment for Idd9.2 was different from that of Idd9.1 and Idd9.3 mice. Nearly 100% of Idd9.1 and Idd9.3 mice while only 56% of Idd9.2 mice developed type 1 diabetes following anti PDL1 treatment. Basal insulitis was also lower in Idd9.2 versus Idd9.1 and Idd9.3 mice. Therefore we sought to determine if any cytokines or chemokines were differentially expressed in these three sub- congenic lines. Real time PCR of pancreas tissue of anti PDL1 treated mice showed that diabetic Idd9.2 mice had lower expression of IFN-c, TNF-a, CCR2, RANTES (CCL5) and MIP-1a (CCL3) as compared to diabetic Idd9.1 and Idd9.3 mice (Figure 3a-f). MIP-1a up-regulation has been associated with progression to type 1 diabetes [39]. Idd9 mice also had lower cytokine and chemokine levels than the Idd9.1 and Idd9.3 substrains. These studies show that a low level of insulitis as observed in Idd9.2 correlates with lower levels of cytokines even Clearly, there seems to be a direct link between basal insulitis levels and the incidence of anti PDL1 induced accelerated diabetes. Interestingly, Idd9.2 mice that turned diabetic showed similarly high insulitis scores (3.3160.25) upon anti PDL1 treatment as treated NOD mice (3.25860.1195), while the Idd9.2 mice which stayed non-diabetic had almost no pancreatic infiltrates and low insulitis scores (0.3860.17) (Figure 2). PLOS ONE \| www.plosone.org February 2014 \| Volume 9 \| Issue 2 \| e89561 4 PDL1 Blockade Reverses Genetic Protection from T1D Proliferation of BDC2.5 Tg CD4+ T cells and NY8.3 CD8+ T cells in pancreatic LN of Idd3/10/18 mice following anti PDL1 treatment Proliferation of BDC2.5 Tg CD4+ T cells and NY8.3 CD8+ T cells in pancreatic LN of Idd3/10/18 mice following anti PDL1 treatment Table 1. Statistical Significance Figure 1. Table 1. Statistical Significance Figure 1. Comparison of Idd Strains p-value Idd5 vs. Idd5.1 p = 0.1679 Idd5 vs. Idd5.2 p = 0.0194 Idd5 vs. Idd5.3 p = 0.0140 Idd5.1 vs. Idd5.2/5.3 p = 0.0539 Idd9 vs. Idd9.1 p,0.0001 Idd9 vs. Idd9.2 p = 0.2029 Idd9 vs. Idd9.3 p,0.0001 Idd9.2 vs. Idd9.1 p = 0.094 Idd9.2 vs. Idd9.3 p = 0.0118 Idd3 vs. Idd10/18 p = 0.0103 Idd3/10/18 vs. p,0.0001 Idd10/18 Idd3 vs. Idd3/10/18 p = 0.2531 Idd3/5 vs Idd3/5.2/5.3 p = 0.2298 doi:10.1371/journal.pone.0089561.t001 We performed an adoptive transfer of transgenic T cells and analyzed their proliferation rates to identify differences between the congenic strains undergoing anti PDL1 blockade. Adoptive transfer of CFSE-labeled BDC2.5 Tg CD4+ T cells into untreated NOD and Idd3/10/18 mice showed similar proliferation rates in the pancreatic LN. With administration of anti PDL1, both NOD (P = 0.0276) and Idd3/10/18 (P = 0.0002) strains showed signifi- cantly higher proliferation of BDC2.5 Tg T cells. No difference was detected in BDC2.5 Tg CD4+ proliferation between untreated and anti PDL1 treated NOD and Idd3/10/18 mice (Figure 4a, 5a). In another set of experiments we adoptively transferred CFSE labeled CD8+ 8.3 TCR Tg+ T cells in Idd3/10/18 mice (Figure 4b, 5b) and analyzed their rate of proliferation following anti PDL1 treatment. NY8.3 CD8+ Tg+ T cells divided more frequently as portrayed by an increase in the number of CFSE-diluted CD8+ T cells in Idd3/10/18 mice that received anti PDL1 antibody as compared to mice that received control IgG. These data show that both auto-reactive CD4 as well as CD8 T cells expand in Idd3/10/ 18 mice following anti PDL1 treatment, similar to that of untreated and anti PDL1 treated NOD mice. doi:10.1371/journal.pone.0089561.t001 when diabetes develops in some of these mice following PDL1 blockade. It remains to be seen if the quality of insulitis in Idd9 and Idd9.2 mice is different from that present in the Idd9.1 and Idd9.3 substrains. when diabetes develops in some of these mice following PDL1 blockade. It remains to be seen if the quality of insulitis in Idd9 and Idd9.2 mice is different from that present in the Idd9.1 and Idd9.3 substrains. PDL1 Blockade Reverses Genetic Protection from T1D Therefore, it was extremely difficult to increase the n of anti PDL1 treated non-diabetic group. doi:10.1371/journal.pone.0089561.g002 Figure 2. Insulitis scores in anti PDL1 treated mice. Idd congenic mice were grouped into control treated (untx, white columns, Idd9.1 n = 7, Idd9.2 n = 10, Idd9.3 n = 9, Idd3 n = 4, Idd10/18 n = 5, Idd3/10/18 n = 18, Idd3/5 n = 3, NOD n = 7), anti PDL1 treated diabetic (Tx-D, checked columns, Idd9.2 n = 4, Idd3 n = 5, Idd10/18 n = 8, Idd3/10/18 n = 6, NOD n = 11) and anti PDL1 treated non-diabetic (Tx-ND, black columns, Idd9.2 n = 5, Idd3 n = 5, Idd10/18 n = 1#, Idd3/10/18 n = 6, Idd3/ 5 n = 17) mice. The antibody treatment was given to 10-week old mice at day 0 (500 mg), and days 2, 4, 6, 8 and 10 (250 mg). The injections were stopped once the mouse had become diabetic and had a glucose reading of .250 mg/dl on two consecutive days. H&E sections of pancreases were scored for degree of infiltrating lymphocytes in islets. Results are expressed as Mean6SD. P-values are expressed as * (P, 0.05), (P,0.01), * (P,0.0001). # In case of the Idd10/18 mice, treatment with anti PDL1 resulted in 94% of diabetes incidence. Therefore, it was extremely difficult to increase the n of anti PDL1 treated non-diabetic group. doi:10.1371/journal.pone.0089561.g002 The Idd9 strain including sub-congenic 9.1, 9.2 and 9.3 shows high levels of pancreatic infiltrates (90%), but does not develop diabetes at a high rate (3%) [21]. Nonetheless, this strain develops insulin autoantibodies [27]. The Idd9.1 region was identified to control type 1 diabetes development through TNF-a [42]. Idd9.2 and Idd9.3 regions were found to be responsible for preventing the expansion of islet specific CD8+ T cells, providing an explanation for the dichotomy of high insulitis incidence and a low rate of actual diabetes development in the Idd9 strain [24]. In the current study we examined the effects of 9 Idd regions, alone and in combination, on accelerated type 1 diabetes following anti PDL1 treatment. These congenic and subcongenic mice have multiple protective alleles that mediate varying degrees of resistance to type 1 diabetes. PDL1 Blockade Reverses Genetic Protection from T1D Figure 2. Insulitis scores in anti PDL1 treated mice. Idd congenic mice were grouped into control treated (untx, white columns, Idd9.1 n = 7, Idd9.2 n = 10, Idd9.3 n = 9, Idd3 n = 4, Idd10/18 n = 5, Idd3/10/18 n = 18, Idd3/5 n = 3, NOD n = 7), anti PDL1 treated diabetic (Tx-D, checked columns, Idd9.2 n = 4, Idd3 n = 5, Idd10/18 n = 8, Idd3/10/18 n = 6, NOD n = 11) and anti PDL1 treated non-diabetic (Tx-ND, black columns, Idd9.2 n = 5, Idd3 n = 5, Idd10/18 n = 1#, Idd3/10/18 n = 6, Idd3/ 5 n = 17) mice. The antibody treatment was given to 10-week old mice at day 0 (500 mg), and days 2, 4, 6, 8 and 10 (250 mg). The injections were stopped once the mouse had become diabetic and had a glucose reading of .250 mg/dl on two consecutive days. H&E sections of pancreases were scored for degree of infiltrating lymphocytes in islets. Results are expressed as Mean6SD. P-values are expressed as * (P, 0.05), (P,0.01), * (P,0.0001). # In case of the Idd10/18 mice, treatment with anti PDL1 resulted in 94% of diabetes incidence. Therefore, it was extremely difficult to increase the n of anti PDL1 treated non-diabetic group. doi:10.1371/journal.pone.0089561.g002 strains. This is probably because Ctla4and Il2both modulate the survival and function of Treg cell population and the blockade of PD1-PDL1 pathway is not enough to limit the ability of these Tregs, and tolerance is maintained in the Idd3/5 congenic mice. It is also worth mentioning that PDL1 and Ctla4mediated tolerance induction functions through two distinct pathways. Allelic interaction of Ctla4 with the other candidate genes in the Idd3/5 strain is able to overcome the effect of anti PDL1 treatment and maintain tolerance in these congenic mice. Slc11a1 plays an important part in antigen presenting function of DCs and may play a role in inducing tolerance to self antigens. Acadl is proposed to have a significant role in T cell function and survival by altering fatty acid metabolism. We therefore suggest that the combination of these four candidate genes and their interaction renders the Idd3/5 congenics resistant to diabetes induction by PDL1 blockade. The Idd3/5 mice have also been shown to be resistant to other experimental autoimmunity [41]. Discussion More than 38 Idd regions from resistant strains that confer protection in the NOD model, have been described to date [7,40]. More than 38 Idd regions from resistant strains that confer protection in the NOD model, have been described to date [7,40]. Table 2. Spontaneous incidence of type 1 diabetes at 7 months of age compared to anti PDL1 treatment of 10 week old mice. Table 2. Spontaneous incidence of type 1 diabetes at 7 months of age compared to anti PDL1 treatment of 10 week old mice. Strain Line Cumulative Incidence of spontaneous type 1 diabetes Cumulative Incidence of type 1 diabetes with aPDL1 treatment starting at 10 weeks NOD.B10-Idd5.1 1094 40% 62.5% Idd5.2 Idd5.3 NOD.B10-Idd5.1 2193 62% 66% NOD.B10-Idd5.2 6146 38% 80% NOD.B10-Idd5.3 6360 69% 90% NOD.B10-Idd5.2 1595 25% 65% Idd5.3 NOD.B10- 905 3% 46% Idd9.1/9.2/9.3 NOD.B10-Idd9.1 1565 35% 95% NOD.B10-Idd9.2 1566 55% 56% NOD.B10-Idd9.3 1106 50% 90% NOD.B6-Idd3 1098 20% 50% NOD.B6- 7754 50% 94% Idd10/Idd18 NOD.B6-Idd3/10/18 1538 9% 31% NOD.B6-Idd3 B10- 6109 1% 0% Idd5 NOD.B6-Idd3 7380 0% 15% Idd5.2/5.3 NOD.B6-Idd3 9245 0% Idd5.2/5.3 NOD 70–90% 93% doi:10.1371/journal.pone.0089561.t002 \| \| \| \| dence of type 1 diabetes at 7 months of age compared to anti PDL1 treatment of 10 week old mice. Table 2. Spontaneous incidence of type 1 diabetes at 7 months of age compared to anti PDL1 treatment of 10 week old mice. February 2014 \| Volume 9 \| Issue 2 \| e89561 PLOS ONE \| www.plosone.org 5 PDL1 Blockade Reverses Genetic Protection from T1D PDL1 Blockade Reverses Genetic Protection from T1D The Idd10/18 strain has an insulitis rate of 78%, and ,50% rate of spontaneous diabetes development [37], in contrast to the Idd3/10/18 strain which shows greater protection, with 19% of mice developing insulitis and 7% developing type 1 diabetes. The Idd3/10/18 strain demonstrates a median protection against anti PDL1 accelerated diabetes development with a 31% cumulative incidence as opposed to a cumulative incidence of 94% in Idd10/ 18, and 0% in the Idd3/5 strain following treatment. The Idd3/ 10/18 strain is almost completely protected from diabetes, similar to the Idd3/5 strain. However, the effect of PDL1 blockade results in significantly different outcomes in respect to diabetes induction. These data also imply that in the absence of negative costimulation by the PD1-PDL1 pathway, CTLA4 possibly maintains the self tolerance with the help of IL-2 (one of the candidate genes in Idd3) in case of the Idd3/5 strain. And IL-2 alone is not sufficient to prevent anti PDL1 mediated accelerated diabetes in case of the Idd3/10/18 strain. Similarly in Idd3/5.2 and Idd3/5.3 strains where the congenic Idd5.1 locus containing the CTLA4 gene is absent, IL-2 alone cannot prevent diabetes induced by PDL1 blockade. Figure 2. Insulitis scores in anti PDL1 treated mice. Idd congenic mice were grouped into control treated (untx, white columns, Idd9.1 n = 7, Idd9.2 n = 10, Idd9.3 n = 9, Idd3 n = 4, Idd10/18 n = 5, Idd3/10/18 n = 18, Idd3/5 n = 3, NOD n = 7), anti PDL1 treated diabetic (Tx-D, checked columns, Idd9.2 n = 4, Idd3 n = 5, Idd10/18 n = 8, Idd3/10/18 n = 6, NOD n = 11) and anti PDL1 treated non-diabetic (Tx-ND, black columns, Idd9.2 n = 5, Idd3 n = 5, Idd10/18 n = 1#, Idd3/10/18 n = 6, Idd3/ 5 n = 17) mice. The antibody treatment was given to 10-week old mice at day 0 (500 mg), and days 2, 4, 6, 8 and 10 (250 mg). The injections were stopped once the mouse had become diabetic and had a glucose reading of .250 mg/dl on two consecutive days. H&E sections of pancreases were scored for degree of infiltrating lymphocytes in islets. Results are expressed as Mean6SD. P-values are expressed as * (P, 0.05), (P,0.01), * (P,0.0001). # In case of the Idd10/18 mice, treatment with anti PDL1 resulted in 94% of diabetes incidence. PDL1 Blockade Reverses Genetic Protection from T1D Among the three subloci of Idd9, Idd9.2 is considered the most potent region in providing protection against the disease by restraining autoreactive CD8+ T cells. Blockade of PDL1 is known to cause CD8+ T cell exhaustion. This explains the similarity in the anti PDL1 induced diabetes incidence (approximately 50%) in the Idd9 and Idd9.2 strains; whereas the Idd9.1 and Idd9.3 strains are completely susceptible (90-95%) to anti PDL1 induced diabetes. We also observe a very low frequency of pancreatic infiltrates in the Idd9.2 strain. This can be explained by the fact that the strains of Idd9 and its sub regions have a low frequency of autoreactive CD8+ T cells in comparison to the NOD mice. Further, the genes in the Idd9 sub regions prevent a massive expansion of these autoreactive CD8+ T cells during disease onset and progression [25]. However, extensive insulitis was observed in the group of Idd9.2 mice that become diabetic after PDL1 blockade suggesting that in some of the mice the low frequency of autoreactive cells can expand when this regulatory pathway is inhibited. The decreased cytokine and chemokine production in Idd9 and Idd9.2 mice may also be related to the low frequency of autoreactive T cells [25] that affects the quality of the infiltrating cells following PDL1 blockade as compared to mice not having protective alleles at Idd9.2. Our study using treatment with anti PDL1 mAb indicates that sufficient numbers of effector cells are present in these congenic strains to mediate type 1 diabetes. The rapid onset of diabetes in some of the Idd congenic strains is probably due to auto-aggressive memory/effector T cells that are suddenly set free when PDL1 is blocked, as has been shown in a study in NOD mice [43]. Further, CD4+ Type II NKT cells were shown as regulators of diabetes and it was shown that these cells were sufficient in down- regulating diabetes, promoting activity of CD4+ BDC2.5 tg T cells in vivo. Interestingly, blockade of ICOS and PDL1 was found to negate the regulatory effect of the CD4+ Type II NKT cells in the pancreatic lymph node leading to a sudden development of diabetes [44]. We used the Idd3/10/18 strain to further dissect anti PDL1 mediated diabetes. BDC2.5 Tg CD4+ T cells were transferred into NOD and Idd3/10/18 mice treated with anti PDL1. Similar rates of T cell proliferation were observed in pancreatic LNs of both strains. PDL1 Blockade Reverses Genetic Protection from T1D The difference in the pathogenicity of the February 2014 \| Volume 9 \| Issue 2 \| e89561 PLOS ONE \| www.plosone.org 6 PDL1 Blockade Reverses Genetic Protection from T1D Figure 3. Quantitative PCR detection of cytokine, chemokine and transcription factor levels in pancreas tissue after anti PDL1 treatment in Idd9 (n = 3) and subcongenics Idd9.1(n = 3), Idd9.2 (n = 5), Idd9.3 (n = 5). Mice (10 weeks old) were treated with 500 mg anti PDL1 on day 0 and 250 mg anti PDL1 on days 2, 4, 6, 8 and 10 by i.p. injection. Pancreas tissue was harvested on day 30 or when mice had turned diabetic. Horizontal lines show median value. P-values are expressed as * (P,0.05), (P,0.01), * (P,0.0001) in figure. a): IFN-c: Idd9.1 vs. Idd9.2 p = 0.0039; Idd9.1 vs. Idd9.3 p = 0.063. b): CCR2: Idd9 vs. Idd9.1 p = 0.0003; Idd9.1 vs. Idd9.2 p,0.0001; Idd9.2 vs. Idd9.3 p = 0.0431; Idd9 vs. Idd9.3 p = 0.0183. c): RANTES: Idd9 vs. Idd9.1 p = 0.022; Idd9.1 vs. Idd9.2 p = 0.0016; Idd9.2 vs. Idd9.3 p = 0.0284; Idd9 vs. Idd9.3 p = 0.0382. d): No significant differences in FoxP3 expression between Idd9 and subcongenics. e): MIP-1a: Idd9 vs. Idd9.1 p = 0.0257; Idd9.2 vs. Idd9.1 p = 0.0017. f): TNF-a:Idd9 vs. Idd9.1 p = 0.019; Idd9.2 vs. Idd9.1 p = 0.0004. doi:10.1371/journal.pone.0089561.g003 Figure 3. Quantitative PCR detection of cytokine, chemokine and transcription factor levels in pancreas tissue after anti PDL1 treatment in Idd9 (n = 3) and subcongenics Idd9.1(n = 3), Idd9.2 (n = 5), Idd9.3 (n = 5). Mice (10 weeks old) were treated with 500 mg anti PDL1 on day 0 and 250 mg anti PDL1 on days 2, 4, 6, 8 and 10 by i.p. injection. Pancreas tissue was harvested on day 30 or when mice had turned diabetic. Horizontal lines show median value. P-values are expressed as * (P,0.05), (P,0.01), * (P,0.0001) in figure. a): IFN-c: Idd9.1 vs. Idd9.2 p = 0.0039; Idd9.1 vs. Idd9.3 p = 0.063. b): CCR2: Idd9 vs. Idd9.1 p = 0.0003; Idd9.1 vs. Idd9.2 p,0.0001; Idd9.2 vs. Idd9.3 p = 0.0431; Idd9 vs. Idd9.3 p = 0.0183. c): RANTES: Idd9 vs. Idd9.1 p = 0.022; Idd9.1 vs. Idd9.2 p = 0.0016; Idd9.2 vs. Idd9.3 p = 0.0284; Idd9 vs. Idd9.3 p = 0.0382. PDL1 Blockade Reverses Genetic Protection from T1D We chose the NOD Idd congenics (Idd3/5, Idd9, Idd3/10/18 and Idd3/5.2/5.3) that are almost completely protected from spontaneous diabetes occurrence due to allelic interactions between the candidate genes present in the loci and subloci. We also looked at mouse strains that are variations of the above-mentioned strains, containing the individ- ual locus or a combination of loci, where the disease progression is either moderate (Idd3, Idd9.1, Idd5) or relatively high but always lower than that of the NOD parental strain (Idd5.1, Idd5.2, Idd5.3, Idd5.2/5.3, Idd9.1, Idd9.2, Idd9.3, Idd10/18). Frequency of diabetes in these mice strains range from ,5% to 65%. Remarkably, a profound increase of diabetes incidence (3% to 50%) was observed after PDL1 blockade in Idd9 congenic mice. We studied the effect of anti PDL1 on Idd9 subcongenics (Idd9.1, Idd9.2 and Idd9.3). Interestingly, almost 100% of Idd9.1 and Idd9.3 mice developed accelerated diabetes following anti PDL1 treat- ment, in contrast to the Idd9.2 strain, which was partially protected (56% became diabetic). Findings in the Idd9 strain suggest that in mice that already have infiltrating lymphocytes in target organs at 10 weeks of age, like Idd9.1 and Idd9.3 strains, diabetes development is exacerbated following anti PDL1 treatment. Idd9.1 and Idd9.3 strains which develop accelerated diabetes, also show slightly higher scores of insulitis. Idd9.2 mice had significantly less pancreatic infiltrates and only 56% developed diabetes after anti PDL1 treatment. PDL1 blockade is known to accelerate diabetes precipitation in NOD mice. Our aim was to determine if the interaction between the protective Idd loci in the different NOD congenics affects diabetes induction by PDL1 blockade. All except one among the NOD congenic strains tested here, Idd3/5, developed accelerated type 1 diabetes following anti PDL1 treatment. Our data show that PDL1 blockade is not enough to induce accelerated diabetes in the NOD Idd3/5 congenic mice strain that contains alleles for Il2, Ctla4, Slc11a1 and Acadl. The interaction between these alleles is able to protect the mice from diabetes induction by PDL1 blockade. We do not observe this in the case of any other congenic The Idd9 mice strain similar to Idd3/5 and Idd3/10/18, is also resistant to spontaneous diabetes occurrence. However, the level of insulitis is much higher in the Idd9 strain in comparison to that of Idd3/5 and Idd3/10/18. PDL1 Blockade Reverses Genetic Protection from T1D d): No significant differences in FoxP3 expression between Idd9 and subcongenics. e): MIP-1a: Idd9 vs. Idd9.1 p = 0.0257; Idd9.2 vs. Idd9.1 p = 0.0017. f): TNF-a:Idd9 vs. Idd9.1 p = 0.019; Idd9.2 vs. Idd9.1 p = 0.0004. doi:10.1371/journal.pone.0089561.g003 disease in the Idd9 strain is attributed to the Th2 type cellular response induced by the TNFR superfamily gene cluster present in the Idd9 locus. The combination of three sub loci Idd9.1, Idd9.2 and Idd 9.3 renders this strain resistant to the disease even though individually the candidate genes are susceptible. PDL1 blockade negates this interaction and in the absence of any other functional negative costimulatory pathway, disease progression is accelerated and pathogenicity is altered. Among the three subloci of Idd9, Idd9.2 is considered the most potent region in providing protection against the disease by restraining autoreactive CD8+ T cells. Blockade of PDL1 is known to cause CD8+ T cell exhaustion. This explains the similarity in the anti PDL1 induced diabetes incidence (approximately 50%) in the Idd9 and Idd9.2 strains; whereas the Idd9.1 and Idd9.3 strains are completely susceptible (90-95%) to anti PDL1 induced diabetes. We also observe a very low frequency of pancreatic infiltrates in the Idd9.2 strain. This can be explained by the fact that the strains of Idd9 and its sub regions have a low frequency of autoreactive CD8+ T cells in comparison to the NOD mice. Further, the genes in the Idd9 sub regions prevent a massive expansion of these autoreactive CD8+ T cells during disease onset and progression [25]. However, extensive insulitis was observed in the group of Idd9.2 mice that become diabetic after PDL1 blockade suggesting that in some of the mice the low frequency of autoreactive cells can expand when this regulatory pathway is inhibited. The decreased cytokine and chemokine production in Idd9 and Idd9.2 mice may also be related to the low frequency of disease in the Idd9 strain is attributed to the Th2 type cellular response induced by the TNFR superfamily gene cluster present in the Idd9 locus. The combination of three sub loci Idd9.1, Idd9.2 and Idd 9.3 renders this strain resistant to the disease even though individually the candidate genes are susceptible. PDL1 blockade negates this interaction and in the absence of any other functional negative costimulatory pathway, disease progression is accelerated and pathogenicity is altered. February 2014 \| Volume 9 \| Issue 2 \| e89561 PDL1 Blockade Reverses Genetic Protection from T1D Corresponding to CD4+ TCR Tg T cells tested above, CD8+ 8.3 TCR Tg+ T cells divided more frequently, as seen by an increase in the number of CFSE-diluted CD8+ T cells in Idd3/10/ 18 mice (and NOD mice) that received anti PDL1 antibody as compared to mice that received control IgG. These data are similar to our findings in regular NOD mice [4] and suggest that February 2014 \| Volume 9 \| Issue 2 \| e89561 7 PLOS ONE \| www.plosone.org PDL1 Blockade Reverses Genetic Protection from T1D Figure 4. Proliferation of adoptively transferred BDC2.5 Tg CD4 T and NY8.3 tg CD8+ T cells in pancreatic LN and spleen of anti PDL1 treated NOD and Idd3/10/18 mice. A) Representative CFSE dilution plot for each group is shown. Cells were gated on CD4+ Vbeta 4+. B) A representative CFSE dilution plot of transferred NY8.3tg T cells for each group is shown. Cells were gated on CD8+ Vbeta 8+. doi:10.1371/journal.pone.0089561.g004 Figure 4. Proliferation of adoptively transferred BDC2.5 Tg CD4 T and NY8.3 tg CD8+ T cells in pancreatic LN and spleen of anti PDL1 treated NOD and Idd3/10/18 mice. A) Representative CFSE dilution plot for each group is shown. Cells were gated on CD4+ Vbeta 4+. B) A representative CFSE dilution plot of transferred NY8.3tg T cells for each group is shown. Cells were gated on CD8+ Vbeta 8+. doi:10.1371/journal.pone.0089561.g004 Figure 4. Proliferation of adoptively transferred BDC2.5 Tg CD4 T and NY8.3 tg CD8+ T cells in pancreatic LN and spleen of anti PDL1 treated NOD and Idd3/10/18 mice. A) Representative CFSE dilution plot for each group is shown. Cells were gated on CD4+ Vbeta 4+. B) A representative CFSE dilution plot of transferred NY8.3tg T cells for each group is shown. Cells were gated on CD8+ Vbeta 8+. doi:10.1371/journal.pone.0089561.g004 the lower susceptibility of Idd3/10/18 mice to develop type 1 diabetes following PDL1 blockade is probably not dependent on expansion of CD4 and CD8 T cells, rather that anti PDL1 treatment likely affects behavior of these cells which contributes to lowering susceptibility for developing disease. The role of PDL1 expression in the pancreas and its effect on resistance in this strain remains to be further investigated. IL-2 levels correlated with higher numbers of DCs and increased T cell stimulation and activation. The cellular mechanism of protection from T1D in Idd3/5 congenic mice strain is already defined by Hamilton-Williams et al. [9]. PDL1 Blockade Reverses Genetic Protection from T1D Further analysis of the Idd3/5 region showed that removal of protective alleles at a subcongenic region from the Idd3/5 region as in the Idd3/5.2/5.3 strain results in a 15% incidence of disease upon anti PDL1 treatment in contrast to no incidence of disease in the Idd3/5 group, which supports the role of Ctla4 at the Idd5.1 locus in preventing diabetes in a concerted interplay with Idd3. Although Idd3/5.2/5.3 mice do not develop diabetes, an increase of insulitis in Idd3/5.2/5.3 mice as compared to Idd3/5 has been reported [33]. These observations support the hypothesis that the ability of PDL1 to accelerate diabetes relies on some minimal amount of effector cell accumulation that is normally manifested as at least a mild insulitis. Correlation between the level of basal insulitis and the development of anti PDL1 induced diabetes also proved to be true in the case of Idd3/5 mice to which anti PDL1 was administered. These mice stayed diabetes free. The Idd3/5 strain exhibits profound resistance, has the lowest spontaneous diabetes incidence, and also shows the lowest levels of insulitis among all Idd congenics. Prominent genes associated with Idd3/5 congenic strain are Il2 and Il21 from Idd3, and Ctla4 from Idd5 regions [37]. The role of IL-2 in diabetes has been previously demonstrated. NOD mice express less IL-2 than diabetes resistant mouse strains [45], and low dose IL-2 administered at the onset of type 1 diabetes can reverse established disease in NOD mice [46]. This mechanism has been attributed to an increase in regulatory T cell numbers in the pancreas, and to increased expression of FoxP3, CD25, CTLA-4, ICOS and GITR [46]. Lower levels of IL-2 were found to have an impact on antigen presenting cells like DCs, since low Conclusion The PD1-PDL1 pathway is currently studied for developing therapy for cancer and infectious diseases including HIV, since blockade of this pathway results in increased immune responses against tumor cells [1,47,48] and infectious agents [3,49,50]. However, we show that blockade of this pathway interrupts critical tolerance mechanisms that operate to prevent autoimmune diabetes. Acceleration of diabetes following PD1-PDL1 pathway blockade to treat disease underscores the need for caution before proceeding to a widespread use of this form of treatment, especially when used in combination with antiCTLA-4 (Ipilimumab) that is currently approved for use in melanoma. A combined blockade of CTLA4 and PD1-PDL1 will in all probability shift the balance from an effective immune response towards autoimmunity. It is important to note that our group had earlier shown that type 1 diabetes resistant NOR mice, which are congenic for the MHC locus to the responsible to prevent disease susceptibility induced by anti PDL1. Increased understanding of the mechanisms of gene-gene interac- tion, and discovering additional traits that play a role in type 1 diabetes will help identify novel treatments of this disease. The PD1-PDL1 pathway is currently studied for developing therapy for cancer and infectious diseases including HIV, since blockade of this pathway results in increased immune responses against tumor cells [1,47,48] and infectious agents [3,49,50]. However, we show that blockade of this pathway interrupts critical tolerance mechanisms that operate to prevent autoimmune diabetes. Acceleration of diabetes following PD1-PDL1 pathway blockade to treat disease underscores the need for caution before proceeding to a widespread use of this form of treatment, especially when used in combination with antiCTLA-4 (Ipilimumab) that is currently approved for use in melanoma. A combined blockade of CTLA4 and PD1-PDL1 will in all probability shift the balance from an effective immune response towards autoimmunity. It is important to note that our group had earlier shown that type 1 diabetes resistant NOR mice, which are congenic for the MHC locus to the Future research should focus on strategies to exploit enhanced immune responses by blocking the PD-PDL1 pathway and at the same time prevent the development of autoimmune disease as a consequence. Conclusion Taken together, our data show that PDL1 blockade destroys the genetic protection mediated by different protective alleles. We show a link between occurrence of insulitis and disease suscepti- bility through a break of tolerance induced by anti PDL1. We suggest that the presence of a functional CTLA4 allele is probably February 2014 \| Volume 9 \| Issue 2 \| e89561 PLOS ONE \| www.plosone.org 8 PDL1 Blockade Reverses Genetic Protection from T1D Figure 5. Proliferation of adoptively transferred BDC2.5 Tg CD4 T and NY8.3 tg CD8+ T cells in pancreatic LN and spleen of anti PDL1 treated NOD and Idd3/10/18 mice. a) Collective data from 3 out of 8 experiments of the percentage of CFSE-dividing cells (gated on CD4+ Vb4+) are shown. Horizontal lines express mean value. For CD4+ T cells pLN, NOD, control vs. treated p = 0.0276; pLN, Idd3/10/18, control vs. treated p = 0.0002. b) A representative experiment from 3 performed is shown. For CD8 T cells pLN, Idd3/10/18 control vs. treated p = 0.0321, spleen Idd3/10/ 18 control vs. treated p = 0.0185. doi:10.1371/journal.pone.0089561.g005 Figure 5. Proliferation of adoptively transferred BDC2.5 Tg CD4 T and NY8.3 tg CD8+ T cells in pancreatic LN and spleen of anti PDL1 treated NOD and Idd3/10/18 mice. a) Collective data from 3 out of 8 experiments of the percentage of CFSE-dividing cells (gated on CD4+ Vb4+) are shown. Horizontal lines express mean value. For CD4+ T cells pLN, NOD, control vs. treated p = 0.0276; pLN, Idd3/10/18, control vs. treated p = 0.0002. b) A representative experiment from 3 performed is shown. For CD8 T cells pLN, Idd3/10/18 control vs. treated p = 0.0321, spleen Idd3/10/ 18 control vs. treated p = 0.0185. doi:10.1371/journal.pone.0089561.g005 NOD mice, did not develop diabetes following anti PDL1 treatment [51]. The fact that these congenic mice were protected against type 1 diabetes post-anti PDL1 treatment suggests that PDL1 blockade may still prove suitable in human patients without HLA alleles associated with autoimmune disease such as type 1 diabetes. responsible to prevent disease susceptibility induced by anti PDL1. Increased understanding of the mechanisms of gene-gene interac- tion, and discovering additional traits that play a role in type 1 diabetes will help identify novel treatments of this disease. References Proc Natl Acad Sci U S A 105: 19857–19862. 37. Pearson T, Markees TG, Wicker LS, Serreze DV, Peterson LB, et al. (2003) NOD congenic mice genetically protected from autoimmune diabetes remain resistant to transplantation tolerance induction. Diabetes 52: 321–326. 14. Rainbow DB, Moule C, Fraser HI, Clark J, Howlett SK, et al. (2011) Evidence that Cd101 is an autoimmune diabetes gene in nonobese diabetic mice. J Immunol 187: 325–336. 38. Hill NJ, Lyons PA, Armitage N, Todd JA, Wicker LS, et al. (2000) NOD Idd5 locus controls insulitis and diabetes and overlaps the orthologous CTLA4/ IDDM12 and NRAMP1 loci in humans. Diabetes 49: 1744–1747. 15. Fraser HI, Dendrou CA, Healy B, Rainbow DB, Howlett S, et al. (2010) Nonobese diabetic congenic strain analysis of autoimmune diabetes reveals genetic complexity of the Idd18 locus and identifies Vav3 as a candidate gene. J Immunol 184: 5075–5084. 39. Cameron MJ, Arreaza GA, Grattan M, Meagher C, Sharif S, et al. (2000) Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes. J Immunol 165: 1102– 1110. J 16. Hamilton-Williams EE, Martinez X, Clark J, Howlett S, Hunter KM, et al. (2009) Expression of diabetes-associated genes by dendritic cells and CD4 T cells drives the loss of tolerance in nonobese diabetic mice. J Immunol 183: 1533– 1541. 40. Ridgway WM, Peterson LB, Todd JA, Rainbow DB, Healy B, et al. (2008) Gene-gene interactions in the NOD mouse model of type 1 diabetes. Adv Immunol 100: 151–175. 41. Bour-Jordan H, Thompson HL, Giampaolo JR, Davini D, Rosenthal W, et al. Distinct genetic control of autoimmune neuropathy and diabetes in the non- obese diabetic background. J Autoimmun 45: 58–67. 17. Dai YD, Marrero IG, Gros P, Zaghouani H, Wicker LS, et al. (2009) Slc11a1 enhances the autoimmune diabetogenic T-cell response by altering processing and presentation of pancreatic islet antigens. Diabetes 58: 156–164. 42. Chamberlain G, Wallberg M, Rainbow D, Hunter K, Wicker LS, et al. (2006) A 20-Mb region of chromosome 4 controls TNF-alpha-mediated CD8+ T cell aggression toward beta cells in type 1 diabetes. J Immunol 177: 5105–5114. 18. Araki M, Chung D, Liu S, Rainbow DB, Chamberlain G, et al. (2009) Genetic evidence that the differential expression of the ligand-independent isoform of CTLA-4 is the molecular basis of the Idd5.1 type 1 diabetes region in nonobese diabetic mice. J Immunol 183: 5146–5157. 43. References Paterson AM, Brown KE, Keir ME, Vanguri VK, Riella LV, et al. (2011) The programmed death-1 ligand 1:B7-1 pathway restrains diabetogenic effector T cells in vivo. J Immunol 187: 1097–1105. 19. Kissler S, Stern P, Takahashi K, Hunter K, Peterson LB, et al. (2006) In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes. Nat Genet 38: 479–483. 44. Kadri N, Korpos E, Gupta S, Briet C, Lofbom L, et al. (2012) CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes. J Immunol 188: 3138–3149. 20. Irie J, Reck B, Wu Y, Wicker LS, Howlett S, et al. (2008) Genome-wide microarray expression analysis of CD4+ T Cells from nonobese diabetic congenic mice identifies Cd55 (Daf1) and Acadl as candidate genes for type 1 diabetes. J Immunol 180: 1071–1079. 45. Tang Q, Adams JY, Penaranda C, Melli K, Piaggio E, et al. (2008) Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity 28: 687–697. 21. Hamilton-Williams EE, Rainbow DB, Cheung J, Christensen M, Lyons PA, et al. (2013) Fine mapping of type 1 diabetes regions Idd9.1 and Idd9.2 reveals genetic complexity. Mamm Genome 24: 358–375. 46. Grinberg-Bleyer Y, Baeyens A, You S, Elhage R, Fourcade G, et al. (2010) IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells. J Exp Med 207: 1871–1878. 22. Lyons PA, Hancock WW, Denny P, Lord CJ, Hill NJ, et al. (2000) The NOD Idd9 genetic interval influences the pathogenicity of insulitis and contains molecular variants of Cd30, Tnfr2, and Cd137. Immunity 13: 107–115. 47. Zhang L, Gajewski TF, Kline J (2009) PD-1/PD-L1 interactions inhibit antitumor immune responses in a murine acute myeloid leukemia model. Blood 114: 1545–1552. 23. Yamanouchi J, Puertas MC, Verdaguer J, Lyons PA, Rainbow DB, et al. (2010) Idd9.1 locus controls the suppressive activity of FoxP3+CD4+CD25+ regulatory T-cells. Diabetes 59: 272–281. 48. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366: 2443–2454. 24. Matsuki N, Stanic AK, Embers ME, Van Kaer L, Morel L, et al. (2003) Genetic dissection of V alpha 14J alpha 18 natural T cell number and function in autoimmune-prone mice. J Immunol 170: 5429–5437. 49. References (2005) Establishment of NOD-Pdcd1-/- mice as an efficient animal model of type I diabetes. Proc Natl Acad Sci U S A 102: 11823–11828. 7. Driver JP, Serreze DV, Chen YG (2011) Mouse models for the study of autoimmune type 1 diabetes: a NOD to similarities and differences to human disease. Semin Immunopathol 33: 67–87. 32. Hunter K, Rainbow D, Plagnol V, Todd JA, Peterson LB, et al. (2007) Interactions between Idd5.1/Ctla4 and other type 1 diabetes genes. J Immunol 179: 8341–8349. 8. Wicker LS, Todd JA, Peterson LB (1995) Genetic control of autoimmune diabetes in the NOD mouse. Annu Rev Immunol 13: 179–200. 33. Lin X, Hamilton-Williams EE, Rainbow DB, Hunter KM, Dai YD, et al. (2013) Genetic interactions among Idd3, Idd5.1, Idd5.2, and Idd5.3 protective loci in the nonobese diabetic mouse model of type 1 diabetes. J Immunol 190: 3109– 3120. 9. Hamilton-Williams EE, Cheung J, Rainbow DB, Hunter KM, Wicker LS, et al. (2012) Cellular mechanisms of restored beta-cell tolerance mediated by protective alleles of Idd3 and Idd5. Diabetes 61: 166–174. 34. Luhder F, Katz J, Benoist C, Mathis D (1998) Major histocompatibility complex class II molecules can protect from diabetes by positively selecting T cells with additional specificities. J Exp Med 187: 379–387. 10. Liu SM, Lee DH, Sullivan JM, Chung D, Jager A, et al. (2011) Differential IL-21 signaling in APCs leads to disparate Th17 differentiation in diabetes-susceptible NOD and diabetes-resistant NOD.Idd3 mice. J Clin Invest 121: 4303–4310. 35. Yamazaki T, Akiba H, Iwai H, Matsuda H, Aoki M, et al. (2002) Expression of programmed death 1 ligands by murine T cells and APC. J Immunol 169: 5538– 5545. 11. Anderson AC, Chandwaskar R, Lee DH, Kuchroo VK (2008) Cutting edge: the Idd3 genetic interval determines regulatory T cell function through CD11b+ CD11c- APC. J Immunol 181: 7449–7452. 36. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, et al. (2000) B7/ CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12: 431–440. 12. Yamanouchi J, Rainbow D, Serra P, Howlett S, Hunter K, et al. (2007) Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity. Nat Genet 39: 329–337. 13. D’Alise AM, Auyeung V, Feuerer M, Nishio J, Fontenot J, et al. (2008) The defect in T-cell regulation in NOD mice is an effect on the T-cell effectors. References 1. Blank C, Mackensen A (2007) Contribution of the PD-L1/PD-1 pathway to T- cell exhaustion: an update on implications for chronic infections and tumor evasion. Cancer Immunol Immunother 56: 739–745. 26. Cannons JL, Chamberlain G, Howson J, Smink LJ, Todd JA, et al. (2005) Genetic and functional association of the immune signaling molecule 4-1BB (CD137/TNFRSF9) with type 1 diabetes. J Autoimmun 25: 13–20. 2. Zhou Q, Munger ME, Highfill SL, Tolar J, Weigel BJ, et al. Program death-1 signaling and regulatory T cells collaborate to resist the function of adoptively transferred cytotoxic T lymphocytes in advanced acute myeloid leukemia. Blood 116: 2484–2493. 27. Waldner H, Sobel RA, Price N, Kuchroo VK (2006) The autoimmune diabetes locus Idd9 regulates development of type 1 diabetes by affecting the homing of islet-specific T cells. J Immunol 176: 5455–5462. 28. Robles DT, Eisenbarth GS, Dailey NJ, Peterson LB, Wicker LS (2003) Insulin autoantibodies are associated with islet inflammation but not always related to diabetes progression in NOD congenic mice. Diabetes 52: 882–886. 3. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, et al. (2006) Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med 12: 1198–1202. y diabetes progression in NOD congenic mice. Diabetes 52: 882–886 29. Podolin PL, Denny P, Armitage N, Lord CJ, Hill NJ, et al. (1998) Localization of two insulin-dependent diabetes (Idd) genes to the Idd10 region on mouse chromosome 3. Mamm Genome 9: 283–286. 4. Guleria I, Gubbels Bupp M, Dada S, Fife B, Tang Q, et al. (2007) Mechanisms of PDL1-mediated regulation of autoimmune diabetes. Clin Immunol 125: 16– 25. 30. Penha-Goncalves C, Moule C, Smink LJ, Howson J, Gregory S, et al. (2003) Identification of a structurally distinct CD101 molecule encoded in the 950-kb Idd10 region of NOD mice. Diabetes 52: 1551–1556. 5. Fife BT, Pauken KE, Eagar TN, Obu T, Wu J, et al. (2009) Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR-induced stop signal. Nat Immunol 10: 1185–1192. Idd10 region of NOD mice. Diabetes 52: 1551–1556 31. Wicker LS, Chamberlain G, Hunter K, Rainbow D, Howlett S, et al. (2004) Fine mapping, gene content, comparative sequencing, and expression analyses support Ctla4 and Nramp1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse. J Immunol 173: 164–173. 6. Wang J, Yoshida T, Nakaki F, Hiai H, Okazaki T, et al. Author Contributions Conceived and designed the experiments: IG. Performed the experiments: NMK AJK BZ LA. Analyzed the data: NMK ST AJK BZ. Wrote the paper: NMK ST. Reviewed and Edited the manuscript: MHS DLG LSW IG DR AR. Guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis: IG. February 2014 \| Volume 9 \| Issue 2 \| e89561 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9 PDL1 Blockade Reverses Genetic Protection from T1D 51. Ansari MJ, Salama AD, Chitnis T, Smith RN, Yagita H, et al. (2003) The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 198: 63–69. PDL1 Blockade Reverses Genetic Protection from T1D References Freeman GJ, Wherry EJ, Ahmed R, Sharpe AH (2006) Reinvigorating exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade. J Exp Med 203: 2223–2227. 25. Hamilton-Williams EE, Wong SB, Martinez X, Rainbow DB, Hunter KM, et al. (2010) Idd9.2 and Idd9.3 protective alleles function in CD4+ T-cells and nonlymphoid cells to prevent expansion of pathogenic islet-specific CD8+ T- cells. Diabetes 59: 1478–1486. 50. Quigley M, Pereyra F, Nilsson B, Porichis F, Fonseca C, et al. (2010) Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF. Nat Med 16: 1147–1151. February 2014 \| Volume 9 \| Issue 2 \| e89561 PLOS ONE \| www.plosone.org 10 PDL1 Blockade Reverses Genetic Protection from T1D February 2014 \| Volume 9 \| Issue 2 \| e89561 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 11
https://openalex.org/W4205740435	https://www.researchsquare.com/article/rs-1256959/latest.pdf	English	null	A cohort study of the effect of SARS-CoV-2 point of care rapid RT-PCR at the Emergency Department on targeted admission	medRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	10,346	A Cohort Study of the Effect of SARS-CoV-2 Point of Care Rapid RT-PCR at the Emergency Department on Targeted Admission Susanne E Mortazavi Lund University Faculty of Medicine: Lunds universitet Medicinska fakulteten Malin Inghammar Lund University Faculty of Medicine: Lunds universitet Medicinska fakulteten Claus Christansen Region Skåne: Region Skane Anne-Katrine Pesola Region Skåne: Region Skane Mikael Stenkilsson Skånes universitetssjukhus Lund: Skanes universitetssjukhus Lund Magnus Paulsson (  magnus.paulsson@med.lu.se ) Lund University Faculty of Medicine: Lunds universitet Medicinska fakulteten https://orcid.org/0000- 0003-1104-2727 Research Article Keywords: Infectious disease, Virology, Emergency medicine, Hospital infection, Microbiology, Medical error, patient safety, quality of care Posted Date: March 7th, 2022 Susanne E Mortazavi Lund University Faculty of Medicine: Lunds universitet Medicinska Malin Inghammar Lund University Faculty of Medicine: Lunds universitet Medicinska Claus Christansen Region Skåne: Region Skane Anne-Katrine Pesola Region Skåne: Region Skane Mikael Stenkilsson Skånes universitetssjukhus Lund: Skanes universitetssjukhus Lun Magnus Paulsson (  magnus.paulsson@med.lu.se ) Lund University Faculty of Medicine: Lunds universitet Medicinska 0003-1104-2727 A Cohort Study of the Effect of SARS-CoV-2 Point of Care Rapid RT-PCR at the Emergency Department on Targeted Admission Susanne E Mortazavi Lund University Faculty of Medicine: Lunds universitet Medicinska fakulteten Malin Inghammar Lund University Faculty of Medicine: Lunds universitet Medicinska fakulteten Claus Christansen Region Skåne: Region Skane Anne-Katrine Pesola Region Skåne: Region Skane Mikael Stenkilsson Skånes universitetssjukhus Lund: Skanes universitetssjukhus Lund Magnus Paulsson (  magnus.paulsson@med.lu.se ) Lund University Faculty of Medicine: Lunds universitet Medicinska fakulteten https://orcid.org/0000- 0003-1104-2727 Research Article Keywords: Infectious disease, Virology, Emergency medicine, Hospital infection, Microbiology, Medical error, patient safety, quality of care Posted Date: March 7th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1256959/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Article Keywords: Infectious disease, Virology, Emergency medicine, Hospital infection, Microbiology, Medical error, patient safety, quality of care Posted Date: March 7th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1256959/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/24 Page 1/24 Abstract Background: To prevent nosocomial transmission of SARS-CoV-2, infection control measures are implemented for patients with symptoms compatible with COVID-19 until reliable test results are available. This delay targeted admission to the most appropriate ward based on the medical condition. SARS-CoV-2 rapid antigen detection (RAD) tests and point of care (POC) rapid RT-PCR were introduced at emergency departments (EDs) in late 2020, but the consequence on targeted admission is unknown. Methods: Patients presenting at the emergency department of a referral hospital (N = 2,940) between 13- Nov-2020 and 12-Jan-2021 were included. The study period was delimited by introduction of rapid antigen tests and VitaPCR. Participant data was collected retrospectively, and outcome variables were Length-of-Stay (LoS), intrahospital transfers and targeted admission to COVID-19 ward. Results: Rapid antigen tests reduced ED Length-of-Stay for participants with positive tests or that were not tested. Negative VitaPCR results reduced mean hospital Length-of-Stay by 1.5 (95%CI: 0.3–2.7) days and admissions to COVID-19 wards from 34.5 (95%CI: 28.9-40.5) to 14.7 (95%CI: 11.1-19.1) per 100 admissions. Introduction of VitaPCR reduced transfers between hospital wards in the first 5 days from 50.0 (95%CI: 45.0-55.0) to 34.0 (95%CI: 30.3-37.9) per 100 admissions. Conclusion: Rapid antigen tests enabled prompt detection of SARS-CoV-2 infection which had pronounced effects on Length-of-Stay at the emergency department. VitaPCR added the possibility of exclusion of the infection which increased targeted admissions, reduced intrahospital transfers and lead to shorter stay at the hospital. Background The coronavirus disease 2019 (COVID-19) pandemic is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged in China in late 2019 (1). According to WHO, on 7 November 2021 over 248,467,363 global cases and 5,027,183 global deaths have been verified (2). Rapid detection and isolation of infected individuals are important to limit the spread of the virus and to protect patients and health care workers (3). Real-time reverse transcription polymerase chain reaction (RT-PCR) is the gold standard for SARS-CoV-2 detection, due to high sensitivity and specificity compared to other diagnostic methods (4). However, RT-PCR is time consuming and requires specialized laboratory settings, personnel, and instruments. As a less expensive and faster point-of-care test method, SARS-CoV-2 rapid antigen detection (RAD) tests became widely available during the autumn of 2020. However, RAD tests are generally inferior to RT-PCR in terms of sensitivity and specificity, which is particularly important when testing asymptomatic patients with low pretest probability when the main objective is to rule out infection (4-6). In late 2020, the optimized point-of-care (POC) RT-PCR VitaPCR SARS-CoV-2 Assay (Credo Diagnostics Biomedical, Singapore) was introduced and implemented at the Skåne University Hospital, Lund, Sweden. The assay utilizes a single tube for collection of the nasopharyngeal swab, cell lysis and nucleotide Page 2/24 extraction. The total analysis time is about 20 min. Sample preparation does not require specialized laboratory setting and the reported sensitivity and specificity for SARS-CoV-2 is 99,3% and 94,7%, respectively, possibly lower in samples with low viral load (7, 8). During 2020, substantial reorganizations were made at Emergency Departments (ED) and hospital wards globally to cope with the extraordinary requirements caused by the SARS-CoV-2 pandemic, including infection prevention and control (IPC) precautions to prevent secondary cases among patients and hospital staff. The study region was largely affected by the second wave of the pandemic with a considerable increase of COVID-19 cases from 1 Sept 2020 – 31 January 2021 (9). Due to the broad clinical manifestations of COVID-19, infection cannot be safely excluded based on clinical symptoms and signs only (10). Before introduction of RAD tests and VitaPCR, all patients with suspected COVID-19 infection were isolated at the ED or admitted to hospital wards dedicated to patients with positive or unknown COVID-19 status until RT-PCR results from the core hospital facility were available. The typical time from sampling to results ranged between 12 and 24 h. Background In the high prevalence setting during the second wave of the COVID-19 pandemic, a large proportion of the patients at the ED met the definition of suspected COVID-19 and were admitted to COVID-19 isolation wards instead of targeted admission to wards specialized on the true medical problem. If the COVID-19 RT-PCR test was negative, IPC precautions were discontinued, and the patient transferred to another hospital ward for continued treatment. Although the sensitivity and specificity of the VitaPCR is superior to that of any RAD test, and time to result is substantially shorter for VitaPCR than for RT-PCR tests analyzed at the core hospital laboratory, the effects of these improvements on patient care are unknown. We hypothesized that introduction of the faster tests in the algorithm facilitated clinical decisions at the ED, limited IPC precautions to when necessary and improved targeted admission. This study evaluates the introduction of RAD tests and VitaPCR based on length-of-stay at the ED and hospital ward, intrahospital transfers the first five days and targeted admissions to COVID-19 ward during the peak of the second wave. Data sources Data on visits to the ED and hospital ward admissions were collected from the hospital records using key word search. Study setting and design This retrospective observational study is based on data from patients presenting at the Emergency Department, Skåne University Hospital, Lund, Sweden between Nov 13, 2020, and Jan 12, 2021. The hospital is a regional referral center, but the ED primarily serves the population of Lund and near surroundings (population of about 300,000). The total annual ED visits in 2020 were 59,000 patients. The present study was divided into three distinct time periods separated by the dates for introduction of RAD tests and VitaPCR, respectively: Period 1 (Nov 13 to Dec 2), Period 2 (Dec 3 to Dec 22) and Period 3 (Dec 23 to Jan 12). The standard of care from the beginning of the pandemic and Period 1 of the present study was analysis of nasopharyngeal samples for SARS-CoV-2 with RT-PCR at a core hospital laboratory facility (Laboratory medicine Skåne, Region Skåne, Lund, Sweden). On Dec 3, 2020 (Period 2 of this Page 3/24 Page 3/24 study), POC rapid SARS-CoV-2 antigen detection test (ClinitestRT; Siemens Healthineers, Erlangen, Germany) was introduced together with an algorithm to select which analysis method that was to be used. On Dec 23, POC testing with VitaPCR was added to the algorithm. The algorithms are presented in Figure 1. The standard of care for suspect or confirmed patients with COVID-19 were unchanged during the study period and no changes were made concerning routines for hospital admissions. The average weekly COVID-19 incidence rate in Skåne county per 100,000 inhabitants was 355 in Period 1, 623 in Period 2 and 630 in Period 3. Variables SARS-CoV-2 test results and test method were recorded and used for grouping of participants into independent variables: “Positive test at the ED” included participants that were sampled at the ED for a test that became positive for SARS-CoV-2 by RT-PCR, RAD test or VitaPCR, whereas “Negative test at the ED” included participants who tested negative at the ED, regardless of when the result of the test was given. Participants with positive SARS-CoV-2 analyzed before presentation to the ED were not sampled for SARS-CoV-2 and labeled “Positive test before admission”. Finally, “Not tested” included participants not tested for SARS-CoV-2 at the ED and the medical records did not specify COVID-19 status. The ICD-10 diagnoses at discharge from the ED and hospital wards were recorded and aggregated into compound variables (Additional file 1). Data on admission and transfer between wards were recorded and wards were grouped in the compound variables “COVID-19 ward” which were designated wards for patients with suspect or diagnosed COVID-19, “Mixed COVID-19/Internal Medicine ward” which were wards with ICP facilities but not dedicated only for patients with COVID-19, “Intensive care unit” (ICU) or “Other”, which included a broad range of specialized hospital wards. For each subject, sex and age were recorded and presented together with diagnoses as descriptive data. In all analyses, study period 1-3 were considered for exposure variables. Outcome variables were ED Length-of-Stay (LoS), discharge to home from ED, admission to hospital ward, hospital LoS, intrahospital transfers and targeted admissions. Participants All adult patients (≥ 18 years) that presented at the ED during the study period were screened for inclusion in the study. Inclusion criterions were any of the following: known COVID-19 infection at presentation to the ED, treatment in isolation room in the ED, emergency alerts labeled “Infection”, or emergency alerts with main complaints marked as “dyspnea”, “fever”, “infection”, “confusion”, “shock”, “cardiac arrest” and “non-specified illness”. All eligible patients during the study period were included. Variables Statistical analyses Continuous variables with normal distribution are expressed as mean ± standard deviation (SD) or 95% confidence intervals (CI). Categorical variables are presented as counts and percentages. Comparisons Page 4/24 were done by contingency tables with 95% confidence intervals (CI) reported, by One-Way-ANOVA with Tukey’s multiple comparison tests and by Fisher’s exact test. Analyses were performed with GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). P-values < 0.05 were considered statistically significant. Missing data is presented in the tables. Study cohort and participant enrollment 9,325 patients visited the Emergency Department during the study period, 2,940 of these met the inclusion criteria and were selected for enrollment in the study. There was a consecutive increase in the total number of patients that visited the ED and in the proportion that met the inclusion criteria: In Period 1: 781 participants out of 3,024 patient visits (25.8%) met the criteria, in Period 2: 988 participants out of 3,149 patient visits (31.4%), and Period 3: 1,171 participants out of 3,152 patient visits (37.2%). The mean age was 60.8 (SD ±20.8) years and 1497 (50.9%) of the participants were women. SARS-CoV-19 testing and test results During the study period, a total of 1,866 (63.5%) participants were tested for COVID-19 at the ED. There was no significant difference in testing percentage between men and women (48.3% vs 51.7%). The mean age among participants that were tested for SARS-CoV-2 was 64.1 (SD ± 20.0) years and the mean age among those not tested was 55.1 (SD ± 21.0) years. As the study periods were defined by changes in testing routines, substantial differences in SARS-CoV-2 analysis methods were observed between the periods. Samples were analyzed with more than one method for 318 of the 2,940 participants (10.8%). The most common combination was RAD and RT-PCR (n = 186, 6.3%), followed by RAD and VitaPCR (n = 75, 2.6%), which is consistent with the testing algorithms in use during Period 2 respectively Period 3 for patients with negative RAD test but high risk of COVID-19 (Figure 1). After introduction of the RAD test and VitaPCR, there was a significant decrease in RT-PCR test analyses, but this method was still used in 9.5% (n = 111) of the 1,171 participants in Period 3 (Table 1). SARS-CoV-19 testing and test results Page 5/24 Page 5/24 Table 1 SARS-CoV-19 testing data Period 1 (N = 781) Period 2 (N = 988) Period 3 (N = 1171) Total (N = 2940) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Analysis method RT-PCR 443 56.7 (53.2 - 60.2) 298 30.2 (27.4 - 33.1) 111 9.5 (7.9 - 11.3) 852 29.0 (27.4 - 30.6) RAD test 1 0.1 (0.007 - 0.7) 506 51.2 (48.1 - 54.3) 395 33.7 (31.1 - 36.5) 902 30.7 (29.0 - 32.4) VitaPCR 0 0.0 (0.0 - 0.5) 4 0.4 (0.2 - 1.0) 435 37.1 (34.4 - 40.0) 439 14.9 (13.7 - 16.3) Total 444 56.9 (53.4 - 60.3) 650 65.8 (62.8 - 68.7) 772 65.9 (63.2 - 68.6) 1866 63.5 (61.7 - 65.2) Participants tested with multiple methods Antigen and RT-PCR 0 0.0 (0.0 - 0.5) 152 15.4 (13.3 - 17.8) 34 2.9 (2.1 - 4.0) 186 6.3 (5.5 - 7.3) Antigen and Vita-PCR 0 0.0 (0.0 - 0.5) 0 0.0 (0.0 - 0.4) 75 6.4 (5.1 - 8.0) 75 2.6 (2.0 - 3.2) VitaPCR and RT-PCR 0 0.0 (0.0 - 0.5) 2 0.2 (0.04 - 0.7) 44 3.8 (2.8 - 5.0) 46 1.6 (1.2 - 2.1) RAD, VitaPCR and RT-PCR 0 0.0 (0.0 - 0.5) 2 0.2 (0.04 - 0.7) 9 0.8 (0.4 - 1.5) 11 0.4 (0.2 - 0.7) (Table legend) SARS-CoV-2 analysis methods used at the Emergency department. The use of Real-time polymerase chain reaction (RT-PCR) at the core laboratory decreased significantly between each study period as the point of care rapid antigen detection (RAD) test and point of care rapid RT-PCR VitaPCR were introduced in Period 2 and 3 respectively. Number of tests are presented with percentages (%) of total participants in each period and 95% confidence intervals (CI). (Table legend) SARS-CoV-2 analysis methods used at the Emergency department. The use of Real-time polymerase chain reaction (RT-PCR) at the core laboratory decreased significantly between each study period as the point of care rapid antigen detection (RAD) test and point of care rapid RT-PCR VitaPCR were introduced in Period 2 and 3 respectively. Number of tests are presented with percentages (%) of total participants in each period and 95% confidence intervals (CI). (Table legend) SARS-CoV-2 analysis methods used at the Emergency department. SARS-CoV-19 testing and test results The use of Real-time polymerase chain reaction (RT-PCR) at the core laboratory decreased significantly between each study period as the point of care rapid antigen detection (RAD) test and point of care rapid RT-PCR VitaPCR were introduced in Period 2 and 3 respectively. Number of tests are presented with percentages (%) of total participants in each period and 95% confidence intervals (CI). (Table legend) SARS-CoV-2 analysis methods used at the Emergency department. The use of Real-time polymerase chain reaction (RT-PCR) at the core laboratory decreased significantly between each study period as the point of care rapid antigen detection (RAD) test and point of care rapid RT-PCR VitaPCR were introduced in Period 2 and 3 respectively. Number of tests are presented with percentages (%) of total participants in each period and 95% confidence intervals (CI). Page 6/24 Of the 2,193 tests that were analyzed, 449 were positive (20.5%). The proportion of positive tests increased significantly during the study period (Period 1: 70 positive of 443 tested, 15.8%, 95% CI 12.7 – 19.4%; Period 2: 156 positive of 808 tested, 19.3%, 95% CI 16.7 – 22.2%; Period 3: 222 positive of 941 tested, 23.6%, 95% CI 21.0 – 26.4%). Of the 2,940 participants, 408 (13.9%) tested positive for SARS-CoV- 2 infection at the ED, 1,458 (49.6%) tested negative, 568 (19%) had had a positive test before admission to the ED, and 506 (17.2%) were not tested for SARS-CoV-2 infection at the ED (Table 2). Of the 2,193 tests that were analyzed, 449 were positive (20.5%). The proportion of positive tests increased significantly during the study period (Period 1: 70 positive of 443 tested, 15.8%, 95% CI 12.7 – 19.4%; Period 2: 156 positive of 808 tested, 19.3%, 95% CI 16.7 – 22.2%; Period 3: 222 positive of 941 tested, 23.6%, 95% CI 21.0 – 26.4%). Of the 2,940 participants, 408 (13.9%) tested positive for SARS-CoV- 2 infection at the ED, 1,458 (49.6%) tested negative, 568 (19%) had had a positive test before admission to the ED, and 506 (17.2%) were not tested for SARS-CoV-2 infection at the ED (Table 2). SARS-CoV-19 testing and test results Page 7/24 Table 2 Participant characteristics at the ED Period 1 (N = 781) Period 2 (N = 988) Period 3 (N = 1171) P - value Total (N = 2940) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Demographic data of study participants Age (mean (±SD)) 61 (±22) 66 (±18) 61 (±20) P = 0.04 61 (±21) Female sex 417 46.6 (43.1 - 50.1) 489 50.5 (47.4 - 53.6) 591 49.5 (46.6 - 52.3) P = 0.27 1497 50.9 (49.1 - 52.7) SARS-CoV-2 test results Positive test at the ED 70 9.0 (7.2 - 11.2) 135 13.7 (11.7 - 15.9) 203 17.3 (15.3 - 19.6) 408 13.9 (12.7 - 15.2) Negative test at the ED 374 47.9 (44.4 - 51.4) 515 52.1 (49.0 - 55.2) 569 48.6 (45.7 - 51.5) 1458 49.6 (47.8 - 51.4) Positive test before admission 92 11.8 (9.7 - 14.2) 193 19.5 (17.2 - 22.1) 283 24.2 (21.8 - 26.7) 568 19.3 (17.9 - 20.8) Not tested 245 31.4 (28.2 - 34.7) 145 14.7 (12.6 - 17.0) 116 9.9 (8.3 - 11.8) 506 17.2 (15.9 - 18.6) ICD-10 Diagnosis at discharge from ED Covid-19 53 6.8 (5.2 – 8.85) 146 14.8 (12.7 – 17.1) 229 19.6 (17.4 – 21.9) 428 14.6 (13.3 - 15.9) Respiratory symptoms 109 14.0 (11.7 - ) 163 16.5 (14.3 - ) 143 12.2 (10.5 - ) 415 14.1 (12.9 Page 8/24 Period 1 (N = 781) Period 2 (N = 988) Period 3 (N = 1171) P - value Total (N = 2940) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Other infection 103 13.3 (11.0 - 15.7) 107 10.8 (9.0 – 12. SARS-CoV-19 testing and test results The (Table legend) Characteristics of all study participants at the Emergency Department (ED). The ( g ) y p p g y p ( ) percentage of participants with positive SARS-CoV-2 tests taken at the ED or before admission to the ED increased during the study period. ICD-10 diagnoses were grouped into compound variables (described in detail in Supplemental figure 1). COVID-19 diagnose increased, but no changes could be seen for other diagnoses. Total number of participants in each period (N=) and in each subgroup (n=) are presented with percentage of N= and 95% confidence interval (CI), or mean value with standard deviation (SD), as indicated. P-values calculated by One-Way-ANOVA with Tukey’s multiple comparison tests. SARS-CoV-19 testing and test results 9) 106 9.1 (7.5 - 10.8) 316 10.7 (9.7 - 11.9) Chest pain or heart disease 95 12.2 (10.1 - 14.6) 95 9.6 (7.9 - 11.6) 107 9.1 (7.6 - 10.9) 297 10.1 (9.1 - 11.2) Abdominal pain or GI symptoms 93 11.9 (9.8 - 14.4) 78 7.9 (6.4 - 9.7) 102 8.7 (7.2 - 10.5) 273 9.3 (8.3 - 10.4) Other Internal Medicine 58 7.4 (5.8 - 9.5) 54 5.5 (4.2 - 7.1) 78 6.77 (5.4 - 8.2) 190 6.5 (5.6 - 7.4) Neurological deficit or symptoms 53 6.4 (5.2 - 8.8) 48 4.9 (3.7 - 6.4) 59 5.0 (3.9 - 6.4) 160 5.4 (4.7 - 6.3) Other respiratory tract infection 35 4.5 (3.2 - 6.2) 30 3.0 (2.1 -4.3) 41 3.5 (2.6 - 4.7) 106 3.6 (3.0 - 4.3) Trauma 25 3.2 (2.2 - 4.7) 34 3.4 (2.5 - 4.8) 29 2.5 (1.7 - 3.5) 88 3.0 (2.4 - 3.7) Other Orthopedic diagnose 20 2.6 (1.7 - 3.9) 16 1.6 (1.0 -2.6) 34 2.9 (2.1 - 4.0) 70 2.4 (1.9 - 3.0) Other surgery or urology 20 2.6 (1.7 - 3.9) 10 1.0 (1.9 -0.6) 17 1.5 (0.9 - 2.3) 47 1.6 (1.2 - 2.1) No diagnosis 91 11.7 (9.6 - 14.1) 173 17.5 (15.3 - 20.0) 180 15.4 (13.4 - 17.6) 444 15.1 (13.9 - 16.4) Other 26 3.3 (2.3 - 4.8) 34 3.4 (2.5 - 4.8) 46 3.9 (3.0 - 5.2) 106 3.6 (3.0 - 4.3) Discharge from ED Discharge to home from ED, total 395 50.6 (47.1 - 54.1) 478 48.4 (45.3 - 51.5) 580 49.5 (46.7 - 52.4) 1453 49.4 (47.6 - 51 2) Page 9/24 Period 1 (N = 781) Period 2 (N = 988) Period 3 (N = 1171) P - value Total (N = 2940) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Positive test at the ED 20 2.6 (1.7 - 3.9) 50 5.1 (3.9 - 6.6) 81 6.9 (5.6 - 8.5) 151 5.1 (4.4 - 6.0) Negative test at the ED 119 15.2 (12.9 - 17.9) 207 21.0 (18.5 - 23.6) 268 22.9 (20.6 - 25.4) 594 20.2 (18.8 - 21.7) Positive test before admission 51 6.5 (5.0 - 8.5) 104 10.5 (8.8 - 12.6) 136 11.6 (9.9 - 13.6) 291 9.9 (8.9 - 11.0) Not tested 205 26.2 (23.3 - 29.4) 117 11.8 (10.0 - 14.0) 95 8.1 (6.7 - 9.8) 417 14.2 (13.0 - 15.5) Hospital admissions, total 386 49.4 (45.9 - 52.9) 510 51.6 (48.5 - 54.7) 591 50.5 (47.6 - 53.3) 1487 50.6 (48.8 - 52.4) Positive test at the ED 50 6.4 (4.9 - 8.3) 85 8.6 (7.0 - 10.5) 122 10.4 (8.8 - 12.3) 257 8.7 (7.8 - 9.8) Negative test at the ED 255 32.7 (29.5 - 36.0) 308 31.2 (28.4 - 34.1) 301 25.7 (23.3 - 28.3) 864 29.4 (27.8 - 31.1) Positive test before admission 41 5.2 (3.9 - 7.0) 89 9.0 (7.4 - 11.0) 147 12.6 (10.8 - 14.6) 277 9.4 (8.4 - 10.5) Not tested 40 5.1 (3.8 - 6.9) 28 3.6 (2.5 - 5.1) 21 1.8 (1.2 - 2.7) 89 3.0 (2.5 - 3.7) ICD-10 diagnosis, death at ED or Page 10/24 Period 1 (N = 781) Period 2 (N = 988) Period 3 (N = 1171) P - value Total (N = 2940) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Cardiovascular disease 15 1.9 (1.2 - 3.1) 6 0.6 (0.3 - 1.3) 17 1.5 (0.9 - 2.3) 38 1.3 (0.9 - 1.8) Neurological disease 7 0.9 (0.4 - 1.8) 2 0.2 (0.04 - 0.7) 9 0.8 (0.4 - 1.5) 18 0.6 (0.4 - 1.0) Malignancy 3 0.4 (0.1 - 1.1) 5 0.5 (0.2 - 1.2) 3 0.3 (0.1 - 0.8) 11 0.4 (0.2 - 0.7) Other 3 0.4 (0.1 - 1.1) 8 0.8 (0.4 - 1.6) 1 0.1 (0.004 - 0.5) 12 0.4 (0.2 - 0.7) Hospital deaths, total 44 5.6 (4.2 - 7.5) 61 6.2 (4.8 - 7.9) 99 8.5 (7.0 - 10.2) 204 6.9 (6.1 - 7.9) (Table legend) Characteristics of all study participants at the Emergency Department (ED). Emergency Department Length-of-Stay The mean Length-of-Stay (LoS) at the ED was 374 (SD ± 269) minutes (Table 3). This did not change significantly between the periods, but we observed a significant reduction in ED LoS between the three periods for participants with “Positive test at the ED” (P = 0.0002) or “Not tested” participants (P < 0.0001). Mean ED LoS for participants with “Positive test at the ED” decreased with 28 minutes (95% CI: 3.0 - 53.0) (P = 0.02) after introduction of RAD test, and another 15 min (95% CI: -7.6 - 37.6) after introduction of the VitaPCR. Participants that were “Not tested” for SARS-CoV-2 at the ED had a LoS at the ED that was reduced by 102 min (95% CI: 76.3 - 127.7) after introduction of the RAD test (P < 0.0001), which further decreased slightly (10 min; (95% CI: -33.16 - 13.16)) in Period 3. Page 11/24 Table 3 Length-of-Stay Period 1 (N = 781) Period 2 (N = 988) Period 3 (N = 1171) P - value Total (N = 2940) ED Length of Stay (min (±SD)) All study participants (mean (±SD)) 383 (±263) 377 (±271) 363 (±263) P = 0.22 374 (±269) Positive test at the ED 393 (±237) 365 (±209) 350 (±225) P = 0.0002 362 (±222) Negative test at the ED 430 (±276) 442 (±307) 423 (±278) P = 0.31 431 (±288) Positive test before admission 296 (±219) 313 (±201) 297 (±228) P = 0.15 302 (±218) Not tested 345 (±251) 243 (±183) 253 (±247) P < 0.0001 295 (±237) Hospital Length of Stay (mean days (±SD)) Period 1 (N = 386) Period 2 (N = 510) Period 3 (N = 591) P - value Total (N =1487) Positive test at the ED 8.5 (± 7.4) 9.3 (± 9.1) 8.0 (± 11) P = 0.08 8.6 (± 11.0) Negative test at the ED 6.6 (± 8.2) 5.8 (± 6.9) 5.1 (± 8) P = 0.01 5.6 (± 8) Positive test before admission 1.4 (± 3.0) 2.7 (± 5.8) 5.9 (± 34.8) P =0.004 3.0 (± 17.3) Not tested 8.2 (± 29.1) 6.1 (± 12.5) 5.8 (± 9.2) P = 0.09 6.7 (± 12.7) All participants 5.0 (± 11.9) 6.0 (± 8.8) 6.7 (± 14.7) P = 0.1 6.1 (± 12.2) (Table legend) Length of stay (LoS) in each study period at the Emergency department (ED) and total hospital stay. Emergency Department Length-of-Stay Numbers indicate mean minutes and standard deviation (SD) for the ED and mean days and SD for hospital LoS. P-value calculated by Fisher’s exact test. Emergency department discharge to home and hospital wards COVID-19 diagnoses at discharge from the ED increased consecutively during the three study periods, from 6.8% in Period 1 (95% CI 5.2% - 8.85%), to 14.8% (95% CI: 12.7 -17.1) in Period 2 and 19.6% (95% CI: Emergency department discharge to home and hospital wards COVID-19 diagnoses at discharge from the ED increased consecutively during the three study periods, from 6.8% in Period 1 (95% CI 5.2% - 8.85%), to 14.8% (95% CI: 12.7 -17.1) in Period 2 and 19.6% (95% CI: 17.4 -21.9) in Period 3. No statistically significant changes were seen for other diagnoses. Of the 2,940 participants 1 487 (50 6%) were admitted to a hospital ward and 1 453 (49 4%) discharged to home The Emergency department discharge to home and hospital wards COVID-19 diagnoses at discharge from the ED increased consecutively during the three study periods, Emergency department discharge to home and hospital wards COVID-19 diagnoses at discharge from the ED increased consecutively during the three study periods, from 6.8% in Period 1 (95% CI 5.2% - 8.85%), to 14.8% (95% CI: 12.7 -17.1) in Period 2 and 19.6% (95% CI: 17.4 -21.9) in Period 3. No statistically significant changes were seen for other diagnoses. Of the 2,940 participants, 1,487 (50.6%) were admitted to a hospital ward and 1,453 (49.4%) discharged to home. The percentage of participants that were discharged to home did not change notably during the study period. Page 12/24 Page 12/24 Page 12/24 However, after introduction of RAD tests and VitaPCR, there was an increase of patients discharged to home from the ED for participants with either positive or negative test result at the ED or with a positive test before admission to the ED (Table 2). In Period 3, 47.1% of participants with a “Negative test at the ED” were discharged to home, an increase from 31.8% in Period 1 (P < 0.001) and 40.2% in Period 2 (P = 0.02). Similarly, 37.0% of participants with a “Positive test at the ED” in Period 2 and 39.9% in Period 3 were discharged to home, compared to 28.6% in Period 1 (P = 0.65 and P = 0.11, respectively). Emergency Department Length-of-Stay Conversely, the percentage of participants with “Positive test at the ED” of all hospital admissions increased from 6.4% (95% CI 4.9% - 8.3%) in Period 1, to 8.6% (95% CI 7.0% - 10.5%) in Period 2, to 10.4% (95% 8.8% - 12.3%) in Period 3 Hospital admissions and Length-of-Stay We hypothesized that the total LoS for patients admitted to wards would be shortened by introduction of faster and more accurate testing methods that could safely exclude SARS-CoV-2 infection at the ED. The mean LoS during the entire study period was 6.1 (SD ± 11.0) days (Table 3). This increased slightly during the study period from 5.0 days in Period 1, 6.0 days in Period 2 to 6.7 days in Period 3 (P = 0.11). This increase was pronounced among participants that had a “Positive test before admission” to the ED for whom the mean hospital LoS increased from 1.4 (SD ± 3.0) days in Period 1 to 2.7 (SD ± 5.8) days in Period 2 and 5.9 (SD ± 34.8) (P < 0.004) days in Period 3. In contrast, there was a significant reduction in mean hospital LoS between the three periods for participants who had a “Negative test at the ED”. Introduction of the RAD test coincided with a reduction from 6.6 to 5.8 days (P = 0.27), and introduction of VitaPCR coincided with a further reduction to 5.1 days (Period 1 vs Period 3: P = 0.008; Period 2 vs Period 3: P = 0.046). Emergency Department Length-of-Stay However, after introduction of RAD tests and VitaPCR, there was an increase of patients discharged to home from the ED for participants with either positive or negative test result at the ED or with a positive test before admission to the ED (Table 2). In Period 3, 47.1% of participants with a “Negative test at the ED” were discharged to home, an increase from 31.8% in Period 1 (P < 0.001) and 40.2% in Period 2 (P = 0.02). Similarly, 37.0% of participants with a “Positive test at the ED” in Period 2 and 39.9% in Period 3 were discharged to home, compared to 28.6% in Period 1 (P = 0.65 and P = 0.11, respectively). Meanwhile, the percentage of hospital ward admissions of participants with “Negative test at the ED” was significantly reduced after introduction of the VitaPCR (Period 1: 32.7% (95% CI: 29.5% - 36.0%); Period 2: 31.2% (CI 95% 28.4% - 34.1%); Period 3: 25.7% (CI 95% 23.3% - 28.3%). Conversely, the percentage of participants with “Positive test at the ED” of all hospital admissions increased from 6.4% (95% CI 4.9% - 8.3%) in Period 1, to 8.6% (95% CI 7.0% - 10.5%) in Period 2, to 10.4% (95% 8.8% - 12.3%) in Period 3 were discharged to home, compared to 28.6% in Period 1 (P 0.65 and P 0.11, respectively). Meanwhile, the percentage of hospital ward admissions of participants with “Negative test at the ED” was significantly reduced after introduction of the VitaPCR (Period 1: 32.7% (95% CI: 29.5% - 36.0%); Period 2: 31.2% (CI 95% 28.4% - 34.1%); Period 3: 25.7% (CI 95% 23.3% - 28.3%). Conversely, the percentage of participants with “Positive test at the ED” of all hospital admissions increased from 6.4% (95% CI 4.9% - 8.3%) in Period 1, to 8.6% (95% CI 7.0% - 10.5%) in Period 2, to 10.4% (95% 8.8% - 12.3%) in Period 3 Meanwhile, the percentage of hospital ward admissions of participants with “Negative test at the ED” was significantly reduced after introduction of the VitaPCR (Period 1: 32.7% (95% CI: 29.5% - 36.0%); Period 2: 31.2% (CI 95% 28.4% - 34.1%); Period 3: 25.7% (CI 95% 23.3% - 28.3%). Targeted admission and intrahospital transfers The local routine was to discharge patients with suspect or confirmed COVID-19 from the ED to wards with facilities for IPC until test results. Patients with symptoms compatible with COVID-19 were transferred to other wards if the RT-PCR test was negative. However, negative RAD tests were not sufficient for termination of IPC measures in cases with high suspicion of COVID-19 as the accuracy of the test was unsatisfactory. While the trend for total hospital LoS was an increase, SARS-CoV-2 negative patients had a significant 1.5-day reduction in LoS after introduction of both tests. Hence, we hypothesized that introduction of the VitaPCR would increase targeted admission of participants with “Negative test at the ED”. Targeted admission and intrahospital transfers Intrahospital transfers between wards the first 5 days after admission from the Emergency Department (ED) decreased after introduction of SARS-CoV-2 rapid antigen detection test (Period 2) and further with the introduction of the point of care rapid RT-PCR VitaPCR (Period 3). Hospital length of stay from admission at the ED to discharge to home from the hospital ward increased, particularly for patients with positive test before admission to the ED. Conversely, negative test at the ED coincided with a shorter length of stay in the latter two periods. Total number of participants in each period (N=) and in each subgroup (n=) are presented with percentage of N= and 95% confidence interval (CI), or mean value with standard deviation (SD), as indicated. P-values calculated by One-Way-ANOVA with Tukey’s multiple comparison tests. (Table legend) The proportion of participants that were admitted different types of wards remained stable throughout the study period. Intrahospital transfers between wards the first 5 days after admission from the Emergency Department (ED) decreased after introduction of SARS-CoV-2 rapid antigen detection test (Period 2) and further with the introduction of the point of care rapid RT-PCR VitaPCR (Period 3). Hospital length of stay from admission at the ED to discharge to home from the hospital ward increased, The proportion of participants that were admitted to COVID-19 wards, Mixed COVID-19/Internal medicine wards, ICU or Other wards did not vary significantly during the study period (Table 4). However, introduction of the algorithms including RAD test and VitaPCR were associated with a significant 32.0% decline in intrahospital transfers the first 5 days after admission (P < 0.0001). The number of participants with a “Negative test at the ED” that were transferred to another ward during the first 5 days of hospital admission decreased from 128 participants out of 386 (33.2%; 95% CI: 28.7% - 38.0%)) in Period 1, 122 participants out of 510 (23.9% (95% CI: 20.4% - 27.8%)) in Period 2 to 94 participants out of 591 (15.9% (95% CI: 13.2% - 19.1%)) in Period 3, a 52.1% reduction (Figure 2). There were no significant changes in intrahospital transfers between the periods for participants with positive tests or participants that were not tested. Targeted admission and intrahospital transfers Page 13/24 Table 4 Descriptive and outcome data at hospital wards Period 1 (N = 386) Period 2 (N = 510) Period 3 (N = 591) Total (N =1487) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Admissions to ward Covid-19 ward 166 43 (38.2 - 48) 229 44.9 (40.6 - 49.2) 274 46.4 (42.4 - 50.4) 669 45 (42.5 - 47.5) Mixed Covid- 19/Internal Medicine ward 86 22.3 (18.4 - 26.7) 91 17.8 (14.8 - 21.4) 98 16.6 (13.8 - 19.8) 275 18.5 (29.8 - 34.6) ICU 10 2.6 (1.4 - 4.7) 7 1.4 (0.7 - 2.8) 6 1 (0.5 - 2.2) 23 1.5 (1 - 2.3) Other 115 29.8 (25.4 - 34.5) 166 32.5 (28.6 - 36.7) 197 33.3 (29.7 - 37.2) 478 32.1 (29.8 - 34.6) Missing data 9 2.3 (1.2 - 4.4) 17 3.3 (2.1 - 5.3) 16 2.7 (1.7 - 4.4) 42 2.8 (2.1 - 3.8) Total 386 100.0 (99.0 - 100.0) 510 100.0 (99.3 - 100.0) 591 100.0 (99.4 - 100.0) 1487 100.0 (99.7 - 100.0) Intrahospital transfers first 5 days Positive test at the ED 35 9.1 (6.6 - 12.3) 45 8.8 (6.7 - 11.6) 55 9.3 (7.2 - 11.9) 135 9.1 (7.7 - 10.6) Negative test at the ED 128 33.2 (28.7 - 38.0) 122 23.9 (20.4 - 27.8) 94 15.9 (13.2 - 19.1) 344 23.1 (21.1 - 25.3) Positive test before admission 25 6.5 (4.4 - 9.4) 27 5.3 (3.7 - 7.6) 49 8.3 (6.3 - 10.8) 101 6.8 (5.6 - 8.2) Not tested 3 0.8 (0.2 - 2.3) 11 2.2 (1.2 - 3.8) 3 0.5 (0.1 - 1.5) 17 1.1 (0.7 - 1.8) Total 191 50.0 (45.0 - 55.0) 205 40.2 (36.0 - 44.5) 201 34.0 (30.3 - 37.9) 597 40.1 (37.7 - 42.7) Page 14/24 Page 14/24 Period 1 (N = 386) Period 2 (N = 510) Period 3 (N = 591) Total (N =1487) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) ICD-10 Diagnosis on discharge Covid-19 81 21.0 (17.2 - 25.3) 161 31.5 (27.6 - 35.7) 241 40.8 (37.0 - 44.9) 298 32.5 (30.1 - 34.9) Other infection 80 20.7 (25.0 - 25.0) 80 15.7 (12.8 - 19.1) 56 9.5 (7.4 - 12.1) 208 14.5 (12.8 - 16.4) Chest pain or heart disease 43 11.1 (8.4 - 14.7) 52 10.2 (7.8 - 13.1) 48 8.1 (6.2 - 10.6) 144 9.6 (8.2 - 11.2) Other respiratory tract infection 38 9.8 (7.3 - 13.2) 50 9.8 (7.5 - 12.7) 49 8.3 (6.3 - 10.8) 136 9.2 (7.8 - 10.8) Surgery or Urology 33 8.5 (6.2 - 11.8) 31 6.1 (4.3 - 8.5) 48 8.1 (6.2 - 10.6) 102 7.5 (6.3 - 9.0) Other Internal Medicine 31 8.0 (5.7 - 11.2) 29 5.7 (4.0 - 8.0) 38 6.4 (4.7 - 8.7) 93 6.6 (5.4 - 8.0) Neurological deficit or symptoms 25 6.5 (4.4 - 9.4) 28 5.5 (3.8 - 7.8) 33 5.6 (4.0 - 7.8) 75 5.8 (4.7 - 7.1) Respiratory symptoms 15 3.9 (2.4 - 6.3) 23 4.5 (3.0 - 6.7) 22 3.7 (2.5 - 5.6) 52 4.0 (3.1 - 5.2) Malignancy 15 3.9 (2.4 - 6.3) 12 2.3 (1.3 - 4.1) 14 2.4 (1.4 - 3.9) 38 2.8 (2.0 - 3.7) Trauma 7 1.8 (0.9 - 3.7) 13 2.5 (1.5 - 4.3) 11 1.9 (1.0 - 3.3) 26 2.1 (1.5 - 2.9) Abdominal pain and GI symptoms 6 1.6 (0.7 - 3.3) 9 1.8 (0.9 - 3.3) 6 1.0 (0.5 - 2.2) 17 1.4 (0.9 - 2.1) Other Orthopedic 0 0.0 (1 - ) 1 0.2 (0.01 ) 2 0.3 (0.1 - ) 13 0.2 ( Page 15/24 Page 15/24 Period 1 (N = 386) Period 2 (N = 510) Period 3 (N = 591) Total (N =1487) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) No diagnosis 9 2.3 (1.2 - 4.4) 11 2.2 (1.2 - 3.8) 12 2.0 (1.2 - 3.5) 30 2.2 (1.5 - 3.0) Other 3 0.8 (0.2 - 2.3) 11 2.2 (1.2 - 3.8) 10 1.7 (0.9 - 3.1) 14 1.6 (1.1 - 2.4) (Table legend) The proportion of participants that were admitted different types of wards remained stable throughout the study period. Targeted admission and intrahospital transfers Intrahospital transfers between wards the first 5 days after admission from the Emergency Department (ED) decreased after introduction of SARS-CoV-2 rapid antigen detection test (Period 2) and further with the introduction of the point of care rapid RT-PCR VitaPCR (Period 3). Hospital length of stay from admission at the ED to discharge to home from the hospital ward increased, particularly for patients with positive test before admission to the ED. Conversely, negative test at the ED coincided with a shorter length of stay in the latter two periods. Total number of participants in each period (N=) and in each subgroup (n=) are presented with percentage of N= and 95% confidence interval (CI), or mean value with standard deviation (SD), as indicated. P-values calculated by One-Way-ANOVA with Tukey’s multiple comparison tests Period 1 (N = 386) Period 2 (N = 510) Period 3 (N = 591) Total (N =1487) n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) No diagnosis 9 2.3 (1.2 - 4.4) 11 2.2 (1.2 - 3.8) 12 2.0 (1.2 - 3.5) 30 2.2 (1.5 - 3.0) Other 3 0.8 (0.2 - 2.3) 11 2.2 (1.2 - 3.8) 10 1.7 (0.9 - 3.1) 14 1.6 (1.1 - 2.4) ( bl l d) h f h d d d ff f d d bl (Table legend) The proportion of participants that were admitted different types of wards remained stable throughout the study period. Intrahospital transfers between wards the first 5 days after admission from the Emergency Department (ED) decreased after introduction of SARS-CoV-2 rapid antigen detection test (Period 2) and further with the introduction of the point of care rapid RT-PCR VitaPCR (Period 3). Hospital length of stay from admission at the ED to discharge to home from the hospital ward increased, particularly for patients with positive test before admission to the ED. Conversely, negative test at the ED coincided with a shorter length of stay in the latter two periods. Total number of participants in each period (N=) and in each subgroup (n=) are presented with percentage of N= and 95% confidence interval (CI), or mean value with standard deviation (SD), as indicated. P-values calculated by One-Way-ANOVA with Tukey’s multiple comparison tests. (Table legend) The proportion of participants that were admitted different types of wards remained stable throughout the study period. Targeted admission and intrahospital transfers Similarly, participants with “Negative test at the ED”, were 57% less likely to be admitted to a COVID-19 ward and 81% more likely to be admitted to a targeted hospital ward after the introduction of both the RAD test and VitaPCR (Table 5). For participants with positive tests before or at the ED, or that were not tested, no significant changes were seen during the study period. Targeted admission and intrahospital transfers However, test-negative patients were increasingly admitted to appropriate specialized hospital wards (“Other”) during Period 2 and 3. Total number of participants in each period (N=) and in each subgroup (n=) are presented with percentage of N= and 95% confidence interval (CI). Period 1 Period 2 Period 3 Total n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Missing data 2 5.0 (0.9 - 16.5) 1 3.6 (0.2 - 17.7) 1 4.8 (0.2 - 22.7) 4 4.5 (1.8 - 11.0) (Table legend) Targeted admission of patients with positive test or suspect SARS-CoV-2 infection to a COVID-19 ward or other ward with infection prevention control (IPC) facilities did not change by the introduction of rapid point of care tests (Period 2: Rapid antigen detection test; Period 3: rapid RT-PCR VitaPCR) at the Emergency Department (ED). However, test-negative patients were increasingly admitted to appropriate specialized hospital wards (“Other”) during Period 2 and 3. Total number of participants in each period (N=) and in each subgroup (n=) are presented with percentage of N= and 95% confidence interval (CI). (Table legend) Targeted admission of patients with positive test or suspect SARS-CoV-2 infection to a COVID-19 ward or other ward with infection prevention control (IPC) facilities did not change by the introduction of rapid point of care tests (Period 2: Rapid antigen detection test; Period 3: rapid RT-PCR VitaPCR) at the Emergency Department (ED). However, test-negative patients were increasingly admitted to appropriate specialized hospital wards (“Other”) during Period 2 and 3. Total number of participants in each period (N=) and in each subgroup (n=) are presented with percentage of N= and 95% confidence interval (CI). Targeted admission and intrahospital transfers Page 16/24 Table 5 Targeted admissions Period 1 Period 2 Period 3 Total n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Positive test at the ED (N = 257) All admissions 50 100 (92.9 - 100.0) 85 100 (95.7 - 100.0) 122 100 (96.9 - 100.0) 257 100 (98.5 - 100.0) Covid-19 ward 41 82.0 (69.2 - 90.2) 62 72.9 (62.7 - 81.2) 101 82.8 (75.1 - 88.5) 204 79.4 (74.0 - 83.9) Mixed Covid- 19/Internal Medicine ward 6 12.0 (5.6 - 23.8) 17 20.0 (12.9 - 29.7) 17 13.9 (8.9 - 21.2) 40 15.6 (11.6 - 20.5) ICU 1 2.0 (0.1 - 10.5) 2 2.4 (0.4 - 8.2) 1 0.8 (0.04 - 4.5) 4 1.6 (0.6 - 3.9) Other 2 4.0 (0.7 - 13.5) 2 2.4 (0.4 - 8.2) 1 0.8 (0.04 - 4.5) 5 1.9 (0.8 - 4.5) Missing data 0 0.0 (0.0 - 7.7) 2 2.4 (0.4 - 8.2) 2 1.6 (0.29 - 5.8) 4 1.6 (0.6 - 3.9) Negative test at the ED (N = 864) All admissions 255 100 (98.5 - 100.0) 308 100 (98.8 - 100.0) 301 100 (98.7 - 100.0) 864 100 (99.6 - 100.0) Covid-19 ward 88 34.5 (28.9 - 40.5) 91 29.5 (24.7 - 34.9) 44 14.7 (11.1 - 19.1) 223 25.8 (23 - 28.9) Mixed Covid- 19/Internal Medicine ward 68 26.7 (21.6 - 32.4) 57 18.5 (14.6 - 23.2) 63 21.0 (16.8 - 26.0) 188 21.8 (19.2 - 24.7) ICU 9 3.5 (1.9 - 6.6) 4 1.3 (0.5 - 3.3) 5 1.7 (0.7 - 3.8) 18 2.1 (1.3 - 3.3) Other 84 32.9 (27.5 - 38.9) 144 46.8 (41.3 - 52.3) 180 59.8 (54.2 - 65.2) 408 47.2 (43.9 - 50.6) Page 17/24 Page 17/24 Period 1 Period 2 Period 3 Total n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Missing data 6 2.4 (1.1 - 5) 12 3.9 (2.2 - 6.7) 9 3.0 (1.6 - 5.6) 27 3.1 (2.2 - 4.5) Positive test before admission (N = 277) All admissions 41 100.0 (91.4 - 100.0) 89 100.0 (95.9 - 100.0) 147 100.0 (97.5 - 100.0) 277 100.0 (98.6 - 100.0) Covid-19 ward 35 85.4 (71.6 - 93.1) 73 82.0 (72.8 - 88.6) 128 87.1 (80.7 - 91.6) 236 85.2 (80.5 - 88.9) Mixed Covid- 19/Internal Medicine ward 4 9.8 (3.9 - 22.5) 12 13.5 (7.9 - 22.1) 13 8.8 (5.2 - 14.5) 29 10.5 (7.4 - 14.6) ICU 0 0.0 (0.0 - 8.6) 0 0.0 (0.0 - 4.1) 0 0.0 (0.0 - 2.5) 0 0.0 (0.0 - 1.4) Other 1 2.4 (0.1 - 12.6) 2 2.2 (0.4 - 7.8) 2 1.4 (0.2 - 4.8) 5 1.8 (0.8 - 4.2) Missing data 1 2.4 (0.1 - 12.6) 2 2.2 (0.4 - 7.8) 4 2.7 (1.1 - 6.8) 7 2.5 (1.2 - 5.1) Not tested (N = 89) All admissions 40 100 (91.2 - 100.0) 28 100 (87.9 - 100.0) 21 100 (84.5 - 100.0) 89 100 (95.9 - 100.0) Covid-19 ward 2 5.0 (0.9 - 16.5) 3 10.7 (3.7 - 27.2) 1 4.8 (0.2 - 22.7) 6 6.7 (3.1 - 13.9) Mixed Covid- 19/Internal Medicine ward 8 20.0 (10.5 - 34.8) 5 17.9 (35.6 - 7.9) 5 23.8 (10.6 - 45.1) 18 20.2 (13.2 - 29.7) ICU 0 0.0 (0.0 - 8.8) 1 3.6 (0.2 - 17.7) 0 0.0 (0.0 - 15.5) 1 1.1 (0.1 - 6.1) O h 28 70 0 (54 6 18 64 3 14 66 7 60 67 4 Page 18/24 Period 1 Period 2 Period 3 Total n = % (95% CI) n = % (95% CI) n = % (95% CI) n = % (95% CI) Missing data 2 5.0 (0.9 - 16.5) 1 3.6 (0.2 - 17.7) 1 4.8 (0.2 - 22.7) 4 4.5 (1.8 - 11.0) (Table legend) Targeted admission of patients with positive test or suspect SARS-CoV-2 infection to a COVID-19 ward or other ward with infection prevention control (IPC) facilities did not change by the introduction of rapid point of care tests (Period 2: Rapid antigen detection test; Period 3: rapid RT-PCR VitaPCR) at the Emergency Department (ED). Discussion In this retrospective study, we have explored the impact of the introduction of SARS-CoV-2 RAD tests and the POC rapid RT-PCR VitaPCR on patient care during a period of high prevalence of SARS-CoV-2 infection. The study included 2,940 participants that visited the ED, who were grouped into three periods to highlight differences between the previous standard of care (Core laboratory RT-PCR) in Period 1, introduction of RAD test in Period 2 and introduction of VitaPCR in Period 3. Importantly, the results reveal that the implementation had a significant effect on length of stay at the ED and hospital, intrahospital transfers first 5 days and targeted admission to wards with IPC facilities, which has implications for the treatment of the individual patient, patient safety, hospital infection control and optimal resource use. Before the introduction of point-of-care rapid SARS-CoV-2 tests at the ED, patients with suspected COVID- 19 infection were admitted to COVID-19 diagnostic and treatment wards with IPC facilities until results from RT-PCR became available. Patients with negative tests were then transferred to the most appropriate ward based on the medical need. While this prevented secondary cases of COVID-19, intrahospital transfers have been related to unfavorable events such as increased falls, medication errors, length-of- stay and hospital-acquired infections (11, 12), as well as increased nurse and doctor work load (13). This study analyzed targeted admission to COVID-19 or non-COVID-19 wards directly, as well as using intrahospital transfers and hospital time of stay as surrogate markers of targeted admission. A significant improvement was seen in the outcome of all these variables that started with the introduction of the RAD test but was further pronounced after addition of the VitaPCR. Negative RAD test led to cessation of IPC at the ED and before admission to a hospital ward for patients without high risk of COVID-19. However, during Period 2, it was still necessary to exclude SARS-CoV-2 infection with RT-PCR at the core laboratory for patients with high risk of the infection. In contrast, in Period 3 a negative VitaPCR was sufficient for cessation of IPC even for patients with symptoms compatible with COVID-19. Hence Page 19/24 Page 19/24 targeted admission was possible for negative patients regardless of symptoms, leading to fewer intrahospital transfers, immediate initiation of appropriate therapy and shorter length-of-stay at the hospital. Discussion Similarly, it is interesting to note that discharges from the ED to home increased during the study period, particularly for participants that were tested for SARS-CoV-2 at the ED. The change was seen after introduction of the RAD tests, possibly because of the faster diagnostic work up that was enabled by the rapid tests. In the algorithms used during the study period, a positive RAD test was enough to confirm COVID-19. Hence, it can be expected that any change in patient care inflicted by the rapid tests for participants that tested positive at the ED would occur already in Period 2 (after introduction of RAD test) and not significantly changed after introduction of the VitaPCR. The study period is unique in that allows for interrogation of the sequential introduction of two rapid POC analysis methods with direct implications for IPC management of patients in a high endemic setting. The study site is the only ED in the area and the hospital treats all kinds of medical emergencies, which resulted in an unbiased adult population that is generalizable to similar ED’s. The study population was sufficiently large for the proposed analysis. The conclusions drawn here may well be applicable in other similar settings, for instance the need for rapid and accurate diagnostic tools for the annual influenza virus epidemic, which put similar demands on ED and hospital wards. Limits of the present study mainly relate to the retrospective study design. At the time of introduction of these diagnostic methods, it was not possible, or ethically defendable, to conduct a prospective randomized study. Conclusions In conclusion, the implementation of the testing algorithm including VitaPCR enabled exclusion of SARS- CoV-2 infection in patients at the ED, which reduced intrahospital transfers, shortened the stay at hospital wards, and increased targeted admissions to an appropriate ward. In addition, early identification of SARS-CoV-2 infection with rapid tests at the ED facilitated decisions at the ED and reduced ED time-of- stay. It would be of great interest to further investigate the health-economic implications of these results. Discussion No significant effect on targeted admission variables could be seen for test positive participants, likely because both suspected and confirmed cases of COVID-19 were admitted to wards with IPC measures. In fact, hospital LoS increased substantially for patients with positive SARS-CoV-2 test before admission to the ED. The reason for this is unknown as no difference in age or in proportion that received ICU care (data n.s.) were seen between the periods. High patient load at the ED puts increased pressure on hospital wards, which can lead to ED crowding and decreased patient safety (14). A key characteristic of crowding is an extended mean ED length-of- stay. The overall ED LoS did not substantially change during the study period, which is notable considering the increased number of patients in the latter periods of the study. Even though the proportion of COVID-19 diagnosis increased in the ED, participants with positive rapid tests at the ED (RAD test or VitaPCR) spent a shorter time at the ED than in Period 1. Similarly, it is interesting to note that discharges from the ED to home increased during the study period, particularly for participants that were tested for SARS-CoV-2 at the ED. The change was seen after introduction of the RAD tests, possibly because of the faster diagnostic work up that was enabled by the rapid tests. In the algorithms used during the study period, a positive RAD test was enough to confirm COVID-19. Hence, it can be expected that any change in patient care inflicted by the rapid tests for participants that tested positive at the ED would occur already in Period 2 (after introduction of RAD test) and not significantly changed after introduction of the VitaPCR. High patient load at the ED puts increased pressure on hospital wards, which can lead to ED crowding and decreased patient safety (14). A key characteristic of crowding is an extended mean ED length-of- stay. The overall ED LoS did not substantially change during the study period, which is notable considering the increased number of patients in the latter periods of the study. Even though the proportion of COVID-19 diagnosis increased in the ED, participants with positive rapid tests at the ED (RAD test or VitaPCR) spent a shorter time at the ED than in Period 1. List Of Abbreviations Page 20/24 ICU Intensive Care Unit Consent for publication Not applicable. Ethics approval and consent to participate The study protocol has been reviewed and approved by the Swedish Ethical Review Authority (Dnr: 2021- 00475). Informed consent was not retrieved according to Swedish legislation. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding This work was supported by the Swedish Research Council (MP #2018-06924,), Swedish Society for Medical Research (MP, MI), the Royal Physiographical Society (MP) and Swedish Heart and Lung Foundation (MI). Authors' contributions Competing interests The authors declare that they have no competing interests. References 1. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020;382(8):727-33. 2. World Health Organization. WHO Coronavirus (COVID-19) Dashboard [Internet]. Geneva: World Health Organization; 2020 [cited 2021 Nov 7]. Available from: https://covid19.who.int. 2. World Health Organization. WHO Coronavirus (COVID-19) Dashboard [Internet]. Geneva: World Health Organization; 2020 [cited 2021 Nov 7]. Available from: https://covid19.who.int. 3. European Centre for Disease Prevention and Control. Infection prevention and control and preparedness for COVID-19 in healthcare settings – Sixth update. Stockholm: ECDC; 2021 Feb. 3. European Centre for Disease Prevention and Control. Infection prevention and control and preparedness for COVID-19 in healthcare settings – Sixth update. Stockholm: ECDC; 2021 Feb. 4. World Health Organization. Recommendations for national SARS-CoV-2 testing strategies and diagnostic capacities. Geneva: World Health Organization; 2021 June. 4. World Health Organization. Recommendations for national SARS-CoV-2 testing strategies and diagnostic capacities. Geneva: World Health Organization; 2021 June. 5. Turcato G, Zaboli A, Pfeifer N, Sibilio S, Tezza G, Bonora A, et al. Rapid antigen test to identify COVID- 19 infected patients with and without symptoms admitted to the Emergency Department. Am J Emerg Med. 2021;51:92-7. 6. Treggiari D, Piubelli C, Caldrer S, Mistretta M, Ragusa A, Orza P, et al. SARS-CoV-2 rapid antigen test in comparison to RT-PCR targeting different genes: A real-life evaluation among unselected patients in a regional hospital of Italy. J Med Virol. 2021. 6. Treggiari D, Piubelli C, Caldrer S, Mistretta M, Ragusa A, Orza P, et al. SARS-CoV-2 rapid antigen test in comparison to RT-PCR targeting different genes: A real-life evaluation among unselected patients in a regional hospital of Italy. J Med Virol. 2021. 7. Fournier PE, Zandotti C, Ninove L, Prudent E, Colson P, Gazin C, et al. Contribution of VitaPCR SARS- CoV-2 to the emergency diagnosis of COVID-19. J Clin Virol. 2020;133:104682. 8. Fitoussi F, Dupont R, Tonen-Wolyec S, Bélec L. Performances of the VitaPCR™ SARS-CoV-2 Assay during the second wave of the COVID-19 epidemic in France. J Med Virol. 2021;93(7):4351-7. 9. Region Skåne. Situation Report COVID-19 in Skåne. [Internet]. Region Skåne; 2020 [cited 2021 Nov 9]. Available from: https://www.skane.se/digitala-rapporter/lagesbild-COVID-19-i-skane/situation-report- in-english/#Cases. 10. World Health Organization. WHO COVID-19: Case defintions. Geneva: World Health Organization; 2020 Dec. 11. Bristol AA, Schneider CE, Lin SY, Brody AA. A Systematic Review of Clinical Outcomes Associated With Intrahospital Transitions. J Healthc Qual. 2020;42(4):175-87. Authors' contributions Page 21/24 Conception and design of study: SM, MP, MI, MS, AP, CC. Acquisition of data: SM, MP. Analysis and/or interpretation of data: SM, MP. Drafting of the manuscript: SM, MP. Revising the manuscript critically for important intellectual content: SM, MP, MI, MS, AP, CC. All authors approved the final version of the manuscript to be published. Acknowledgments he authors would like to thank “Forum Söder” for their help in retrieving data The authors would like to thank “Forum Söder” for their help in retrieving data. References 12. Webster J, New K, Fenn M, Batch M, Eastgate A, Webber S, et al. Effects of frequent PATient moves on patient outcomes in a large tertiary Hospital (the PATH study): a prospective cohort study. Aust Health Rev. 2016;40(3):324-9. Page 22/24 Page 22/24 13. Blay N, Roche MA, Duffield C, Gallagher R. Intrahospital transfers and the impact on nursing workload. J Clin Nurs. 2017;26(23-24):4822-9. 14. Morley C, Unwin M, Peterson GM, Stankovich J, Kinsman L. Emergency department crowding: A systematic review of causes, consequences and solutions. PLoS One. 2018;13(8):e0203316. Supplemental Figure Supplemental Figure 1 is not available with this version. Figures g Figure 1 Algorithms for SARS-CoV-2 testing during the study Period 1, Period 2 and Period 3. At the Emergency Department during Period 1 (A), patients with symptoms of COVID-19 were tested with RT-PCR and Infection prevention and control (IPC) precautions continued. During Period 2 (B), a rapid antigen detection (RAD) test for SARS-CoV-2 was added, which enabled diagnosis of SARS-CoV-2 infection or discontinuation of IPC precautions for patients with low risk of SARS-CoV-2 infection and negative test result. During Period 3 (C), the rapid POC RT-PCR Vita-PCR was introduced, and COVID-19 specific IPC precautions were ended for Vita-PCR negative patients regardless of symptoms. * newly developed upper or lower respiratory tract symptoms, fever, nausea, diarrhoea, malaise, anosmia or recent close contact with a person with confirmed SARS-CoV-2 infection. anosmia, fever with unknown origin, dyspnoea with unknown origin, cough or other respiratory symptoms and known contact with a COVID-19 case previous 7 days Figure 2 Intrahospital transfers first 5 days after hospital admission. Percentage of admitted participants that were transferred between hospital wards during the first 5 days of admission from the emergency department (ED). Participants that had a negative SARS-CoV-2 test at the ED were less likely to be transferred in Period 2 and 3, after introduction of the SARS-CoV-2 rapid antigen detection test and VitaPCR respectively. Bar shows mean value in percent and bars 95 % confidence interval. indicate P ≤ 0.01, * P ≤ 0.001 and ** P ≤ 0.0001, calculated with Fisher’s exact test. Figure 1 Algorithms for SARS-CoV-2 testing during the study Period 1, Period 2 a Algorithms for SARS-CoV-2 testing during the study Period 1, Period 2 and Period 3. At the Emergency Department during Period 1 (A), patients with symptoms of COVID-19 were tested with RT-PCR and Infection prevention and control (IPC) precautions continued. During Period 2 (B), a rapid antigen detection (RAD) test for SARS-CoV-2 was added, which enabled diagnosis of SARS-CoV-2 infection or discontinuation of IPC precautions for patients with low risk of SARS-CoV-2 infection and negative test result. During Period 3 (C), the rapid POC RT-PCR Vita-PCR was introduced, and COVID-19 specific IPC precautions were ended for Vita-PCR negative patients regardless of symptoms. * newly developed upper or lower respiratory tract symptoms, fever, nausea, diarrhoea, malaise, anosmia or recent close contact with a person with confirmed SARS-CoV-2 infection. ** anosmia, fever with unknown origin, dyspnoea with unknown origin, cough or other respiratory symptoms and known contact with a COVID-19 case previous 7 days. Page 23/24 Figure 2 Additionalfile1.xlsx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 24/24
https://openalex.org/W2756113743	https://jnp.chitkara.edu.in/index.php/jnp/article/download/67/42	English	null	Application of a CdTe Detector for Measurements of Mammographic X-ray Spectra	Journal of nuclear physics, materials sciences, radiation and applications	2,017	cc-by	5,360	1 Instituto de Ciencias Nucleares, UNAM, 04510 Ciudad de México, México 2 Instituto de Física, Benemérita Universidad Autónoma de Puebla, Puebla, A. Postal J-48 Puebla, Puebla 72750, Mexico 2 Instituto de Física, Benemérita Universidad Autónoma de Puebla, Puebla, A. Postal J-48 Puebla, Puebla 72750, Mexico 3 Facultad de Ciencias, UNAM, 04510 Ciudad de México, México 3 Facultad de Ciencias, UNAM, 04510 Ciudad de México, México 4 The Advanced Light Source, Lawrence Berkeley Laboratory, Berke 4 The Advanced Light Source, Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA The Advanced Light Source, Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA Email: jimenez@nucleares.unam.mx Email: jimenez@nucleares.unam.mx Published online: August 07, 2017 Published online: August 07, 2017 The Author(s) 2017. This article is published with open access at www.chitkara.edu. in/publications Abstract Resonant inelastic x-ray scattering (RIXS) is used to study the electronic structure of NiF2, which is the most ionic of the nickel compounds. RIXS can be viewed as a coherent two-steps process involving the absorption and the emission of x-rays. The soft x-ray absorption spectrum (XAS) at the metal L2,3 edge indicate the importance of atomic multiplet effects. RIXS spectra at L2,3 contain clearly defined emission peaks corresponding to d-excited states of Ni2+ at energies few eV below the elastic emission, which is strongly suppressed. These results are confirmed by atomic multiplet calculations using the Kramers-Heisenberg formula for RIXS processes. For larger energy losses, the emission spectra have a broad charge-transfer peak that results from the decay of hybridized Ni(3d)-F(2p) valence states. This is confirmed by comparison of the absorption and emission spectra recorded at the nickel L and fluorine K edges with F p and Ni d partial density of states using LDA + U calculations. Keywords: Core-level spectroscopies. RIXS, Nickel difluoride, Electronic structure Journal of Nuclear Physics, Material Sciences, Radiation and Applications Vol-5 No-1 August 2017 pp. 1–13 Journal of Nuclear Physics, Material Sciences, Radiation and Applications Vol-5 No-1 August 2017 pp. 1–13 https://doi.org/10.15415/jnp.2017.51001 https://doi.org/10.15415/jnp.2017.51001 Journal of Nuclear Physics, Material Sciences, Radiation and Applications Vol-5 No-1 August 2017 pp. 1–13 1. INTRODUCTION Transition metal (TM) compounds play a major role in condensed matter physics and materials science. A complex interplay between the charge, orbital, 1 spin and lattice degrees of freedom gives birth to emergent phenomena like high temperature superconductivity (HTSC), colossal/giant magneto resistivity (C/GMR) and thermoelectricity in copper, manganese and cobalt oxides, respectively [5]. Understanding the origin of these emergent phenomena has prompted theoretical and experimental research which points to the crucial role of 3d electronic correlations. Indeed, the insulating Mott state and its related metal to insulator transitions (MITs) are a living classic in contemporary condensed matter physics [12]. The advance in the understanding of these phenomena requires combined efforts between theory and experiment. Therefore, systematic investigations on model systems are crucial to establish solid foundations in this regard. For example, the simplest 3d transition metal oxides (TMOs), e.g. MnO, CoO, and NiO have allowed us to reach the current understanding of the electronic structure of these materials and their insulating bandgap either as Mott-Hubbard or as charge-transfer (CT) insulators, as stated by the Zaanen-Sawatzky-Allen (ZSA) diagram [25]. However, it took more than two decades to have a direct experimental proof of the ZSA theory and it came from a combined experiment of X-ray absorption (XAS) and emission (XES) spectroscopies on the most ionic TM compounds, the difluorides TMF2 (Cr-Zn) at the TM L2,3 and F K edges [18]. ( ) 2, g [ ] Among the simplest TMOs, NiO is the test ground of the interplay between theory and experiment. Although there is a comparative study between NiO and NiF2 [15], overall the latter has been marginally studied. However several parallelisms can be established: both solids are antiferromagnetic insulators with the Ni2+ ions at the center of a perfect octahedra (Oh symmetry in NiO) and slightly distorted one (D4h symmetry in NiF2) surrounded by six ligands (O or F). Interestingly, one important difference, consequence of the strong ionicity in NiF2, is the fact that according to ZSA theory NiO is in the intermediate region between Mott-Hubbard and charge-transfer insulator regimes while NiF2 is a clear Mott-Hubbard insulator [18]. This indicates that replacing O by F induces profound changes in the electronic structure of NiF2. These changes naturally should be reflected in the absorption (XAS), emission (XES), photoelectron (XPS) and resonant inelastic scattering (RIXS) x-ray spectra. Jiménez-Mier, J Olalde-Velasco, P de la Mora, P Yang, W -L Denlinger, J 1. INTRODUCTION For instance, the XAS spectra of Ni2+ in NiO and NiF2 were analyzed by van der Laan et al. [15] and Olalde et al. [19] using, respectively, many-body calculations including charge transfer (CT) effects and ligand field atomic multiplet calculations. From the analysis it was shown that CT effects were more important to describe completely all the features in the NiL2,3 XAS spectrum in NiO as compared to NiF2. On the other hand, in XES spectroscopy, CT effects, from ligand (F or O) to metal and vice versa were also observed in TMF2 including NiF2[18]. At the Ni L2,3 edges, RIXS grants access to neutral 2 optically forbidden Ni dd and F to Ni CT excitations. In RIXS, the ligand to metal CT effects at the Ni L3 [7] and M2,3 [3] edges have also been observed by RIXS in NiO and NiCl2, but to the best of our knowledge not in NiF2. Thus, a systematic RIXS study at the NiL2,3 edges in NiF2 should in principle, in combination with all the previous spectroscopic results, provide a more complete picture of the electronic structure of the material. Atomic Multiplet and Charge Transfer Effects in the Resonant Inelastic X-Ray Scattering (RIXS) Spectra at the Nickel L2,3 Edge of NiF2 Theoretically, the electronic band structure of NiF2 has been studied using the full potential linearized augmented plane wave (LAPW) method, with the exchange and correlation effects treated in the local spin-density approximation (LSDA) [6]. This calculation correctly predicts the insulating ground state in NiF2 but the bandgap is largely underestimated as compared to the experimental determination obtained from soft X-ray spectroscopies [18]. In practical terms, NiF2 has recently received attention for various reasons. It was used to produce 3-D nanoporous flexible electrodes for commercial applications [24]. It shows potential as a cathode material for rechargeable Li ion batteries [16, 26] which in a NiO-doped NiF2-C version of the previous cathodes was investigated by hard X-ray absorption spectroscopy [17]. It has also been used as catalyst for the dehydrogenation of ammonia borane [23], a proposed material for hydrogen storage. Therefore, from the fundamental physics point of view as well as for the applicability of NiF2 as a practical material in energy storage and catalysis, it is crucial to pin down the energy levels of Ni and F in this material. 2. X-RAY ABSORPTION, X-RAY EMISSION AND RIXS SPECTROSCOPIES Soft x-ray spectroscopies are very well suited to study the electronic structure of transition metal compounds [9]. In the case of nickel fluoride, the fluorine K-edge is at 685 eV and the nickel L2,3 edges are at 850 eV (L3) and 867 eV (L2). In x-ray absorption spectroscopy (XAS) a photon is used to excite a core electron into the conduction band. The core hole can then be filled by an electromagnetic transition involving an electron in the valence band, resulting in x-ray emission spectra (XES). Resonant inelastic x-ray scattering (RIXS) is a coherent second order process in which an incoming photon of energy hν1 is scattered, resulting in an outgoing photon with energy hν2. The energy lost in RIXS, given by hν1 - hν2, is transferred to the sample. Therefore, RIXS spectra plotted as functions of the energy loss give information about excited states of the compound that is studied. X-Ray absorption and emission are local processes at the atomic level. Fluorine XAS and XES spectra probe conduction and valence states projected 3 3 Jiménez-Mier, J Olalde-Velasco, P de la Mora, P Yang, W -L Denlinger, J at this atom site. By the same token, nickel XAS and XES spectra give information about unoccupied and occupied states at the metal. Furthermore, the transitions are governed by the electric-dipole selection rules, and therefore the incoming and outgoing x-ray photons give information about conduction and valence states of specific orbital symmetry. In fluorine XAS a 1s electron is excited to 2p conduction states, and the 1s hole is filled by valence 2p electrons. In nickel XAS a 2p electron is promoted to conduction states with 3d symmetry. In RIXS at the nickel L2,3 edges, the metal emission that follows resonant excitation of a 2p electron produces d-excited states of the ground configuration. Jiménez-Mier, J Olalde-Velasco, P de la Mora, P Yang, W -L Denlinger, J A. Atomic multiplet calculations The calculation allows interaction of the original ionic configurations and these charge transfer configurations through the inclusion of new, adjustable parameters [21]. These are the Hubbard energy U, which is the energy necessary for a Ni 3d electron to move from one metal site to another; the nickel 2p — 3d core hole potential Vpd that represents the shift in energy levels introduced by the 2p hole in the metal, the charge transfer energy ∆, which is the energy needed to move the F 2p electron into the Ni ion, and the nickel site to nickel site hopping energy T. In this paper we present two Ni2+ x-ray absorption spectra. One was calculated using only the crystal field part of the program, and and for the other we also included charge transfer effects. The parameters used in the calculation are shown in Table I. p The RIXS process is described by the Kramers-Heisenberg equation σ ε ε δ v v f r i i r g E E hv i E E h i g p i f f i 1 2 2 1 1 2 2 2 , / ( ) < ⋅ >< ⋅ > − − − − − ∑ ∑ ∝ Γ v v 1 2 − ( ) ( ) (1) (1) In this formula \|g >, \|i > and \|f > are the wavefunctions of the initial, intermediate and final states of the system, with energies Eg, Ei and Ef, respectively; ε 1 ⋅r and ε 2 ⋅r are the electric dipole transition operators; and Γ2p is the 2p core hole width. The output from the crystal field program, without any charge transfer effects, provides the values of the transition matrix elements and also for the state energies. This information was used to calculate Ni2+ RIXS spectra for all values of the incoming photon energy used in the experiment. For the RIXS calculation we used the crystal field parameters given in Table I. A. Atomic multiplet calculations A theoretical description of the electronic structure of transition metal compounds is complicated by the fact that the 3d orbitals are spatially localized like those of shallow core-orbitals, but at the same time their energies are low, making them to participate in chemical bonding. The atomic multiplet approach [10, 11] begins with a description of the nickel electronic structure at the free- ion level. The ground state configuration for Ni2+ is 3d8 which is excited by the incoming photon to states in the 2p5 3d9 configuration. The calculation includes the electron-electron Coulomb interaction through the F and G Slater integrals, and also the ζ2p and ζ3d spin-orbit interaction parameters. These parameters are obtained directly from a Hartree-Fock calculation for the free- ion [4]. The two 3d holes in the ground configuration result in a 3F ground term. Including spin-orbit interaction of the 3d subshell gives a ground total angular momentum of J = 4. At room temperature only this 3F4 ground state can be excited by electric dipole transitions into states of the 2p5 3d9 configuration. All other terms will not be excited, but they can be produced as result of x-ray emission. Decay into these 3d excited states plays a very important role in the RIXS process. Besides the 3F, the excited terms in the 3d8 configuration are the singlets and triplets 1D, 3P, 1G and 1S, listed in order of increasing energy.ii The crystal field splits the 3d levels. In the case of an octahedral field the atomic 3d orbitals are split into three degenerate t2g orbitals (xy, yz and zx) and two degenerate eg orbitals (z2 and x2 - y2) separated by an energy 10Dq. These degeneracies are further removed in lower symmetries by inclusion of more crystal field parameters. For the distorted octahedron for the spatial structure of NiF2 the corresponding symmetry is tetragonal, and one has to introduce the parameters Ds and Dt [21]. In this work the program CTM4XAS [21] was used to calculate the x-ray absorption spectrum of NiF2 at the nickel L2,3 edges. 4 Table I: Atomic multiplet, crystal field and charge transfer parameters (in eV) used in the XAS calculation. For the RIXS calculation the same crystal field parameters were used. For the meaning of the parameters see the text. A. Atomic multiplet calculations Atomic Multiplet and Charge Transfer Effects in the Resonant Inelastic X-Ray Scattering (RIXS) Spectra at the Nickel L2,3 Edge of NiF2 Atomic M and Transfe in the R Inelasti Scattering Spec Nickel L2,3 Table I: Atomic multiplet, crystal field and charge transfer parameters (in eV) used in the XAS calculation. For the RIXS calculation the same crystal field parameters were used. For the meaning of the parameters see the text. Parameter 10Dq 1.0 Dt 0.03 Ds 0.12 Γ2p 0.24 U 5.5 Vpd 6.5 ∆ 7.0 T 1.0 Charge transfer effects can also be included in the ionic calculation [10, 11, 21]. In this case the program also considers a configuration in which a ligand electron (F-2p in NiF2) is transferred to the metal. One denotes as 3d9 L the state with an extra electron in the Ni 3d subshell, and a ligand hole denoted by L. After x-ray absorption this configuration is excited into the 2p5 3d10L state. The calculation allows interaction of the original ionic configurations and these charge transfer configurations through the inclusion of new, adjustable parameters [21]. These are the Hubbard energy U, which is the energy necessary for a Ni 3d electron to move from one metal site to another; the nickel 2p — 3d core hole potential Vpd that represents the shift in energy levels introduced by the 2p hole in the metal, the charge transfer energy ∆, which is the energy needed to move the F 2p electron into the Ni ion, and the nickel site to nickel site hopping energy T. In this paper we present two Ni2+ x-ray absorption spectra. One was calculated using only the crystal field part of the program, and and for the other we also included charge transfer effects. The parameters used in the calculation are shown in Table I. Parameter 10Dq 1.0 Dt 0.03 Ds 0.12 Γ2p 0.24 U 5.5 Vpd 6.5 ∆ 7.0 T 1.0 Charge transfer effects can also be included in the ionic calculation [10, 11, 21]. In this case the program also considers a configuration in which a ligand electron (F-2p in NiF2) is transferred to the metal. One denotes as 3d9 L the state with an extra electron in the Ni 3d subshell, and a ligand hole denoted by L. After x-ray absorption this configuration is excited into the 2p5 3d10L state. 4. EXPERIMENT The experiment took place at beamline 8.0.1 of The Advanced Light Source in Berkeley. The experimental x-ray absorption spectrum was obtained by recording the sample current as the photon energy of the incoming x-rays was scanned. Resonant x-ray emission spectra were recorded in a soft x-ray fluorescence spectrometer, which is a grazing incidence instrument with a fixed entrance slit and a position sensitive area detector [13]. Emission spectra were obtained at selected values of the incoming photon energy. Fresh, commercially available polycrystalline NiF2 from Sigma Aldrich with nominal purity greater than 99.9% was prepared inside a N2 flow bag and introduced inside the UHV experimental chamber. All spectra presented in this work were recorded at room temperature. B. Density of states calculation To perform the electronic structure calculations, the WIEN2k code was used [2], which is based on the Density Functional Theory. This code is an all- 5 electron scheme based on the full-potential Linearized Augmented Plane Wave + local orbitals method. The band gaps calculated using the Kohn-Sham eigenvalues are known to be underestimated [8], an issue that can be fixed by using the GW approximation or a hybrid functional. However, these would be quite expensive, and an alternative solution incorporating the Becke-Johnson potential (BJ) [1] was used in this work. This potential is less demanding in computer time and it shows an agreement of the same order with the experiment as that obtained with the GW approximation, for wide band gap insulators, sp semiconductors and strongly correlated 3d transition metal oxides [1, 22]. In considering the exchange-correlation interactions, the generalized gradient approximation of Perdew, Burke and Ernzerhof (PBE) [20] was used, and it was then substituted after convergence, by the Becke-Johnson potential. For the number of plane waves used, the considered criterion was RMT min × Kmax = 7, where RMT is the Muffin-Tin radius and Kmax is the maximum value of the k plane waves. Within the code used, RMT min × Kmax is a measure of the quality of the basis set, and 1000 k-points were used. Jiménez-Mier, J Olalde-Velasco, P de la Mora, P Yang, W -L Denlinger, J 5. RESULTS AND DISCUSSION The x-ray absorption spectrum at the nickel L2,3 edge of NiF2 is shown in Fig. 1. The features around 850 eV result from production of a 2p3/2 core hole and define the L3 edge. The smaller features beginning at 867 eV correspond to the L2 edge, which corresponds to excitation of a 2p1/2 electron. A straight line background was subtracted from the raw spectrum to make the comparison with the multiplet calculation. The labels give the energies of the incoming photon that were used for the RIXS spectra. Results of two calculations are presented. 6 6 Atomic Mu and C Transfer E in the Res Inelastic X Scattering (R Spectra Nickel L2,3 E Figure 1: X-Ray absorption spectrum at the Ni L2,3 edge of NiF2. Gray dots: raw spectrum. Black dots: spectrum after subtraction of a straight line background (dashed gray line). The continuous lines are the results of the multiplet calculation. Dashed blue with crystal field only. Red: with charge transfer effects. Labels a through k give the excitation energies that were used to record RIXS spectra.ii Figure 1: X-Ray absorption spectrum at the Ni L2,3 edge of NiF2. Gray dots: raw spectrum. Black dots: spectrum after subtraction of a straight line background (dashed gray line). The continuous lines are the results of the multiplet calculation. Dashed blue with crystal field only. Red: with charge transfer effects. Labels a through k give the excitation energies that were used to record RIXS spectra. In the first one (dashed blue line) only crystal field effects were included. For the second calculation charge transfer effects were also considered. The main difference between the two calculations is the presence of a small, broad peak at energy (g) that can therefore be identified as a charge transfer peak. The nickel RIXS spectra are dominated by peaks resulting in decay into excited states of the metal 3d8 ground configuration. The energy of these peaks puts them close to the L3 edge in absorption. The outgoing photons with energies above the L3 edge are efficiently absorbed by the sample and therefore the spectra must be corrected for this self-absorption effect. Figure 2 illustrates this effect for the emission spectrum obtained with excitation energy (d). There is a clear overlap between the raw emission spectrum and the absorption spectrum. Self-absorption results in a reduction of the intensity of the main emission peaks y and z. 5. RESULTS AND DISCUSSION Once the self-absorption correction is made [14] these two peaks grow in intensity. All emission spectra presented in this article were corrected for self-absorption. Figure 2 also serves to introduce the features present in the RIXS spectra. Elastic emission E corresponds to an outgoing photon with the same energy of the incoming photon. This means that no energy was absorbed by the sample, and therefore the final state of the 7 Figure 2: Example of self-absorption effects in an emission spectrum. The shaded gray is the raw spectrum and the red lines give the corrected spectrum. Bottom: RIXS spectrum recorded with an excitation energy (d). Top: XAS spectrum. The d-d excitations are indicated by x, y and z. E gives the position of the expected elastic peak. CT is the charge transfer peak. nez-Mier, J de-Velasco, P Mora, P g, W -L inger, J Figure 2: Example of self-absorption effects in an emission spectrum. The shaded gray is the raw spectrum and the red lines give the corrected spectrum. Bottom: RIXS spectrum recorded with an excitation energy (d). Top: XAS spectrum. The d-d excitations are indicated by x, y and z. E gives the position of the expected elastic peak. CT is the charge transfer peak. scattering process is the ground state of the nickel ion. The spectrum shown in Fig. 2 has a very small elastic peak. This indicates that decay proceeds mainly into excited states of the 3d8 configuration. This results in emission peaks x, y and z. This suppression of elastic emission is common to all the nickel RIXS spectra of NiF2. Another feature present in most of the spectra is the broad peak indicated by CT in Fig. 2. This results from decay leading to production of the 3d9 L charge transfer state with an energy loss centered at 10.2 eV. A comparison between experiment and the multiplet calculation for several representative emission spectra is made in Fig. 3. All experimental spectra have peaks resulting from decay into d-excited states, a small, charge transfer peak that stays 10.2 eV below the position of the expected elastic peak. The spectra recorded at the L2 edge also show a strong normal emission peak (N). The agreement between experiment and theory is better for the spectra recorded at the L3 edge, for which the calculation give relative intensities close to the observed ones. 5. RESULTS AND DISCUSSION The spectra obtained with excitation energies (j) and (k) are also dominated by the d-excited states. However, the experimental peaks are significantly broadened, which is an indication of the fast decay of the 8 Ato T in In Sca Nick Figure 3: RIXS spectra recorded at excitation energies (b), (d), (j) and (k). Dots: experimental data. Blue line: calculated spectra. N: normal emission. CT: charge transfer emission. E: expected position of the elastic peak. Figure 3: RIXS spectra recorded at excitation energies (b), (d), (j) and (k). Dots: experimental data. Blue line: calculated spectra. N: normal emission. CT: charge transfer emission. E: expected position of the elastic peak. 2p1/2 hole by a non-radiative Coster-Kronig process. The agreement with the calculation is better for spectrum (j), but for (k) the calculation gives most of the intensity to the lower d-excited states while the peaks around 865 eV, with larger energy losses, have most of the intensity in the experimental data. g gy y p Figure 4 shows the emission spectrum recorded at the excitation energy (g). Recall that this is the only feature in the absorption spectrum that is not predicted by the simple crystal field calculation but requires the inclusion of charge transfer effects. The raw spectrum is given by the shaded area, and the spectrum obtained after the self-absorption correction is indicated by the red line. This spectrum has two separated features. A broad hump between 857 and 860 eV is the one that corresponds to the suppressed elastic emission and to d-excited states. The structure between 845 and 852 eV is composed of two peaks. There is a broad normal emission peak centered at 850 eV that results from decay after excitation of the 2p electron into the continuum. This therefore corresponds to decay from the 2p5 3d8 configuration of Ni3+. The other broad peak is centered at 848 eV, and results from decay of the resonantly produced charge transfer state of Ni2+. g These results are nicely rounded with a comparison between experiment and the density of states calculation. The comparison is made in Fig. 5. The 9 Figure 4: RIXS spectrum for the charge transfer excitation energy (g). Shaded gray area: raw spectrum. Red line: spectrum corrected for self-absorption. Jiménez-Mier, J Olalde-Velasco, P de la Mora, P Yang, W -L Denlinger, J Figure 4: RIXS spectrum for the charge transfer excitation energy (g). 6. CONCLUSIONS The results presented in this work show that x-ray spectroscopies combined with a theoretical analysis can be very useful in the study of the electronic structure of transition metal compounds. These techniques were used to interpret x-ray absorption and emission at the fluorine K-edge and the nickel L2,3 edges of the most ionic nickel compound, namely NiF2. Nickel L2,3 RIXS spectra are dominated by d-d excitations of the 3d8 configuration, but there is also indication of charge transfer emission. Fluorine x-ray absorption and emission are well explained by a density of states calculation that also shows strong hybridization between fluorine 2p states and nickel 3d states at the metal site. By a systematic approach combinig atomic like charge transfer mutlitplet calculations to describe the XAS and RIXS spectra of Ni2+ at the L2,3 edges and a band approach using LDA+U calculations to describe the F K XAS and XES spectra, we reach a comprehensive understanding of the electronic structure of NiF2. 5. RESULTS AND DISCUSSION Shaded gray area: raw spectrum. Red line: spectrum corrected for self-absorption. Figure 5: Comparison between experiment and the calculated projected density of states (DOS). Bottom: experimental XAS (open circles) and XES (dots) spectra at the fluorine K-edge. The continuous yellow line is the calculated F 2p DOS. The conduction band data was multiplied by a a factor of 20. Top: experimental Ni L3 absorption (open circles). The continuous blue line is the calculated Ni 3d DOS. Figure 5: Comparison between experiment and the calculated projected density of states (DOS). Bottom: experimental XAS (open circles) and XES (dots) spectra at the fluorine K-edge. The continuous yellow line is the calculated F 2p DOS. The conduction band data was multiplied by a a factor of 20. Top: experimental Ni L3 absorption (open circles). The continuous blue line is the calculated Ni 3d DOS. 10 experimental data shown here are the absorption spectra at the L3 edge in nickel, and the fluorine absorption and emission spectra at the fluorine K edge. The figure also shows the result of a convolution of projected density of states with a Gaussian window whose width is 0.5 eV. This figure illustrates the information that can be directly obtained from XAS and XES spectra. Both absorption spectra are aligned at the peak at 5.5 eV. The DOS calculation indicates that this feature corresponds to excitation into a narrow conduction sub-band predominantly Ni 3d in character. The fluorine emission spectrum is then given by decay of the valence band which is mainly of F 2p character. The calculation clearly overestimates the band gap, but confirms this band assignment, and is also consistent with the ZSA theory [18]. It is really remarkable that the shape of the fluorine emission spectrum is in very good agreement with the calculated F 2p density of states, once the theoretical curve is shifted upwards. It gives an accurate prediction of the intensity ratio between the fluorine 2p and nickel 3d DOS ratios projected at the fluorine atom. Atomic Multiplet and Charge Transfer Effects in the Resonant Inelastic X-Ray Scattering (RIXS) Spectra at the Nickel L2,3 Edge of NiF2 [1] Becke, A.D. & Johnson, E.R. (2006) A simple effective potential for exchange. J. Chem. Phys. 24, #221101. ACKNOWLEDGMENTS This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. Atomic Multiplet and Charge Transfer Effects in the Resonant Inelastic X-Ray Scattering (RIXS) Spectra at the Nickel L2,3 Edge of NiF2 REFERENCE 11 iménez-Mier, J Olalde-Velasco, P de la Mora, P [2] Blaha, P. et al. WIEN2k an Augmented Plane Wave Plus Local Orbital Program for Calculating Crystal Properties, Vienna University of Technology, Vienna, 2001. [3] Chiuzb˘aian, S. G. et al. (2005) Localized Electronic Excitations in NiO Studied with Resonant Inelastic X-Ray Scattering at the Ni M Threshold: Evidence of Spin Flip. Phys. Rev. Lett. 95, #197402 [4] Cowan, R.D. (1981). The Theory of Atomic Structure and Spectra, Berkeley: University of California Press. [5] Dagotto, E. (2005) Complexity in Strongly Correlated Electronic Systems, Science 309, 257–262. [6] Dufek, P., Schwarz, K. & Blaha, P. (1993) Electronic structure of MnF2 and NiF2, Phys. Rev. B 48, 12672–12681. [7] Ghiringhelli, G. et al. (2009) Observation of Two Nondispersive Magnetic Excitations in NiO by Resonant Inelastic Soft-X-Ray Scattering, Phys. Rev. Lett. 102, #027401. [8] Godby, R., Schlu¨ter, M. & Sham, L. (1986) Accurate Exchange-Correlation Potential for Silicon and Its Discontinuity on Addition of an Electron. Phys. Rev. Lett. 56, 2415–2418. [9] de Groot, F. & Kotani, A. (2008) Core Level Spectroscopy of Solids, Boca Raton, Fl, CRC Press. [10] de Groot, F. (2001) High-Resolution X-ray Emission and X-ray Absorption Spectroscopy. Chemistry Review 101, 1779–1808. [11] de Groot, F. (2005) Multiplet effects in X-ray spectroscopy. Coordination Chemistry Reviews 249, 31–63. [12] Imada, M., Fujimori, A. & Tokura, Y. (1998) Metal-insulator transitions, Rev. Mod. Phys. 70, 1039–1263. [13] Jia, J.J. et al. (1995) First experimental results from IBM/TENN/TULANE/ LLNL/LBL undulator beamline at the advanced light source. Rev. Sci. Instrum. 66, 1394–1397. [14] Jiménez-Mier, J., Ederer, D.L. & Schuler, T. (2005) X-ray Raman scattering at the manganese L edge of MnF2: Valence emission of Mn2+. Phys. Rev. A 72, #022502. [15] van der Laan, G. et al. (1986) Comparison of X-ray absorption with x-ray photoemission of nickel diahlides and NiO, Phys. Rev. B, 33, 4253–4263.l [16] Lee, D. H. et al. (2012) Conversion mechanism of nickel fluoride and NiO-doped nickel fluoride in Li ion batteries. Electrochimica Acta 59, 213–221. [17] Lee, D.H. et al. (2014) Understanding improved electrochemical properties of NiO-doped NiF2- C composite conversion materials by X-ray absorption spectroscopy and pair distribution function analysis, Phys. Chem. Chem. Phys. 16, 3095–3102. [18] Olalde-Velasco, P. et al. (2011) Direct probe of Mott-Hubbard to charge-transfer insulator transition and electronic structure evolution in transition-metal systems, Phys. Rev B. 83, #241102(R). 12 [19] Olalde-Velasco, P., Jiménez-Mier, J., Denlinger, J., & Yang, W.-L. [19] Olalde-Velasco, P., Jiménez-Mier, J., Denlinger, J., & Yang, W.-L. (2013) Atomic multiplets at the L2,3 edge of 3d transition metals and the ligand K edge in x-ray absorption spectroscopy of ionic systems, Phys. Rev. B 87, #245136. REFERENCE (2013) Atomic multiplets at the L2,3 edge of 3d transition metals and the ligand K edge in x-ray absorption spectroscopy of ionic systems, Phys. Rev. B 87, #245136. Atomic Multiplet and Charge Transfer Effects in the Resonant Inelastic X-Ray Scattering (RIXS) Spectra at the Nickel L2,3 Edge of NiF2 [20] Perdew J.P., Burke, K & Ernzerhof, M. (1996) Generalized Gradient Approximation Made Simple. Phys. Rev. Lett. 77, 3865–3868. [21] Stavitski, E. & de Groot, F.M.F. (2008) The CTM4XAS program for EELS and XAS spectral shape analysis of transition metal L edges. Micron 41, 687–694. Scattering (RIXS) Spectra at the [22] Tran, F. & Blaha, P. (2009) Accurate Band Gaps of Semiconductors and Insulators with a Semilocal Exchange-Correlation Potential. Phys. Rev. Lett. 102, #226401. [23] Yang, L., Luo, W. & Chen, G.-Z. (2013) In Situ Synthesis of Ni(o) Catalysts Derived from Nickel Halides for Hydrolytic Dehy-drogenation of Ammonia Borane. Catalysis Letters, 143, 873–880. [24] Yang, Y. et al. (2014) Flexible Three-Dimensional Nanoporous Metal-Based Energy Devices. J. Am. Chem. Soc. 136, 6187–6190. [25] Zaanen, J., Sawatzky, G.A. & Allen, J.W. (1985) Band Gaps and Electronic Structure of Transition-Metal Compounds, Phys. Rev. Lett. 55, 418–421. [26] Zhang, H., Zhou, Y,-N., Sun, Q., & Fu, Z,-W. (2008) Nanostructured nickel fluoride thin film as a new Li storage material, Solid State Sciences 10, 1166–1172. 13
https://openalex.org/W2328575004	https://dash.harvard.edu/bitstream/1/32072014/1/5349091.pdf	English	null	Control of the gut microbiome by fecal microRNA	Microbial cell	2,016	cc-by	1,457	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:32072014 Citation Liu, Shirong, and Howard L. Weiner. 2016. “Control of the gut microbiome by fecal microRNA.” Microbial Cell 3 (4): 176-177. doi:10.15698/mic2016.04.492. http://dx.doi.org/10.15698/ mic2016.04.492. Fecal miRNAs Since their discovery in the early 90s, microRNAs (miRNAs), small non-coding RNAs, have mainly been associated with posttranscriptional regulation of gene expression on a cell-autonomous level. Recent evi- dence has extended this role by adding inter-species communication to the manifold functional range. In our latest study [Liu S, et al., 2016, Cell Host & Mi- crobe], we identified miRNAs in gut lumen and feces of both mice and humans. We found that intestinal epi- thelial cells (IEC) and Hopx+ cells were the two main sources of fecal miRNA. Deficiency of IEC-miRNA re- sulted in gut dysbiosis and WT fecal miRNA transplan- tation restored the gut microbiota. We investigated potential mechanisms for this effect and found that miRNAs were able to regulate the gut microbiome. By culturing bacteria with miRNAs, we found that host miRNAs were able to enter bacteria, specifically regu- late bacterial gene transcripts and affect bacterial growth. Oral administration of synthetic miRNA mim- ics affected specific bacteria in the gut. Our findings describe a previously unknown pathway by which the gut microbiome is regulated by the host and raises the possibility that miRNAs may be used therapeutically to manipulate the microbiome for the treatment of dis- ease. Studies have shown that miRNAs can exist extracellularly in the body and might be indicative of specific diseases. Hence, our primary research focus are extracellular miR- NAs in the circulation as biomarkers of multiple sclerosis. We then asked whether extracellular miRNAs were present in the feces as well, and found that miRNAs were part of the normal composition of feces both in mice and humans. We compared the relative abundance of miRNAs in the upper level and lower level of the gut, in germ-free mice and in antibiotic treated mice. Intriguingly, we found that the abundance of miRNA was inversely correlated with the abundance of microbes. This suggested that microbes might take up miRNAs and that these miRNAs might in turn affect microbes. Microreview www.microbialcell.com g Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, B t MA 02115 USA * Corresponding Author: Howard L. Weiner, E-mail: hweiner@rics.bwh.harvard.edu Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Normal gut microbiota is essential for health Normal gut microbiota is essential for health It is known that the mammal gut harbors trillions of commensal microbes, which are host specific and are important for health. Disturbances of the microbiome have been reported to have consequences for immune development, metabolism, and a variety of diseases states including autoimmune diseases, autism and cancer. Thus, it is important to understand how the microbiome is regulated and to identify strategies to manipulate it. MICROREVIEW on: Liu S, da Cunha AP, Rezende RM, Cialic R, Wei Z, Bry L, Comstock LE, Gandhi R, Weiner HL (2016). The Host Shapes the Gut Microbiota via Fecal microRNA. Cell Host & Microbe 19: 32-43. doi: 10.1016/j.chom.2015.12.005. doi: 10.15698/mic2016.04.492 Received 10.02.2016, Accepted 16.02.2016, Published 09.03.2016. Keywords: microbiota, microRNA, host-microbe interaction, dysbiosis, colitis. miRNA affects bacterial gene expression and bacterial growth We cultured bacteria with fluorescence labeled miRNA mimics and observed that the miRNAs entered bacteria. Specifically, they co-localized with bacterial nucleic acids, which provide the temporal and spatial basis for bacterial gene expression regulation. We then aligned the bacterial gene sequences to the miRNA database. Unexpectedly, we found that all bacterial gene sequences we queried were found to pair with different miRNAs, and these miRNAs were derived from host species including C. elegans, Dro- sophila, and mammals (mice and humans). To ask whether miRNAs altered bacterial gene expression, we synthesized miRNA mimics and added them to the culture of bacteria such as F. nucleatum and E. coli. We found that the tran- scripts of the bacterial genes were specifically altered upon miRNA treatment and that bacterial growth was affected by miRNA treatment. Control of the gut microbiome by fecal microRNA Shirong Liu and Howard L. Weiner* Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA. miRNA shapes gut microbiota miRNA shapes gut microbiota We then asked whether this novel mechanism of regula- doi: 10.15698/mic2016.04.492 Received 10.02.2016, Accepted 16.02.2016, Published 09.03.2016. Keywords: microbiota, microRNA, host-microbe interaction, dysbiosis, colitis. Microbial Cell \| April 2016 \| Vol. 3 Nr. 4 176 OPEN ACCESS \| www.microbialcell.com S. Liu and H.L. Weiner (2016) microRNAs shape microbiota FIGURE 1: Microbe-host interactions in the gut lumen. (A) Bacterial to host mediators. (B) Non-specific host to bacterial mediators. (C) Spe- cific host to bacteria mediators. FIGURE 1: Microbe-host interactions in the gut lumen. (A) Bacterial to host mediators. (B) Non-specific host to bacterial mediators. (C) Spe- cific host to bacteria mediators. -host interactions in the gut lumen. (A) Bacterial to host mediators. (B) Non-specific host to bacterial mediators. (C) Spe- a mediators. of the host on microbes includes antimicrobial peptides (AMPs) such as RegIII-γ, defensin. These mechanisms are largely non-specific (Figure 1, B). Secreted IgA is involved in maintaining the microbiota, but its importance is not clear since IgA immunodeficiency had little effect on the compo- sition of the gut flora. Here, we have identified host miR- NAs that are able to specifically modulate the gut microbi- ome (Figure 1, C). Our findings identify a novel mechanism by which one could specifically manipulate the gut micro- biome for the treatment of disease. tion could shape the composition of gut microbiome in the host. To address this, we first identified the primary sources of fecal miRNAs to be intestinal epithelial cells (IECs) and Hopx-expressing cells by generating mice, with a cell-type-specific deficiency in the miRNA processing en- zyme, Dicer. Data from 16S rDNA sequencing suggested that IEC-specific miRNA knockout (Dicer1∆IEC) caused dysbiosis of the gut microbiota. This was accompanied by the worsening of colitis in a dextran sulfate sodium (DSS) model. When we isolated the fecal miRNA from wild-type mice and transferred them to Dicer1∆IEC recipient mice by gavage, the gut microbiota in the recipient mice was re- stored to the wild-type phenotype and the colitis was ame- liorated. Finally, we synthesized the miRNA mimic for miR- 1226 and found that it affected growth of E.coli in vitro and when given orally to mice also affected E.coli in vivo. tion could shape the composition of gut microbiome in the host. miRNA shapes gut microbiota To address this, we first identified the primary sources of fecal miRNAs to be intestinal epithelial cells (IECs) and Hopx-expressing cells by generating mice, with a cell-type-specific deficiency in the miRNA processing en- zyme, Dicer. Data from 16S rDNA sequencing suggested that IEC-specific miRNA knockout (Dicer1∆IEC) caused dysbiosis of the gut microbiota. This was accompanied by the worsening of colitis in a dextran sulfate sodium (DSS) model. When we isolated the fecal miRNA from wild-type mice and transferred them to Dicer1∆IEC recipient mice by gavage, the gut microbiota in the recipient mice was re- stored to the wild-type phenotype and the colitis was ame- liorated. Finally, we synthesized the miRNA mimic for miR- 1226 and found that it affected growth of E.coli in vitro and when given orally to mice also affected E.coli in vivo. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant R01 AI43458 to H.L.W. COPYRIGHT The gut microbiome plays an important role in health and disease. Commensals reside in an external environment (i.e., the intestinal lumen), and they interact with the envi- ronment and host through mechanisms that are funda- mentally different from systemic compartments. The host- microbe interaction is a two-way interaction (Figure 1) conditioned by diet, bowel peristalsis and digestive en- zymes. Studies to date have focused on how microbes af- fect the host through factors such as microbial compo- nents, metabolites such as LPS, short chain fatty acids (SCFAs) and polysaccharide A (PSA) (Figure 1, A). The effect © 2016 Liu and Weiner. This is an open-access article re- leased under the terms of the Creative Commons Attribu- tion (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged. Please cite this article as: Shirong Liu and Howard L. Weiner (2016). Control of the gut microbiome by fecal microRNA. Microbial Cell 3(4): 176-177. doi: 10.15698/mic2016.04.492 Microbial Cell \| April 2016 \| Vol. 3 Nr. 4 OPEN ACCESS \| www.microbialcell.com 177
https://openalex.org/W4391532585	https://www.jca.org.br/jca/article/download/3548/3533	English	null	Effects and parameters of taVNS in individuals with atrial fibrillation: An infographic	Journal of Cardiac Arrhythmias	2,024	cc-by	366	https://doi.org/10.24207/ABEC.2023.22380 https://doi.org/10.24207/ABEC.2023.22380 Effects and parameters of taVNS in individuals with atrial fibrillation: An infographic Maria Williane de Sousa Ribeiro1, Nicole Beatriz Nascimento Miranda1, Ferna Dantas Estevam1, Tiago da Silva Lopes2 e de Sousa Ribeiro1, Nicole Beatriz Nascimento Miranda1, Fernanda Leite Dias m1, Tiago da Silva Lopes2 1. Bahia Adventist College – Cachoeira (BA), Brazil 2. Center for Mathematics, Computing and Cognition – Federal University of ABC – Santo André (SP), Brazil 1. Bahia Adventist College – Cachoeira (BA), Brazil 2. Center for Mathematics, Computing and Cognition – Federal University of ABC – Santo André (SP), Brazil 1. Bahia Adventist College – Cachoeira (BA), Brazil 2. Center for Mathematics, Computing and Cognition – Federal University of ABC – Santo André (SP), Brazil Introduction: Transcutaneous auricular vagus nerve stimulation (taVNS) is a non-invasive neuromodulatory technique used to modulate autonomic excitability through electrical stimulation of the vagus nerve. Studies have demonstrated that taVNS can be used as a therapeutic strategy for managing arterial fibrillation (AF). This infographic aims to highlight the evidence on the effect and parameters of taVNS in individuals with AF. Method: We conducted a review using the combination of descriptors in PubMed (Transcutaneous Electric Nerve Stimulation) AND (Nerve Stimulation, Vagal) OR (Nerve Stimulation, Vagus) OR (Nerve Stimulations, Vagal) AND (Atrial Fibrillation), which included six clinical trials of human adults over 18 years old using taVNS. Results: taVNS can promote an increase in the PQ interval on the electrocardiogram by increasing parasympathetic tone. Furthermore, taVNS may prevent AF progression through anti-arrhythmogenic effects, such as anti-adrenergic neural remodeling mechanisms, decreased pro-inflammatory cytokines, and decreased AF burden. Discussion: The acute effect of taVNS may increase P wave alternation (PWA), which is believed to be related to AF. However, the chronic effect of taVNS for six months promotes a decrease in PWA and AF burden. Furthermore, taVNS may alter cardiac variability ratio parameters by increasing the HF/LF ratio in individuals with AF, which may be a sign of sympathovagal rebalancing. However, taVNS may improve autonomic balance by decreasing the HF/LH ratio in healthy individuals. Conclusion: taVNS may be a useful therapeutic tool in the management of individuals with AF due to its anti-arrhythmogenic effects. J. Cardiac Arrythmias, São Paulo, 2023
https://openalex.org/W4253259458	https://osf.io/rzv2w/download	English	null	Correcting Misperceptions: The Causal Role of Motivation in Corrective Science Communication about Vaccine and Food Safety	null	2,019	public-domain	11,558	ttps://doi.org/10.1177/1075547019898256 Science Communication 1–30 © The Author(s) 2020 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/1075547019898256 journals.sagepub.com/home/scx 8256 SCXXXX10.1177/1075547019898256Science Communicationvan Stekelenburg et al. 8256 SCXXXX10.1177/1075547019898256Science Communicationvan Stekelenburg et al. 256 SCXXXX10.1177/1075547019898256Science Communicationvan Stekelenburg et al. Research Article Correcting Misperceptions: The Causal Role of Motivation in Corrective Science Communication About Vaccine and Food Safety Correcting Misperceptions: The Causal Role of Motivation in Corrective Science Communication About Vaccine and Food Safety Aart van Stekelenburg1 , Gabi Schaap1, Harm Veling1, and Moniek Buijzen1 Corresponding Author: Aart van Stekelenburg, Communication & Media, Behavioural Science Institute, Radboud University, Montessorilaan 3, Nijmegen 6525 HR, Netherlands. Email: a.vanstekelenburg@bsi.ru.nl Abstract Some people stick to beliefs that do not align with scientific consensus when faced with science communication that contradicts those misperceptions. Two preregistered experiments (total N = 1,256) investigated the causal role of motivated reasoning in the effectiveness of correcting misperceptions. In both experiments, accuracy-driven reasoning led to a larger corrective effect of a science communication message than reasoning driven by directional motivation. Individuals’ default reasoning made them just as receptive to the correction as accuracy-driven reasoning. This finding supports a more optimistic view of human receptivity to science communication than often found in the literature. 1Radboud University, Nijmegen, Netherlands Keywords misperceptions, motivated reasoning, science communication, vaccination, food safety Science Communication 00(0) 2 Some people hold beliefs that do not align with the scientific consensus. And some of them stick to these misperceptions, even when they are faced with evidence that directly contradicts their beliefs (e.g., Holman & Lay, 2019; Nyhan et al., 2013; Nyhan & Reifler, 2010). The current research investi- gated the causal role of motivation in holding on to misperceptions about scientific facts in the face of corrective science communication. To what extent is sticking to misperceptions driven by directional motivated reason- ing? And can reasoning be influenced to increase the effectiveness of correc- tive information? Vaccine and Food Safety Misperceptions are factual beliefs that are false or contradict the best avail- able evidence in the public domain (Flynn et al., 2017). They can be very harmful, for example, when they are related to health issues. Two health issues that suffer from misperceptions are vaccination and food safety. While vaccination programs are one of the most successful health interventions in the history of humankind, a notable proportion of the general public is skepti- cal of vaccines (Larson et al., 2016). This skepticism influences the decision to vaccinate (Joslyn & Sylvester, 2019), which can have very harmful conse- quences. For instance, preliminary data from 2019 indicate that measles cases have spread fast among clusters of unvaccinated people in countries with high overall vaccination coverage (World Health Organization, 2019). Furthermore, in the United States, vaccine refusal has been linked with out- breaks of vaccine-preventable diseases (Phadke et al., 2016). Trends such as these have led the World Health Organization to classify vaccine hesitancy as one of the most important threats to global health in 2019, together with threats such as climate change and HIV (World Health Organization, n.d.). Focusing on the second issue, food safety, a primary example of harmful misperceptions can be found in beliefs about E numbers. E numbers are used in Europe to identify food additives and were introduced to reassure consum- ers that additives indicated with these numbers are safe to consume. Food additives indicated with E numbers can have important functions, such as preventing the growth of harmful bacteria that cause potentially fatal diseases like botulism (Food Standards Agency, n.d.). Paradoxically though, E num- bers have evolved into a cause of worry among consumers over the safety of these additives (Haen, 2014; Saltmarsh, 2015). The misperception that these additives are unsafe can have serious negative consequences for food produc- tion, with food producers forced to look for costly replacements for safe and tested food additives. Moreover, consumers’ food choices likely suffer from avoiding food products that are actually safe to eat (Shim et al., 2011). van Stekelenburg et al. 3 In the current research, we focused on correcting one misperception related to vaccination and one misperception related to food safety. By study- ing two different topics, we tested whether our findings are generalizable to diverse topics of misperceptions. Vaccine and Food Safety In the first experiment, we provided correc- tive information to the misperception that childhood vaccines can “overload” a child’s immune system. In the second experiment, we provided corrective information to the misperception that food additives indicated with E num- bers are unsafe to consume. Correcting such misperceptions is important because these beliefs inform opinions on important policy, such as policy about unvaccinated children going to day care, and behavioral intentions, such as the intention to avoid safe to consume food products. These beliefs may even be determinants of behavior (e.g., Joslyn & Sylvester, 2019). In addition, having an informed citizenry is a valid goal in itself. Motivated Reasoning Misperceptions can be difficult to correct because people are biased infor- mation processors. One important psychological source for bias is called directional motivated reasoning (Kunda, 1990). This means that one has a directional goal in reasoning, for instance, a preferred outcome. A direc- tional goal can lead to biased reasoning in support of that goal. This type of reasoning explains the heartfelt supporter of the free market who believes that climate change is a hoax, because the preferred outcome of minimum climate change regulation biases reasoning toward believing climate change is not real. Similarly, directional motivated reasoning can explain the heavy smoker who rejects the evidence for a link between smoking and lung can- cer, because one prefers to be able to smoke without having to worry about negative health effects. It can even explain paradoxical findings of correc- tive messages leading to “backfire” effects (Hart & Nisbet, 2012; Nyhan et al., 2013; Nyhan & Reifler, 2010; Zhou, 2016): Directional motivation can make one counterargue the corrective message, thereby strengthening the original misperception. While numerous studies have focused on identifying sources of direc- tional motivated reasoning, evidence for a causal role of directional moti- vated reasoning in holding on to misperceptions in the face of corrective information is to date limited. Previous research identified prior beliefs as a general source of directional motivated reasoning (e.g., Lord et al., 1979; Taber & Lodge, 2006), as well as more specific sources such as partisanship (Bolsen et al., 2014; Gaines et al., 2007; McCright & Dunlap, 2011) and related sources such as identity protection (Kahan et al., 2017; Lyons, 2018), conspiracy ideation and worldviews (Lewandowsky et al., 2013), and Science Communication 00(0) 4 solution aversion (Campbell & Kay, 2014). Research on the causal effect of directional motivated reasoning in sticking to misperceptions, however, is difficult because one cannot easily manipulate an individual’s political ide- ology or worldview. To test a causal link between directional motivation and holding on to misperceptions, experiments where motivations are manipu- lated are needed to disentangle the effects of motivation from other pro- cesses (Leeper & Slothuus, 2014). This can be done, for instance, by seeking ways to experimentally induce directional motivated reasoning (Nyhan & Zeitzoff, 2018). There is some experimental research on the causal effect of directional motivated reasoning on opinion formation (Bolsen & Druckman, 2015; Bolsen et al., 2014). Motivated Reasoning This effect is demonstrated by comparing directional motivated reasoning to accuracy motivated reasoning. In contrast to direc- tional motivated reasoning, an accuracy-motivated individual has the goal to come to what is believed to be the most accurate conclusion (Kunda, 1990). This experimental research has shown that inducing accuracy-driven reason- ing has the potential to reduce the biasing effect of directional motivation on opinion formation (Druckman, 2012), for instance, regarding support for energy policy (Bolsen et al., 2014) and emergent energy technologies (Bolsen & Druckman, 2015). However, it is unclear whether these effects on opinion formation generalize to factual beliefs. Opinions are more subjective than factual beliefs, possibly making them more receptive to effects of motivated reasoning than, for instance, factual beliefs about the safety of vaccines. Nonetheless, these findings suggest that inducing accuracy motivation might be a suitable intervention to increase the effectiveness of corrective science communication in the face of misperceptions. Yet, there is a lack of studies on inducing accuracy motivation. Understanding when accuracy-driven reason- ing overtakes directional reasoning is important in studying misperceptions (Druckman & McGrath, 2019; Flynn et al., 2017). The current research aims to fill these gaps. The aim was to study the causal role of directional motivated reasoning in holding on to mispercep- tions in the face of corrective science communication and to investigate whether inducing accuracy motivation can aid in correcting misperceptions. We explored, not hypothesized, potential effects of directional motivation on policy opinions and intentions, because results in the literature regarding these second order outcomes are mixed (Flynn et al., 2017; Nyhan & Reifler, 2015). We conducted two preregistered experiments in which a science com- munication message was used to correct a misperception. In both experi- ments, the message itself was effective in reducing endorsement of the misperception. More important, type of motivation in processing of the mes- sage affected how strong the corrective effect of the messages was, with van Stekelenburg et al. 5 accuracy-driven reasoning leading to a larger correction than directional motivated reasoning. Our findings also partially demonstrated that the influ- ence of motivation was not limited to the misperception itself but extends to second order outcomes policy support and intentions. Method Among others, the hypotheses, sampling procedure, main analyses, and exclusion criteria were preregistered on the Open Science Framework (OSF; https://bit.ly/2P2EGdP). Initially, successful completion of an instructional manipulation check (IMC; see Measures section) was included as one of the exclusion criteria. IMCs are used to detect whether participants read the instructions and can increase statistical power and reliability of the data (Oppenheimer et al., 2009). After collecting data from 38 participants, we decided that the IMC was too sensitive. Based on the IMC, we needed to exclude 31 participants (81.58%), who actually appeared to complete the experiment seriously, as indicated by completion times (M = 474.23 sec- onds), reading time of the corrective information (M = 83.88 seconds) and coherence of responses to open questions. We believe that the overly sensi- tive IMC may be due to the instructions of the IMC being irrelevant to the question that was asked, not due to a lack of effort on the side of the partici- pants. Therefore, this exclusion criterion was dropped. The preregistration was updated to reflect this new exclusion criterion (https://bit.ly/2Ry9SU4). We chose to keep the data for exploratory analyses (total N = 404), for ethi- cal reasons (i.e., not wasting participants’ serious responses). The manipula- tion check and main analysis (reported below) included only those participants who participated in the experiment after the preregistration was updated (n = 372). All data and the R script for the analysis can be found on the project page on the OSF (https://bit.ly/2Pv1mlY). Both Experiments 1 and 2 (see below) are part of a research project that was reviewed and approved by the Ethics Committee Social Science at Radboud University (ref. ECSW-2018-056). Participants and Design. Participants were screened and recruited using online crowdsourcing platform Prolific. Prolific has been demonstrated to yield high-quality data and more diverse participants than student samples or other major crowdsourcing platforms (Peer et al., 2017). We screened participants prior to the experiment on whether they endorsed the misperception. They were presented with a list of five statements, including the misperceptions from Experiment 1 and Experiment 2 (see below) and were asked to select all of the statements they believed to be true. Only participants who indicated the statement “Giving young children multiple vaccinations overloads their immune system” was true were eligible for participation in the experiment. Experiment 1: Vaccine Safety In the first experiment, a vaccine-related misperception was corrected. By measuring endorsement of the misperception before (prior) and after (poste- rior) motivation was manipulated and corrective information was presented, we could expose all participants to the corrective message while investigat- ing the influence of motivation. Based on previous research on motivated reasoning (Bolsen & Druckman, 2015; Bolsen et al., 2014), we believed that an accuracy-motivated individual would be more susceptible to corrective information than a directionally motivated individual. Therefore, we expected that inducing accuracy motivation would lead to lower posterior endorsement of the misperception than inducing directional motivation, while controlling for prior belief certainty. Hypothesis 1: Inducing accuracy motivation will lead to lower posterior endorsement of the misperception than inducing directional motivation, while controlling for prior belief certainty. Although accuracy motivation may amplify the corrective effect of a science communication message, the effect of motivation on a belief is sub- ject to reality constraints (Kunda, 1990; Molden & Higgins, 2005). When one holds a belief about reality with high certainty, the degree to which motivation can affect the interpretation of new information regarding that belief is constrained. For instance, you can prefer to be 20 cm taller than you actually are, but the certainty with which one’s height can be deter- mined should inhibit this effect of motivation. Therefore, we expected that when individuals were very certain of their prior belief the effect of motiva- tion would be limited, while under uncertainty the effect of motivation should be larger. Specifically, we expected that motivation would interact with prior belief certainty, such that the more certain the prior belief was, the smaller the corrective effect of accuracy motivation on the posterior belief would be. Hypothesis 2: Motivation will interact with prior belief certainty, such that the more certain the prior belief is, the smaller the corrective effect of accuracy motivation on the posterior belief will be. Science Communication 00(0) 6 Method Following a Bayesian sequential sampling procedure with optional stop- ping and maximum N (Schönbrodt et al., 2017), 542 participants (U.K. nationals) were recruited. Participants each received £0.85 for participating van Stekelenburg et al. 7 in the experiment. During the sequential sampling procedure, we checked the Bayes factor (BF) at predetermined intervals to evaluate the evidence in the data for or against the first hypothesis. The advantage of this procedure is that it allows for efficient data collection. No more or less data are collected than necessary for a specified level of certainty, since the BF indicates both evi- dence for an effect and evidence in favor of no effect. We continued data collection until there was strong evidence (0.1 > BF01 > 10; Schönbrodt et al., 2017) in favor or against our first hypothesis. We started checking the BF for the hypothesized effect when 171 participants (enough to detect a medium effect size with 0.90 power and α = .05; Faul et al., 2009) completed the experiment and continued to check it after every set of 50 new partici- pants. If at any time the BF reached the required level of evidence for or against the hypothesis, data collection would be stopped. This was never the case, therefore data collection continued until the maximum N of 500 partici- pants (excluding participants before the updated preregistration). We collected data from a total of 542 participants. Three participants who completed the experiment too fast and one who did not participate through Prolific were excluded (not preregistered). This resulted in the planned 538 participants, of which 500 participated after the updated pre- registration. At the start of the experiment, participants indicated their endorsement of the misperception. Even though we screened participants before the experiment on whether they held the misperception, only 407 (75.65%) indicated that they endorsed the misperception at the beginning of the experiment (score > 0 on the misperception measure; see Measures section). As we are interested in correcting misperceptions, only these par- ticipants were included in the analyses. Three participants who did not understand the misperception measure were identified by means of an open answer question in the experiment. Their response to the open answer ques- tion indicated a large correction in their endorsement of the misperception, but their response on the misperception measure conflicted with this. They were excluded post hoc. Method This resulted in 404 participants (269 female, 135 male, Mage = 38.95 years, SDage = 12.73) in the total data set, of which 372 participated after the updated preregistration. The experiment consisted of a one-factor (motivation in reasoning; accu- racy-driven reasoning vs. directional reasoning) between-subjects design. In one condition, accuracy motivation in reasoning was induced (n = 209), in the other condition, directional motivation in reasoning was induced (n = 195). Materials and Procedure. The experiment was conducted using Qualtrics survey software. First, prior endorsement of the misperception Science Communication 00(0) 8 was measured. Subsequently, participants were randomly assigned to two conditions. They were instructed to read a text either in a way that induces directional motivation or in a way that induces accuracy motivation, based on Bolsen et al. (2014). We piloted an earlier version of the manipulation in a preregistered experiment (https://bit.ly/2rwk2Ka). Based on the results of that pilot, we made some improvements to the manipulation. The instruc- tions in the directional condition (85 words) included telling participants that we were interested in their judgment because they believed that vac- cines can overload a child’s immune system and asking participants to be aware of this belief when reading the upcoming text. Furthermore, we asked them to apply their perspective, and to think of what would confirm their initial belief. The instructions in the accuracy condition (79 words) included telling participants we were interested in their judgment because we studied how people process information and come to conclusions and asking participants to be evenhanded. Furthermore, we asked them to apply various perspectives and to think of what would disprove their initial belief (for the full texts, see Appendix A). Participants were required to stay on the page with the instructions for at least 10 seconds. After 10 seconds, the button to continue to the next page appeared, which included the text “I will be aware of my belief and view the information from my perspective. I will try to think of what could confirm my initial belief” (directional condition) or “I will view the information in an evenhanded way and from various perspectives. I will try to think of what could disprove my initial belief” (accuracy condition). ( y ) Both groups were then presented with the science communication mes- sage (386 words), which contained information correcting the misperception. Method The information was based on information from Science magazine (Hickok, 2018), the National Health Service (NHS; 2016), the University of Oxford Vaccine Knowledge Project (n.d.), and the American Academy of Pediatrics (2008). The text explained that vaccines do not overload children’s immune system and that this knowledge is based on many scientific studies. One recent study was explained in more detail and an explanation was given of why vaccines do not overload the immune system. A graph was included in the text (see Appendix B for the full message). Both groups were then presented with the science communication mes- sage (386 words), which contained information correcting the misperception. After reading the corrective text, participants’ endorsement of the misper- ception was measured again (the posterior belief). We explained to partici- pants that this second measure was not a test, but that we were interested in their belief. The remaining variables were measured, among which was an open answer question in which participants were asked to give a short justifi- cation for their answer on the measure of posterior endorsement of the misperception. van Stekelenburg et al. 9 Measures. Endorsement of the misperception was measured twice, once at the beginning of the experiment and once after the motivation manipulation and corrective message. This not only increased power to detect an effect but also allowed us to investigate both the corrective effect of the message itself and the effect of motivation on receptivity to this message. Endorsement of the misperception was measured by asking participants to what extent they believed the following statement to be true: “Giving young children multiple vaccinations overloads their immune system.” Their response was measured on a visual analogue scale ranging from I am 100% certain this is false (−100) to I am 100% certain this is true (100) with I don’t know in the middle (0). Since we included only those participants who endorsed the misperception at the beginning of the experiment, the prior score on endorsement of the misperception is simply a score of how certain they were of their belief in the statement (i.e., prior belief certainty). The manipulation check consisted of six statements. Method Half of these state- ments reflected the instructions from the directional motivation condition (e.g., “While reading the information, I tried to view the information from my perspective”), the other half were in line with the accuracy motivation instruc- tions (e.g., “While reading the information, I tried to view the information from various perspectives”). Responses were measured on a 7-point scale rang- ing from strongly disagree to strongly agree. Average scores were calculated for following the directional and accuracy motivation instructions separately. Several exploratory variables were measured at the end of the experiment. Perceived change in belief certainty was measured by asking participants if they became more or less certain of their initial belief, measured on a visual analogue scale ranging from I am much less certain now (−50) to I am much more certain now (50), with my certainty has not changed in the middle (0). Support for policy aimed at stimulating people to vaccinate their children and intention to vaccinate one’s children were measured with responses to single statements. Open-minded cognition (OMC) was measured using the OMC scale developed and validated by Price et al. (2015). Trust in the NHS, trust in scientists, perceived reliability of the information provided to the participant, perceived knowledge of vaccines, and importance of the topic were measured with single response items. All of these items were measured on 7-point scales. Additionally, an IMC (Oppenheimer et al., 2009) was included in a short text that introduced the demographic questions, to detect participants who were not following the instructions. The text instructed participants to ignore the first question, which was about political parties, and instead to mark the “Other” box and write “I read the instructions.” Religiosity and belief in com- plementary or alternative medicine were measured using a discrete (yes or no) response. Finally, age, gender, and education were asked. For the complete 10 Science Communication 00(0) wording of all the questions, see the supplemental material. Not all variables were included in the analyses, because they were not of direct interest to the current research. Some background variables (e.g., trust) were measured because they could be relevant to compare the current research to existing research (e.g., Nyhan et al., 2014), others (e.g., OMC) were measured because they could provide useful insight for future research on correcting mispercep- tions. All data are available on the OSF (https://bit.ly/2Pv1mlY). Data Analysis. Method One analysis was conducted to test both hypotheses simultane- ously: an analysis of covariance (ANCOVA) with posterior belief as the dependent variable and motivation condition as the independent variable, prior belief certainty as continuous predictor, including the interaction term between prior belief certainty and motivation condition. The first hypothesis regarded the main effect of motivation on posterior endorsement of the misper- ception, the second regarded the interaction between motivation and prior belief certainty. In the confirmatory analyses, complementary BF are reported. Results Manipulation Check. Two one-tailed, independent-samples t tests were con- ducted to test whether the motivation manipulation had the expected effect on participants’ motivation in reasoning about the corrective information. As expected, participants in the directional condition (M = 5.38, SD = 1.06) scored significantly higher on the questions measuring directional motivation than participants in the accuracy condition (M = 4.96, SD = 1.18), t(369.75) = 3.57, p < .001, d = 0.37, 95% confidence interval (CI) [0.16, 0.58]. Also as expected, participants in the accuracy condition (M = 5.61, SD = 1.02) scored significantly higher on the questions measuring accuracy motivation than participants in the directional condition (M = 5.04, SD = 1.22), t(348.47) = 4.86, p < .001, d = 0.51, 95% CI [0.30, 0.72]. Confirmatory Analyses. In support of the first hypothesis, the main effect of motivation condition on posterior endorsement of the misperception while controlling for prior belief certainty was significant, F(1, 368) = 4.01, p = .046, ηp 2 = .011, 90% CI [.000, .035], BF01 = 0.79. Posterior endorse- ment of the misperception was significantly lower in the accuracy condition (M = 12.98, SD = 56.86) than in the directional condition (M = 22.77, SD = 54.39), indicating that the corrective science communication message was more effective for accuracy-motivated participants than directionally motivated participants. See Figure 1 for the prior and posterior scores per motivation condition. The interaction effect between prior belief certainty and van Stekelenburg et al. 11 Figure 1. Prior and posterior endorsement of the vaccine misperception separated by motivation condition. Note. Error bars indicate standard errors of the mean. Figure 1. Prior and posterior endorsement of the vaccine misperception separated by motivation condition. Note. Error bars indicate standard errors of the mean. motivation condition was not statistically significant (p = .408, BF01 = 0.16), meaning that the effect of motivation on posterior endorsement of the misper- ception was not moderated by prior belief certainty. Thus, the second hypoth- esis was not supported. Analysis of the standardized residuals of the ANCOVA model indicated that four observations were classified as model outliers at > 3 SD. Further investigation showed that these four participants all changed their belief in the most extreme way possible; from 100 to −100. Although not preregis- tered, another ANCOVA was conducted to examine the effect of these four observations on the results. Discussion Experiment 1 provided support for a causal effect of motivated reasoning on the effectiveness of correcting a vaccine-related misperception. In the face of a corrective science communication message, directional motivated reason- ing made participants stick to a misperception more than accuracy motivated reasoning. Contrary to our expectations, the influence of motivation on the effectiveness of the correction was not moderated by certainty of the prior belief. Motivation seems to play a role in the effectiveness of a corrective message, regardless of how certain an individual was of a misperception. Exploratory analyses indicated that there was no effect of motivation on sup- port for policy aimed at increasing vaccination or intention to vaccinate. This experiment is the first to demonstrate a causal role of motivated reasoning in sticking to misperceptions in the face of corrective information. However, there are two limitations. First, the design of the experiment did not allow us to draw conclusions about the effectiveness of inducing accu- racy motivation in correcting misperceptions because we could only com- pare the accuracy condition to the directional condition. It was unclear whether accuracy motivation would increase the effectiveness of the correc- tion compared with a more natural (no motivational instructions) situation. Second, this experiment addressed one topic: vaccination. It was unclear whether the results would generalize to other topics. Both limitations were addressed in Experiment 2. Results This second ANCOVA, excluding the four model outliers, also yielded a significant main effect of motivation condition on posterior endorsement of the misperception after controlling for prior belief certainty, F(1, 364) = 6.20, p = .013, ηp 2 = .017, 90% CI [.002, .045], BF01 = 2.22. Again, the interaction effect was not statistically signifi- cant (p = .771, BF01 = 0.11). Exploratory Analyses. In addition to the hypotheses tests, exploratory analyses were conducted. In all these analyses, model outliers based on standardized residuals >3 SD were removed from the model. For the complete (preregis- tered) exploratory analyses, see the supplemental material. 12 Science Communication 00(0) The first of the exploratory analyses was similar to the main analysis, but included all participants, not just those who participated after the updated preregistration. The ANCOVA indicated that the main effect of motivation condition on posterior endorsement of the misperception found in the main analysis was again significant when all observations were included, F(1, 397) = 8.88, p = .003, ηp 2 = .022, 90% CI [.004, .051]. p Second, we explored differences between the two conditions on policy support for vaccination and intention to vaccinate. Two ANCOVAs, with motivation condition as the independent variable, policy support or intention as the dependent variable, and controlling for prior belief certainty, yielded no significant effect of motivation condition (both ps > .09). Experiment 2: Food Safety In the second experiment, a food-related misperception was corrected and a control condition was added. First, as in Experiment 1, we expected that inducing accuracy motivation would lead to lower posterior endorsement of van Stekelenburg et al. 13 the misperception than inducing directional motivation, while controlling for prior belief certainty. the misperception than inducing directional motivation, while controlling for prior belief certainty. Hypothesis 1: Inducing accuracy motivation will lead to lower posterior endorsement of the misperception than inducing directional motivation, while controlling for prior belief certainty. Second, research on motivated reasoning has shown that directional rea- soning is likely to be the default reasoning style in information processing (Nyhan & Reifler, 2019). Therefore, we expected that inducing accuracy moti- vation would lead to lower posterior endorsement of the misperception than not inducing any motivation, while controlling for prior belief certainty. Hypothesis 2: Inducing accuracy motivation will lead to lower posterior endorsement of the misperception than not inducing any motivation, while controlling for prior belief certainty. The second hypothesis from Experiment 1, regarding the interaction between motivation and prior belief certainty, was dropped because of a lack of support for this hypothesis in Experiment 1. Method The setup was similar to that of Experiment 1, with the addition of a control condition in which we did not manipulate participants’ motivation in reason- ing about the corrective message. Furthermore, the IMC was replaced by an instructed-response item (see Materials and Procedure section). Just as Experiment 1, the experiment was preregistered on the OSF (https://bit. ly/2Ryop21) and all data and the R script for the analysis can be found on the project page on the OSF (https://bit.ly/2Pv1mlY). Participants and Design. Again, participants were screened prior to participat- ing in the experiment. We selected only those that indicated the statement “Food additives indicated with an E number are unsafe to consume” was true. Following a Bayesian sequential sampling procedure with optional stopping and maximum N (Schönbrodt et al., 2017), 1,142 participants (U.K. nation- als) were recruited. As in Experiment 1, they each received £0.85 for partici- pating in the experiment. Again, we checked the BF at predetermined intervals during the sequential sampling procedure. We started checking the BF for the hypothesized effect when 50% of the maximum number of partici- pants completed the experiment and continued to check it after every set of 14 Science Communication 00(0) 111 (10% of max sample) new participants. If the BF for both the accuracy- directional contrast and the accuracy-default contrast indicated there was strong evidence (0.1 > BF01 > 10; Schönbrodt et al., 2017), data collection would be stopped. Since this was never the case, data collection continued until the maximum N of 1,114 participants, which yielded ~0.95 power to replicate the main effect of Experiment 1 (ηp 2 = .022) with α = .05 and a successful screening rate of ~75% (Faul et al., 2009). We collected data from a total of 1,142 participants, because 21 failed an attention check, five did not meet the inclusion criterion related to national- ity, and one completed the experiment too fast. In accordance with the pre- registration, these participants were excluded from the analyses. Of the remaining 1,115 participants (one more than planned), 854 (76.59 %) indi- cated that they endorsed the misperception at the beginning of the experi- ment. Finally, and just as in Experiment 1, two participants who did not understand the misperception measure were identified post hoc and removed from the data. Method This resulted in 852 participants (596 female, 253 male, 2 nonbinary, 1 unidentified, Mage = 35.27 years, SDage = 12.29) in the total data set. The experiment consisted of a one-factor (motivation in reasoning) between-subjects design with three conditions. The directional motivation and accuracy motivation conditions (n = 285 and n = 277, respectively) were the same as in Experiment 1. We added a “default” motivation condition (n = 290), in which we did not manipulate participants’ motivation. Materials and Procedure. The procedure was similar to that of Experiment 1. The instructions in the default condition (17 words) were to read the text like one normally would. In contrast to the other two conditions, no time restric- tions were applied to reading the instructions and the next button did not include any text. The corrective science communication message (362 words) was based on information from the Food Standards Agency (n.d.) and an article published in Food Quality and Preference (Bearth et al., 2014). The text explained that food additives indicated with E numbers are safe to con- sume, that an E number actually means that the additive passed safety tests, a short background about food additives, and how an E number is assigned to an additive. An edited graphic about the development of E numbers from The Netherlands Nutrition Centre (n.d.) was included in the text (see Appendix B for the full message). The IMC was replaced by an instructed-response item, which was placed in the OMC scale. Instructed-response items are useful to identify careless responding (Meade & Craig, 2012). The statement read “To demonstrate you are paying attention, please answer ‘2’.” van Stekelenburg et al. 15 Measures. The measures were the same as in Experiment 1, but edited to reflect the topic at hand. We added a question measuring participants’ belief that what is natural is good, measured by agreement with the statement “In general, I consider what is natural to be good” on a 7-point scale from strongly disagree to strongly agree. In addition, we asked them to report their nation- ality, as a means of checking the screening criterion for nationality. For the complete wording of all the questions, see the supplemental material. Data Analysis. Both hypotheses were tested by conducting an ANCOVA with posterior belief as the dependent variable, condition as the indepen- dent variable, and prior belief certainty as continuous predictor. Method The first hypothesis, comparing accuracy motivation to directional motivation, was tested with the subsample of participants in the accuracy and directional conditions (n = 562). The second hypothesis, comparing accuracy motiva- tion to the default motivation, was tested with a subsample of participants in the accuracy and default conditions (n = 567). Again, BF are reported in the confirmatory analyses. Results Manipulation Check. Similar to Experiment 1, one-tailed, independent-sam- ples t tests were conducted to test whether the motivation manipulation had the expected effect on participants’ motivation in reasoning about the correc- tive text. As expected, on the questions measuring accuracy motivation par- ticipants in the accuracy condition (M = 5.50, SD = 1.00) scored significantly higher than participants in the directional condition (M = 4.93, SD = 1.16), t(552.49) = 6.29, p < .001, d = 0.53, 95% CI [0.36, 0.70], and the default condition (M = 5.00, SD = 0.96), t(560.75) = 6.07, p < .001, d = 0.51, 95% CI [0.34, 0.68]. Also as expected, on the questions measuring directional motivation participants in the directional condition (M = 5.06, SD = 1.14) scored significantly higher than participants in the accuracy condition (M = 4.47, SD = 1.20), t(556.57) = 5.96, p < .001, d = 0.50, 95% CI [0.33, 0.67], and the default condition (M = 4.67, SD = 1.09), t(571.05) = 4.18, p < .001, d = 0.35, 95% CI [0.18, 0.51]. Confirmatory Analyses. Replicating the results from Experiment 1 and support- ing the first hypothesis, the main effect of motivation condition on posterior endorsement of the misperception, comparing the accuracy and directional conditions, was significant, F(1, 559) = 14.11, p < .001, ηp 2 = .025, 90% CI [.008, .050], BF01 = 83.59. Posterior endorsement of the misperception was lower in the accuracy condition (M = −16.71, SD = 55.21) than in the Science Communication 00(0) 16 Figure 2. Prior and posterior endorsement of the food misperception separated by motivation condition. Note. Error bars indicate standard errors of the mean. Figure 2. Prior and posterior endorsement of the food misperception separated by motivation condition. Note. Error bars indicate standard errors of the mean. directional condition (M = 1.44, SD = 55.41), again indicating that the cor- rective science communication message was more effective for accuracy- motivated participants than directionally motivated participants. Contrary to the second hypothesis, comparing the accuracy condition to the default con- dition yielded no significant effect of motivation (p = .397, BF01 = 0.13), indicating that participants who did not receive motivation instructions were just as receptive to the corrective message as accuracy-motivated partici- pants. See Figure 2 for prior and posterior belief scores per condition. Results directional condition (M = 1.44, SD = 55.41), again indicating that the cor- rective science communication message was more effective for accuracy- motivated participants than directionally motivated participants. Contrary to the second hypothesis, comparing the accuracy condition to the default con- dition yielded no significant effect of motivation (p = .397, BF01 = 0.13), indicating that participants who did not receive motivation instructions were just as receptive to the corrective message as accuracy-motivated partici- pants. See Figure 2 for prior and posterior belief scores per condition. Exploratory Analyses. Again, in all these analyses, model outliers based on stan- dardized residuals > 3 SD were removed from the model. The complete (pre- registered) exploratory analyses can be found in the supplemental material. First, also as per the preregistration, we explored the remaining contrast by comparing posterior endorsement of the misperception in the directional condition to the default condition (n = 575). An ANCOVA, similar to the main analysis, indicated that participants in the default condition (M = −11.12, SD = 58.11) displayed significantly lower posterior endorsement of the misperception than participants in the directional condition (M = 1.44, SD = 55.41), F(1, 572) = 8.15, p = .004, ηp 2 = .014, 90% CI [.003, .034], van Stekelenburg et al. 17 indicating that participants who did not receive motivation instructions were more receptive to the corrective message than participants with a directional motivation. indicating that participants who did not receive motivation instructions were more receptive to the corrective message than participants with a directional motivation. Second, we explored differences between the accuracy and the direc- tional conditions on (1) support for policy aimed at reducing the use of E numbers in food products and (2) intention to avoid the consumption of food products with E numbers in them. Both ANCOVAs, with condition as the independent variable, policy support or intention as the dependent variable, and controlling for prior belief certainty, yielded a significant effect of moti- vation condition. Regarding policy against E numbers, participants in the accuracy condition (M = 4.81, SD = 1.51) scored significantly lower than participants in the directional condition (M = 5.30, SD = 1.52), F(1, 559) = 13.60, p < .001, ηp 2 = .024, 90% CI [.007, .048]. Results Mediation analysis using bootstrapping procedures (N = 10.000; Imai et al., 2010) indicated that this effect was mediated by posterior endorsement of the misperception (β = 0.24, 95% CI [0.11, 0.37], p < .001). Of the total effect of motivation on policy support, 52.62% was explained by posterior endorsement of the misperception, leaving a marginally significant average direct effect (β = 0.21, 95% CI [<0.00, 0.42], p = .052). Similarly, regarding intention to avoid E numbers, participants in the accuracy condition (M = 3.86, SD = 1.55) scored significantly lower than participants in the directional condition (M = 4.21, SD = 1.56), F(1, 559) = 6.17, p = .013, ηp 2 = .011, 90% CI [.001, .030]. This effect was also mediated by posterior endorsement of the misperception (β = 0.27, 95% CI [0.12, 0.41], p < .001). Of the total effect of motivation on intention to avoid E numbers, 78.93% was explained by posterior endorsement of the misperception, leaving a nonsignificant aver- age direct effect (p = .509). These results demonstrate that the effect of motivation is not limited to the misperception. Finally, we explored the hypothesized interaction effect between prior belief certainty and motivation condition from Experiment 1. We conducted the same ANCOVA as in Experiment 1 with only the accuracy and directional conditions. Again, the ANCOVA yielded a nonsignificant interaction effect (p = .430), meaning that the effect of motivation on posterior endorsement of the misperception was not moderated by prior belief certainty. Discussion Experiment 2 replicated and extended the findings from Experiment 1. Again, we found support for a causal role of motivated reasoning in the effectiveness of correcting a misperception, this time for a misperception related to food safety, thereby supporting the generalizability of these results. Contrary to Science Communication 00(0) 18 our expectations, we did not find that inducing accuracy motivation strength- ened the corrective effect of the science communication message compared to the default motivation. Instead, exploratory analyses indicated that the default motivation lead to lower posterior endorsement of the misperception than directional motivation. In contrast to Experiment 1, the results indicated that the effect of motivation on posterior endorsement of the misperception was reflected in a difference in policy support and intention. Accuracy- motivated participants reported lower support for policy aimed at reducing the use of E numbers in food products and intention to avoid the consumption of food products with E numbers in them than directionally motivated partici- pants. This difference was (partly) explained by the change in endorsement of the misperception. General Discussion Only when we induced a directional motiva- tion did participants stick to the misperception more than participants would by default. Moreover, even individuals who were induced to have a direc- tional motivation on average reduced their endorsement of the mispercep- tion, rather than increasing it. did find that directional reasoning might reduce the corrective effect of a science communication message, we found no evidence for the prevailing assumption in research on motivated reasoning that corrections often back- fire (see Hart & Nisbet, 2012; Nyhan et al., 2013; Nyhan & Reifler, 2010; Zhou, 2016). There has been some debate about this backfire effect (Druckman & McGrath, 2019). In line with other research (Garrett et al., 2013; Guess & Coppock, 2018; Hill, 2017; van der Linden et al., 2018; Wood & Porter, 2019), we found no evidence of such a polarizing effect of our science com- munication message. To the contrary, our findings demonstrated that indi- viduals are not automatically predisposed to defend their prior beliefs in the face of corrective information. Only when we induced a directional motiva- tion did participants stick to the misperception more than participants would by default. Moreover, even individuals who were induced to have a direc- tional motivation on average reduced their endorsement of the mispercep- tion, rather than increasing it. How can these contrasting findings be explained? As suggested in the original work on motivated reasoning by Kunda (1990), the influence of directional reasoning is limited by people’s perceptions of reality and plausi- bility. Clear, one-sided information regarding factual beliefs is unlikely to lead to backfire effects (Flynn et al., 2017). In line with this, research demon- strates that biased updating requires, in addition to motivation, at least some level of ambiguity or balance in the information (Dixon & Clarke, 2013; Dixon et al., 2015; Sharot & Garrett, 2016) or should concern “softer” out- comes such as candidate favorability (Nyhan et al., 2019). Instead of backfir- ing, corrective information can be very effective if it “hits [people] between the eyes” (Kuklinski et al., 2000). In line with this idea, our message directly and very specifically contradicted the misperception. The current research demonstrated that a clear corrective message regarding a factual belief is likely to be effective in correcting misperceptions. General Discussion The current research investigated the role of motivated reasoning in the effectiveness of correcting misperceptions about scientific facts. We found evidence for a causal role of motivated reasoning in the effectiveness of corrections, such that individuals who were driven by accuracy motivation were more receptive to the corrective information than individuals driven by directional motivation. Contrary to what we expected, the effect of moti- vation was not moderated by prior belief certainty. Whether in great doubt or very certain, motivation in reasoning about corrective information seems to affect how amenable one is to this information. Also contrary to our expectations, we found that in the case of a food-related misperception, individuals’ default motivation made them just as receptive to the correc- tive information as accuracy motivation. Directional reasoning made indi- viduals stick to the misperception more than accuracy-driven reasoning or default reasoning. Finally, our findings demonstrated that the influence of motivation was not limited to the misperception itself. Specifically, the dif- ference in posterior endorsement of the misperception between accuracy and directionally motivated individuals was reflected in second order out- comes: Support for policy aimed at reducing E numbers in food products and intention to consume less E numbers were lower among accuracy moti- vated individuals than among directional motivated individuals. This was not the case for Experiment 1, which might be explained by lower power as well as the fact that the average correction was larger in Experiment 2 than in Experiment 1. These findings are cause for a more optimistic view of human receptivity to science communication than often found in the literature. Although we van Stekelenburg et al. 19 did find that directional reasoning might reduce the corrective effect of a science communication message, we found no evidence for the prevailing assumption in research on motivated reasoning that corrections often back- fire (see Hart & Nisbet, 2012; Nyhan et al., 2013; Nyhan & Reifler, 2010; Zhou, 2016). There has been some debate about this backfire effect (Druckman & McGrath, 2019). In line with other research (Garrett et al., 2013; Guess & Coppock, 2018; Hill, 2017; van der Linden et al., 2018; Wood & Porter, 2019), we found no evidence of such a polarizing effect of our science com- munication message. To the contrary, our findings demonstrated that indi- viduals are not automatically predisposed to defend their prior beliefs in the face of corrective information. General Discussion The current study provided the first step in investigating the causal role of motivated reasoning in sticking to misperceptions and in finding out whether this role is similar for different contentious issues (i.e., vaccine and food safety). There are, however, some limitations. First, because of demand char- acteristics in the motivation manipulation, part of the difference in posterior belief between the experimental conditions might be a result of response bias. Participants may have chosen to satisfy the researcher’s expectation, thereby biasing the main results of the experiments. As with most research relying on self-report measures, this cannot be ruled out completely (though see Mummolo & Peterson, 2019). However, we have reason to believe that demand characteristics do not explain the current findings. Participants were 20 Science Communication 00(0) told, upon measure of their posterior endorsement of the misperception, that we were interested in their judgment and that this was not a test. If anything, a participant motivated to satisfy the researcher should in this case answer as honestly as possible. Furthermore, Prolific is known as a platform that treats their participants fairly. Participants knew that they would be paid for partici- pating in research, regardless of whether they satisfied the researchers. Finally, there was no interaction between participant and researcher in the experiments, which would further reduce demand characteristics. told, upon measure of their posterior endorsement of the misperception, that we were interested in their judgment and that this was not a test. If anything, a participant motivated to satisfy the researcher should in this case answer as honestly as possible. Furthermore, Prolific is known as a platform that treats their participants fairly. Participants knew that they would be paid for partici- pating in research, regardless of whether they satisfied the researchers. Finally, there was no interaction between participant and researcher in the experiments, which would further reduce demand characteristics. Then, there are a number of smaller limitations. The first considers the ecological validity of our motivation manipulation. We simply asked partici- pants to process the information in such a way that it resembled either accu- racy or directional motivated reasoning. This is not a real-life situation. Future research could make the motivation manipulation more ecologically valid, for instance, by investigating an appeal to accuracy motivation as part of the corrective message or by investigating directional motivation primes in a more complex information environment. General Discussion Second, in the design of the cur- rent experiments we assumed that participants would be exposed to correc- tive information. However, a part of motivated reasoning is motivated selection of information (Knobloch-Westerwick & Meng, 2009). In research that manipulated motivation in information selection, researchers found dif- ferent preferences for information as a product of accuracy motivation and defense motivation (similar to directional motivation; Winter et al., 2016). Although the debate on selective exposure is far from settled (cf. Garrett, 2009), the first challenge is to get people to read corrective information. The current research provides information only on what happens when this expo- sure is achieved in the first place. The current study gives lead to some directions for future research. First, regarding the development of an intervention fostering accuracy motivation, it would be valuable to know which part of the motivation manipulation led to a difference in the corrective effect of the message. Was it being even- handed, considering the opposite view (also see Lord et al., 1984), looking for disconfirmation, or a combination of all of those things? Second, a test of the “between the eyes”-effect of a correction would provide more insight into the conditions of belief polarization. Including both an ambiguous and clear correction in a paradigm such as the one we used should be of great interest for the debate on backfire and boomerang effects. Finally, research on moti- vated reasoning could benefit from a measure of the type of motivation in reasoning. Currently, indicators of motivation such as political ideology and worldview are often used. These indirect measures are useful, but a direct measure of motivation should be more valuable. Ideally, this measure could van Stekelenburg et al. 21 also be used to investigate individuals’ default response to different types of information, potentially uncovering a motivated reasoning “trait.” also be used to investigate individuals’ default response to different types of information, potentially uncovering a motivated reasoning “trait.” Misperceptions can cause serious problems. This research focused on vac- cine and food safety, but the results are expected to also apply to other hotly debated topics, such as climate change, gun control, or genetic modification of food. If there is a motive to hold on to a misperception, it will play an important role in correcting misperceptions. At the same time, this research supports an optimistic view of people’s receptivity to science communica- tion. General Discussion By default, we are open to new information and are likely to change our beliefs when information leads us to. Appendix A Motivation Manipulation Corrective Messages Corrective Messages Corrective Messages Experiment 1. The following text is based on material that was published by the NHS and Science, one of the world’s top scientific journals. Motivation Manipulation Directional Condition. “You will now read a short text about (vaccines and the immune system/E numbers). We are interested in your judgment, because you believe that (multiple vaccines can overload a young child’s immune system/food additives indi- cated with E numbers are unsafe to consume). While reading the text, please try to be aware of this belief and view the information from your perspective. For instance, try to think of what information could confirm your initial belief. So please •• be aware of your belief •• apply your perspective •• think of what would confirm your initial belief” Accuracy Condition. “You will now read a short text about (vaccines and the immune system/E numbers). Accuracy Condition. “You will now read a short text about (vaccines and the immune system/E numbers). We are interested in your judgment, because we study how people process information and come to conclusions. While reading the text, please try to view the information in an evenhanded way and from various perspectives. For instance, try to think of what information could disprove your initial belief. So please •• be evenhanded •• apply various perspectives •• think of what would disprove your initial belief” 22 Science Communication 00(0) Default Condition (Experiment 2 Only). “You will now read a short text about E numbers. Please read it like you normally would.” Vaccines won’t overload your child’s immune system Some parents are concerned that giving too many vaccines at the same time will “overload” a child’s immune system, especially at 1 year of age, when four injections are given in a single session. But this is not the case. Over and over, studies have shown there are no harmful effects from giving multiple injections or vaccines in one session. Now, a new study provides even further evidence for this. Researchers examined the medical records of more than 900 infants from six hospitals and clinics across the western United States between 2003 and 2013. The team compared children who had contracted diseases not covered by vaccinations with those who did not. There was no link between vaccines given before the age of 2 years and other infections from ages 2 to 4 years. This indicates that, looking at any disease other than what is vaccinated against, children who receive the vaccine are not more likely to become ill than children who did not receive the vaccine. The results are not surprising. First of all, this is in line with the scientific consensus. Years of research have demonstrated that vaccines cannot over- load a child’s immune system. Second, we know why the immune system can handle a vaccine. As soon as a baby is born they come into contact with a huge number of different bacteria and viruses every day, having gone from a sterile womb straight into to our bacteria-filled environment. Their immune system copes with them and becomes stronger as a result. The immune sys- tem challenge from bacteria and viruses in vaccines pales in comparison. The bacteria and viruses used in vaccines are weakened or killed, and there are far fewer of them than the natural bugs that babies and children come into con- tact with (see figure below). Immunization helps improve protection against life-threatening diseases at the very earliest opportunity. van Stekelenburg et al. 23 According to the University of Oxford Vaccine Knowledge Project vac- cines for babies and children contained just over 60 antigens (molecules capable of eliciting an immune response) in total, in 2012. According to the American Academy of Pediatrics, the amount of antigens that children fight every day ranges from 2,000 to 6,000. Experiment 2. The following text is based on material that was published by the Food Standards Agency and scientific research published in the academic journal Food Quality and Preference. Declaration of Conflicting Interests Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research authorship, and/or publication of this article. Food additives indicated with E numbers are safe to consume Some consumers are concerned that food additives that are indicated with an E number are dangerous chemicals that are not safe to consume. But this is not the case. All the foods we eat consist of chemicals in one form or another. An E number just indicates that a food additive has passed safety tests and is approved for use in the United Kingdom and in the EU as a whole. Food additives have a long history of consumption and are used in many traditional foods. For example, wines including Champagne contain sulfites and bacon contains the preservatives nitrates and nitrites to prevent the growth of harmful bacteria, such as those that cause potentially fatal diseases like botulism. Many food additives are chemicals which exist in nature such as antioxidants ascorbic acid (vitamin C) or citric acid, found in citrus fruits. Even oxygen can be used as an additive (E number 948). Due to techno- logical advancements, many additives are now manmade to perform certain technological functions, such as preventing food from spoiling. Whether or 24 Science Communication 00(0) not the chemicals used in additives exist in nature, they are subject to the same safety evaluations by the Food Standards Agency. not the chemicals used in additives exist in nature, they are subject to the same safety evaluations by the Food Standards Agency. The safety of food additives is tested in scientific studies investigating acute toxicity, short-term exposure at various doses, and life-time exposure over several generations. If not generally considered safe, a maximum dose is set for use in specific foods: the acceptable daily intake. This is the dose that can be safely consumed daily over a lifetime without causing an effect in humans. Only when all the safety tests are passed, does the additive get the E number (for all rules, see graphic below). Even when an E number is assigned, the additive gets re-evaluated regularly to investigate whether it is safe, based on the latest scientific research. The E numbers on labels on food products are meant as a reassurance, to indicate that the ingredients have been tested and found safe. ORCID iD Aart van Stekelenburg https://orcid.org/0000-0002-9978-0224 Funding The author(s) received no financial support for the research, authorship, and/or publi- cation of this article. 25 van Stekelenburg et al. Supplemental Material Supplemental material for this article is available online at http://journals.sagepub. com/doi/suppl/10.1177/1075547019898256 References American Academy of Pediatrics. (2008). The childhood immunization schedule: Why is it like that? https://www.aap.org/en-us/advocacy-and-policy/Documents/ Vaccineschedule.pdf Bearth, A., Cousin, M. E., & Siegrist, M. (2014, December). The consumer’s per- ception of artificial food additives: Influences on acceptance, risk and benefit perceptions. Food Quality and Preference, 38, 14–23. https://doi.org/10.1016/j. foodqual.2014.05.008 Bolsen, T., & Druckman, J. N. (2015). Counteracting the politicization of science. Journal of Communication, 65(5), 745–769. https://doi.org/10.1111/jcom.12171 Bolsen, T., Druckman, J. N., & Cook, F. L. (2014). The influence of partisan moti- vated reasoning on public opinion. Political Behavior, 36(2), 235–262. https:// doi.org/10.1007/s11109-013-9238-0 Campbell, T. H., & Kay, A. C. (2014). Solution aversion: On the relation between ideology and motivated disbelief. Journal of Personality and Social Psychology, 107(5), 809–824. https://doi.org/10.1037/a0037963 Dixon, G. N., & Clarke, C. E. (2013). Heightening uncertainty around certain science: Media coverage, false balance, and the autism-vaccine controversy. Science Communication, 35(3), 358–382. https://doi.org/10.1177/1075547012458290 Dixon, G. N., Mckeever, B. W., Holton, A. E., Clarke, C., & Eosco, G. (2015). The power of a picture: Overcoming scientific misinformation by commu- nicating weight-of-evidence information with visual exemplars. Journal of Communication, 65(4), 639–659. https://doi.org/10.1111/jcom.12159 Druckman, J. N. (2012). The politics of motivation. Critical Review, 24(2), 199–216. https://doi.org/10.1080/08913811.2012.711022 Druckman, J. N., & McGrath, M. C. (2019). The evidence for motivated reasoning in climate change preference formation. Nature Climate Change, 9(2), 111–119. https://doi.org/10.1038/s41558-018-0360-1 Faul, F., Erdfelder, E., Buchner, A., & Lang, A.-G. (2009). Statistical power analy- ses using G*Power 3.1: Tests for correlation and regression analyses. Behavior Research Methods, 41(4), 1149–1160. https://doi.org/10.3758/BRM.41. 4.1149 Flynn, D. J., Nyhan, B., & Reifler, J. (2017). The nature and origins of misperceptions: Understanding false and unsupported beliefs about politics. Political Psychology, 38(S1), 127–150. https://doi.org/10.1111/pops.12394 Food Standards Agency. (n.d.). Food additives. Retrieved November 16, 2018, from https://www.food.gov.uk/safety-hygiene/food-additives Science Communication 00(0) 26 Gaines, B. J., Kuklinski, J. H., Quirk, P. J., Peyton, B., & Verkuilen, J. (2007). Same facts, different interpretations: Partisan motivation and opinion on Iraq. Journal of Politics, 69(4), 957–974. https://doi.org/10.1111/j.1468-2508.2007.00601.x Gaines, B. J., Kuklinski, J. H., Quirk, P. J., Peyton, B., & Verkuilen, J. (2007). Same facts, different interpretations: Partisan motivation and opinion on Iraq. Journal of Politics, 69(4), 957–974. https://doi.org/10.1111/j.1468-2508.2007.00601.x Garrett, R. K. (2009). Politically motivated reinforcement seeking: Reframing the selective exposure debate. Journal of Communication, 59(4), 676–699. https:// doi.org/10.1111/j.1460-2466.2009.01452.x Garrett, R. K., Nisbet, E. C., & Lynch, E. K. (2013). Undermining the corrective effects of media-based political fact checking? References The role of contextual cues and naïve theory. Journal of Communication, 63(4), 617–637. https://doi. org/10.1111/jcom.12038 Guess, A., & Coppock, A. (2018). Does counter-attitudinal information cause backlash? Results from three large survey experiments. British Journal of Political Science. Advance online publication. https://doi.org/10.1017/S0007123418000327 Haen, D. (2014). The paradox of E-numbers: Ethical, aesthetic, and cultural con- cerns in the Dutch discourse on food additives. Journal of Agricultural & Environmental Ethics, 27(1), 27–42. https://doi.org/10.1007/s10806-013-9440-4 Hart, P. S., & Nisbet, E. C. (2012). Boomerang effects in science communication: How motivated reasoning and identity cues amplify opinion polarization about climate mitigation policies. Communication Research, 39(6), 701–723. https:// doi.org/10.1177/0093650211416646 Hickok, K. (2018, September 19). Vaccines won’t overload your child’s immune system—or increase their risk of other infections. Science. https://doi. org/10.1126/science.aat5104 Hill, S. J. (2017). Learning together slowly: Bayesian learning about political facts. Journal of Politics, 79(4), 1403–1418. https://doi.org/10.1086/692739 Holman, M. R., & Lay, J. C. (2019). They see dead people (voting): Correcting misperceptions about voter fraud in the 2016 U.S. presidential election. Journal of Political Marketing, 18(1–2), 31–68. https://doi.org/10.1080/15377857.2018. 1478656 Imai, K., Keele, L., & Tingley, D. (2010). A general approach to causal media- tion analysis. Psychological Methods, 15(4), 309–334. https://doi.org/10.1037/ a0020761 Joslyn, M. R., & Sylvester, S. M. (2019). The determinants and consequences of accu- rate beliefs about childhood vaccinations. American Politics Research, 47(3), 628–649. https://doi.org/10.1177/1532673X17745342 Kahan, D. M., Peters, E., Dawson, E. C., & Slovic, P. (2017). Motivated numer- acy and enlightened self-government. Behavioural Public Policy, 1(1), 54–86. https://doi.org/10.1017/bpp.2016.2 Knobloch-Westerwick, S., & Meng, J. (2009). Looking the other way: Selective exposure to attitude-consistent and counterattitudinal political information. Communication Research, 36(3), 426–448. https://doi.org/10.1177/0093650209333030 Kuklinski, J. H., Quirk, P. J., Jerit, J., Schwieder, D., & Rich, R. F. (2000). Misinformation and the currency of democratic citizenship. Journal of Politics, 62(3), 790–816. https://doi.org/10.1111/0022-3816.00033 van Stekelenburg et al. 27 Kunda, Z. (1990). The case for motivated reasoning. Psychological Bulletin, 108(3), 480–498. https://doi.org/10.1037/0033-2909.108.3.480 Larson, H. J., de Figueiredo, A., Xiahong, Z., Schulz, W. S., Verger, P., Johnston, I. G., Cook, A. R., & Jones, N. S. (2016, October). The state of vaccine confidence 2016: Global insights through a 67-country survey. EBioMedicine, 12, 295–301. https://doi.org/10.1016/j.ebiom.2016.08.042 Leeper, T. J., & Slothuus, R. (2014). Political parties, motivated reasoning, and public opinion formation. Political Psychology, 35(S1), 129–156. https://doi. org/10.1111/pops.12164 Lewandowsky, S., Gignac, G. E., & Oberauer, K. (2013). The role of conspiracist ideation and worldviews in predicting rejection of science. References PLoS ONE, 8(10), Article e0134773. https://doi.org/10.1371/journal.pone.0075637 Lord, C. G., Lepper, M. R., & Preston, E. (1984). Considering the opposite: A correc- tive strategy for social judgment. Journal of Personality and Social Psychology, 47(6), 1231–1243. https://doi.org/10.1037/0022-3514.47.6.1231 Lord, C. G., Ross, L., & Lepper, M. R. (1979). Biased assimilation and attitude polarization: The effects of prior theories on subsequently considered evidence. Journal of Personality and Social Psychology, 37(11), 2098–2109. https://doi. org/10.1037/0022-3514.37.11.2098 Lyons, B. (2018). Reducing group alignment in factual disputes? The limited effects of social identity interventions. Science Communication, 40(6), 789–807. https:// doi.org/10.1177/1075547018804826 McCright, A. M., & Dunlap, R. E. (2011). The politicization of climate change and polarization in the American public’s views of global warming, 2001-2010. Sociological Quarterly, 52(2), 155–194. https://doi.org/10.1111/j.1533-8525 .2011.01198.x Meade, A. W., & Craig, S. B. (2012). Identifying careless responses in survey data. Psychological Methods, 17(3), 437–455. https://doi.org/10.1037/a0028085 Molden, D. C., & Higgins, E. T. (2005). Motivated thinking. In K. J. Holyoak & R. G. Morrison (Eds.), The Cambridge handbook of thinking and reasoning (pp. 295–317). Cambridge University Press. https://mudarwan.files.wordpress. com/2015/09/the-cambridge-handbook-of-thinking-and-reasoning_2005.pdf Mummolo, J., & Peterson, E. (2019). Demand effects in survey experiments: An empirical assessment. American Political Science Review, 113(2), 517–529. https://doi.org/10.1017/S0003055418000837 National Health Service. (2016). How vaccines work. https://www.nhs.uk/conditions/ vaccinations/how-vaccines-work/ The Netherlands Nutrition Centre. (n.d.). E-nummers. Retrieved November 16, 2018, from https://www.voedingscentrum.nl/Assets/Uploads/voedingscentrum/ Documents/Consumenten/Mijnboodshappen/Veiligeten/e-nummersinfo graphicjpg.pdf Nyhan, B., Porter, E., Reifler, J., & Wood, T. J. (2019). Taking fact-checks literally but not seriously? The effects of journalistic fact-checking on factual beliefs and candidate favorability. Political Behavior. Advance online publication. https:// doi.org/10.1007/s11109-019-09528-x Science Communication 00(0) 28 0(0) Nyhan, B., & Reifler, J. (2010). When corrections fail: The persistence of political misperceptions. Political Behavior, 32(2), 303–330. https://doi.org/10.1007/ s11109-010-9112-2 Nyhan, B., & Reifler, J. (2015). Does correcting myths about the flu vaccine work? An experimental evaluation of the effects of corrective information. Vaccine, 33(3), 459–464. https://doi.org/10.1016/j.vaccine.2014.11.017 Nyhan, B., & Reifler, J. (2019). The roles of information deficits and identity threat in the prevalence of misperceptions. Journal of Elections, Public Opinion and Parties, 29(2), 222–244. https://doi.org/10.1080/17457289.2018.1465061 Nyhan, B., Reifler, J., Richey, S., & Freed, G. L. (2014). Effective messages in vac- cine promotion: A randomized trial. Pediatrics, 133(4), e835–e842. https://doi. org/10.1542/peds.2013-2365 Nyhan, B., Reifler, J., & Ubel, P. A. (2013). The hazards of correcting myths about health care reform. Medical Care, 51(2), 127–132. https://doi.org/10.1097/ MLR.0b013e318279486b Nyhan, B., & Zeitzoff, T. (2018). Conspiracy and misperception belief in the Middle East and North Africa. References Journal of Politics, 80(4), 1400–1404. https://doi. org/10.1086/698663 Oppenheimer, D. M., Meyvis, T., & Davidenko, N. (2009). Instructional manipu- lation checks: Detecting satisficing to increase statistical power. Journal of Experimental Social Psychology, 45(4), 867–872. https://doi.org/10.1016/j. jesp.2009.03.009 Peer, E., Brandimarte, L., Samat, S., & Acquisti, A. (2017). Beyond the Turk: Alternative platforms for crowdsourcing behavioral research. Journal of Experimental Social Psychology, 70, 153–163. https://doi.org/10.1016/j.jesp.2017.01.006 Phadke, V. K., Bednarczyk, R. A., Salmon, D. A., & Omer, S. B. (2016). Association between vaccine refusal and vaccine-preventable diseases in the United States. JAMA Journal of the American Medical Association, 315(11), 1149–1158. https://doi.org/10.1001/jama.2016.1353 Price, E., Ottati, V., Wilson, C., & Kim, S. (2015). Open-minded cognition. Personality and Social Psychology Bulletin, 41(11), 1488–1504. https://doi. org/10.1177/0146167215600528 Saltmarsh, M. (2015). Recent trends in the use of food additives in the United Kingdom. Journal of the Science of Food and Agriculture, 95(4), 649–652. https://doi.org/10.1002/jsfa.6715 Schönbrodt, F. D., Wagenmakers, E.-J., Zehetleitner, M., & Perugini, M. (2017). Sequential hypothesis testing with Bayes factors: Efficiently testing mean dif- ferences. Psychological Methods, 22(2), 322–339. https://doi.org/10.1037/ met0000061 Sharot, T., & Garrett, N. (2016). Forming beliefs: Why valence matters. Trends in Cognitive Sciences, 20(1), 25–33. https://doi.org/10.1016/j.tics.2015.11.002 Shim, S.-M., Seo, S. H., Lee, Y., Moon, G.-I., Kim, M.-S., & Park, J.-H. (2011). Consumers’ knowledge and safety perceptions of food additives: Evaluation on the effectiveness of transmitting information on preservatives. Food Control, 22(7), 1054–1060. https://doi.org/10.1016/j.foodcont.2011.01.001 van Stekelenburg et al. 29 Taber, C. S., & Lodge, M. (2006). Motivated skepticism in the evaluation of politi- cal beliefs. American Journal of Political Science, 50(3), 755–769. https://doi. org/10.1111/j.1540-5907.2006.00214.x Vaccine Knowledge Project. (n.d.). Combination vaccines and multiple vaccinations. Retrieved September 19, 2018, from http://vk.ovg.ox.ac.uk/combination-vac- cines-and-multiple-vaccinations van der Linden, S., Leiserowitz, A., & Maibach, E. (2018). Scientific agreement can neutralize politicization of facts. Nature Human Behaviour, 2(1), 2–3. https://doi. org/10.1038/s41562-017-0259-2 Winter, S., Metzger, M. J., & Flanagin, A. J. (2016). Selective use of news cues: A multiple-motive perspective on information selection in social media envi- ronments. Journal of Communication, 66(4), 669–693. https://doi.org/10.1111/ jcom.12241 Wood, T., & Porter, E. (2019). The elusive backfire effect: Mass attitudes’ steadfast factual adherence. Political Behavior, 41(1), 135–163. https://doi.org/10.1007/ s11109-018-9443-y World Health Organization. (n.d.). Ten threats to global health in 2019. Retrieved February 12, 2019, from https://www.who.int/emergencies/ten-threats-to-global- health-in-2019 World Health Organization. (2019). New measles surveillance data for 2019. https:// www.who.int/immunization/newsroom/measles-data-2019/en/ Zhou, J. (2016). Boomerangs versus javelins: How polarization constrains communi- cation on climate change. Environmental Politics, 25(5), 788–811. References https://doi.org /10.1080/09644016.2016.1166602 Author Biographies Aart van Stekelenburg is a PhD student. He is interested in science communication. His PhD project has two main goals: The first is to find out how people stick to scien- tifically incorrect beliefs when they are confronted with scientific information. The second is to find out how scientific information can be communicated to give it a fair chance in discussion of controversial topics, like vaccination, genetically engineered food, and climate change. Gabi Schaap is an assistant professor. His research topic is “The Digital Mind”: what are the consequences of the ever growing pervasiveness and dependency on digital and mobile technologies in our daily lives? He is particularly interested in how phe- nomena such as media multitasking, second screen usage and outsourcing of our minds to technology (“extended mind”) influence our and coming generations’ pro- cessing of information and thinking. Harm Veling is an associate professor interested in behavior change, preregistra- tion, and food choice. His research is focused on changing habitual and impulsive behavior that is very hard to change by means of the traditional educational approach (e.g., changing unhealthy eating behavior). To accomplish this goal, new interven- tions are developed to stop or change automatic processes that would otherwise elicit Science Communication 00(0) 30 the undesired habits. He also has a strong interest in preregistration and open science research practices in general. the undesired habits. He also has a strong interest in preregistration and open science research practices in general. Moniek Buijzen is a professor of persuasive and strategic communication. Her work focuses on the young (media) consumer, within the paradigm of positive communica- tion research. She strives to enrich and apply communication scientific knowledge to improve young people’s well-being. In 2019, she received a prestigious Vici grant from the Netherlands Organisation for Scientific Research (NWO) for a 5-year research project investigating the implementation of health campaigns via youths’ online social networks.
https://openalex.org/W2036868154	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0102758&type=printable	English	null	Oxaliplatin Neurotoxicity Involves Peroxisome Alterations. PPARγ Agonism as Preventive Pharmacological Approach	PloS one	2,014	cc-by	11,184	Matteo Zanardelli, Laura Micheli, Lorenzo Cinci, Paola Failli, Carla Ghelardini, Lorenzo Di Cesare Mannelli* Dept. of Neuroscience, Psychology, Drug Research and Child Health - Neurofarba - Pharmacology and Toxicology Section, University of Florence, Florence, Italy Dept. of Neuroscience, Psychology, Drug Research and Child Health - Neurofarba - Pharmacology and Toxicology Section, University o 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. nding: This research was funded by the Italian Ministry of Instruction, University and Research (MIUR) and by the University of Floren e in study design, data collection and analysis, decision to publish, or preparation of the manuscript. was funded by the Italian Ministry of Instruction, University and Research (MIUR) and by the University of Florence. The funders had no a collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: lorenzo.mannelli@unifi.it Abstract The development of neuropathic syndromes is an important, dose limiting side effect of anticancer agents like platinum derivates, taxanes and vinca alkaloids. The causes of neurotoxicity are still unclear but the impairment of the oxidative equilibrium is strictly related to pain. Two intracellular organelles, mitochondria and peroxisomes cooperate to the maintaining of the redox cellular state. Whereas a relationship between chemotherapy-dependent mitochondrial alteration and neuropathy has been established, the role of peroxisome is poor explored. In order to study the mechanisms of oxaliplatin-induced neurotoxicity, peroxisomal involvement was evaluated in vitro and in vivo. In primary rat astrocyte cell culture, oxaliplatin (10 mM for 48 h or 1 mM for 5 days) increased the number of peroxisomes, nevertheless expression and functionality of catalase, the most important antioxidant defense enzyme in mammalian peroxisomes, were significantly reduced. Five day incubation with the selective Peroxisome Proliferator Activated Receptor-c (PPAR-c) antagonist G3335 (30 mM) induced a similar peroxisomal impairment suggesting a relationship between PPARc signaling and oxaliplatin neurotoxicity. The PPARc agonist rosiglitazone (10 mM) reduced the harmful effects induced both by G3335 and oxaliplatin. In vivo, in a rat model of oxaliplatin induced neuropathy, a repeated treatment with rosiglitazone (3 and 10 mg kg21 per os) significantly reduced neuropathic pain evoked by noxious (Paw pressure test) and non-noxious (Cold plate test) stimuli. The behavioral effect paralleled with the prevention of catalase impairment induced by oxaliplatin in dorsal root ganglia. In the spinal cord, catalase protection was showed by the lower rosiglitazone dosage without effect on the astrocyte density increase induced by oxaliplatin. Rosiglitazone did not alter the oxaliplatin-induced mortality of the human colon cancer cell line HT-29. These results highlight the role of peroxisomes in oxaliplatin-dependent nervous damage and suggest PPARc stimulation as a candidate to counteract oxaliplatin neurotoxicity. Citation: Zanardelli M, Micheli L, Cinci L, Failli P, Ghelardini C, et al. (2014) Oxaliplatin Neurotoxicity Involves Peroxisome Alterations. PPARc Agonism as Preventive Pharmacological Approach. PLoS ONE 9(7): e102758. doi:10.1371/journal.pone.0102758 Editor: Christian Scho¨nbach, Nazarbayev University, Kazakhstan Citation: Zanardelli M, Micheli L, Cinci L, Failli P, Ghelardini C, et al. (2014) Oxaliplatin Neurotoxicity Involves Peroxisome Alterations. PPARc Agonism as Preventive Pharmacological Approach. PLoS ONE 9(7): e102758. doi:10.1371/journal.pone.0102758 Editor: Christian Scho¨nbach, Nazarbayev University, Kazakhstan Received February 25, 2014; Accepted June 23, 2014; Published July 18, 2014 Copyright: 2014 Zanardelli et al. July 2014 \| Volume 9 \| Issue 7 \| e102758 Citation: Zanardelli M, Micheli L, Cinci L, Failli P, Ghelardini C, et al. (2014) Oxaliplatin Neurotoxicity Involves Peroxisome Alterations. PPARc Agonism as Preventive Pharmacological Approach. PLoS ONE 9(7): e102758. doi:10.1371/journal.pone.0102758 Oxaliplatin Neurotoxicity Involves Peroxisome Alterations. PPARc Agonism as Preventive Pharmacological Approach Matteo Zanardelli, Laura Micheli, Lorenzo Cinci, Paola Failli, Carla Ghelardini, Lorenzo Di Cesare Mannelli* Materials and Methods In mouse liver was originally cloned a nuclear receptor subfamily of ligand-activated transcription factors, the Peroxisome Proliferator-Activated Receptors (PPARs) [23]. PPARs may activate genes with a PPAR response element (PPRE) in their promoter regions [24]. Girnun et al. [25] highlighted that PPARc stimulation increases the expression and activity of catalase, a heme-containing peroxisomal enzyme that breaks down hydrogen peroxide to water and oxygen [26,27]. Recently, agonists of the c subtype of PPARs received considerable attention as potential therapeutic agents for a wide range of neurological diseases, including neurodegenerative diseases, traumatic injuries, stroke and demyelinating diseases [28–38]. Introduction Moreover, peroxisomes were recently involved in the development and progression of specific degenerative diseases [18,20–22]. Introduction cultures of astrocytes [8], a glial cell type activated in vivo by oxaliplatin treatment [9]. Since oxaliplatin does not possess direct oxidative properties [8], redox unbalance seems due to a cell- mediated effect able to alter the oxidative machinery. Oxaliplatin is a chemotherapeutic compound widely used for treating colorectal cancer [1]. The development of sensory neuropathy is the most important, dose-limiting side effect. Platinum-induced peripheral neuropathy is characterized by distal paresthesias and mild muscle contractions for at least 80% of oncologic patients after few hours to days from the first oxaliplatin infusion [2,3]. Moreover, oxaliplatin repeated treatment induces severe peripheral neuropathy that can affect approximately 50% of the patients receiving cumulative doses higher than 1000 mg/ m2 [4,5]. Anti-hyperalgesic compounds currently used to treat chemotherapy-induced pain, like antiepileptics or antidepressant, are weakly effective [6]. The therapeutic failure reflects the lack of knowledge about the molecular bases of neuropathies. In a rat model of oxaliplatin-induced neuropathy we previously identified oxidative stress as a main biomolecular dysfunction showing a relationship between oxidative damage of the nervous system and pain [7]. The ‘‘oxidative hypothesis’’ was confirmed in primary y After oxaliplatin treatment, mitochondria are modified in morphology and impaired in function [10]. Less inquired is the role of the other intracellular organelle strongly implied in redox processes: the peroxisome. Peroxisomes are the last among the subcellular organelles to be identified [11]. The discovery of the co-localization of catalase with H2O2-generating oxidases in peroxisomes was the first indication of their involvement in the metabolism of oxygen metabolites [11]. The high peroxisomal consumption of O2, the demonstration of the production of H2O2, O2 2, ?OH, and more recently of ?NO [11–14], as well as the discovery of several ROS metabolizing enzymes in peroxisomes has supported the notion that these ubiquitous organelles play a key role in both the production and scavenging of ROS in the cell [15]. In the nervous system, the functional relevance of these organelles is dramatically highlighted by peroxisomal disorders. 1 July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org Oxaliplatin Neuropathy and Peroxisome Impairment pharmacological approaches to control oxaliplatin-induced neu- ropathy. Severe demyelination, axonal degeneration and neuroinflamma- tion are induced by genetic deficit of peroxisome [16–19]. Moreover, peroxisomes were recently involved in the development and progression of specific degenerative diseases [18,20–22]. Severe demyelination, axonal degeneration and neuroinflamma- tion are induced by genetic deficit of peroxisome [16–19]. Cell treatments On day 21, astrocytes were plated in 12-wells cell culture (2?105/well; Corning, Tewksbury MA, USA), or on polylysine- coated slides (5?104/well) and experiments were performed after 48 h. Cells were treated with 10-and 1 mM oxaliplatin (Sequoia Research Products, Pangbourne, UK) for 2 or 5 days respectively. Rosiglitazone (10 mM; Sequoia Research Products, Pangbourne, Cell viability assay HT-29 cell viability was evaluated by the reduction of 3-(4,5- dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an index of mitochondrial compartment functionality. Cells were plated into 96-well cell culture plates, and treated after 48 h. Oxaliplatin, at various concentrations, was incubated in DMEM in the presence of 10 mM rosiglitazone for 48 h and 5 days. After extensive washing, 1 mg/ml MTT was added into each well and incubated for 30 minutes at 37uC. After washing, the formazan crystals were dissolved in 150 ml dimethyl sulfoxide. The absorbance was measured at 550 nm. Experiments were per- formed in quadruplicate on at least three different cell batches. The human colon cancer cell line HT-29 was obtained from American Type Culture Collection (Rockville, MD). HT-29 were cultured in DMEM high glucose with 20% FBS in 5% CO2 atmosphere at 37uC. Media contained 2 mM L-glutamine, 1% essential aminoacid mix, 100 IU ml21 penicillin and 100 mg ml21 streptomycin (Sigma, Milan, Italy). Oxaliplatin Neuropathy and Peroxisome Impairment Monza, Italy). Nuclei were stained with 4,6-diamidino-2-pheny- lindole dihydrochloride. 90% of cells in astrocyte cultures were GFAP-positive. Experiments were performed 21 days after cell isolation. Formal approval to conduct the experiments described was obtained from the Animal Subjects Review Board of the University of Florence. The ethics policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996; University of Florence Assurance No. A5278-01). UK) and 30 mM G3335 (BioVision Incorporated, Milpitas, CA, USA) were used in the presence or absence of oxaliplatin for 2 or 5 days. The chosen concentrations are in accord with previous published data [8,40,41] and, as regards oxaliplatin, with plasmatic concentration of treated rats. UK) and 30 mM G3335 (BioVision Incorporated, Milpitas, CA, USA) were used in the presence or absence of oxaliplatin for 2 or 5 days. The chosen concentrations are in accord with previous published data [8,40,41] and, as regards oxaliplatin, with plasmatic concentration of treated rats. HT-29 cells were plated in 96-wells cell culture (1?104/well) and, 48 h after, treated as described above. Astrocyte cultures y Primary cultures of astrocytes were obtained according to the method described by McCarthy and de Vellis [39]. Briefly, the cerebral cortex of newborn (P1–P3) Sprague–Dawley rats (Harlan, Italy) was dissociated in Hanks’ balanced salt solution containing 0.5% trypsin/EDTA and 1% DNase (Sigma-Aldrich, Milan, Italy) for 30 min at 37uC. The suspension was mechanically homoge- nized and filtered. Cells were plated in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS, Gibco, Invitrogen, Milan, Italy). Confluent primary glial cultures were used to isolate astrocytes, removing microglia and oligodendrocytes by shaking. The purity of astrocyte cultures was determined immunocytochemically by staining for GFAP (Dako, Glostrup, Denmark). Cells were fixed in 4% paraformaldehyde, then incubated with the antibody (1:200), and visualized using Alexa Fluor-conjugated secondary antibody (Life Technologies, Aimed to characterize the oxaliplatin neurotoxicity, we studied the peroxisome-related signal in vitro, in astrocyte cell culture, and in vivo in a rat model. Peroxisome stimulation by the PPARc agonist rosiglitazone was analyzed to individuate new possible Figure 1. Catalase immunostaining in primary astrocytes. Cells (5?104 cells/well) were incubated for 5 days with 10 mM rosiglitazone (B), 1 mM oxaliplatin (C), 1 mM oxaliplatin+10 mM rosiglitazone (D), 30 mM G3335 (E), negative control (F) in comparison to control condition (A). Representative images are shown in the left panel. Scale bar 50 mm. The measurements of the number of peroxisomes/mm2 and the catalase optical density per number of peroxisomes are shown in the upper and lower graphs, respectively. P,0.01 vs control; ‘P,0.01 vs 1 mM oxaliplatin. doi:10.1371/journal.pone.0102758.g001 Figure 1. Catalase immunostaining in primary astrocytes. Cells (5?104 cells/well) were incubated for 5 days with 10 mM rosiglitazone (B), 1 mM oxaliplatin (C), 1 mM oxaliplatin+10 mM rosiglitazone (D), 30 mM G3335 (E), negative control (F) in comparison to control condition (A). Representative images are shown in the left panel. Scale bar 50 mm. The measurements of the number of peroxisomes/mm2 and the catalase optical density per number of peroxisomes are shown in the upper and lower graphs, respectively. P,0.01 vs control; ‘P,0.01 vs 1 mM oxaliplatin. doi:10.1371/journal.pone.0102758.g001 July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 2 Oxaliplatin Neuropathy and Peroxisome Impairment Animals For all the experiments described below, male Sprague-Dawley rats (Harlan, Varese, Italy) weighing approximately 200 to 250 g Figure 2. Expression and activity of catalase in astrocyte cell culture. Astrocytes (5?105 cells/well) were treated with the PPARc antagonist G3335 (30 mM) or with oxaliplatin (1 mM) in the absence or in the presence of the PPARc agonist rosiglitazone (10 mM). Expression and activity were measured after 48 h- (A and B, respectively) or 5 day-treatment (C and D, respectively). GAPDH normalization was performed for each sample. Values are expressed as the mean 6 S.E.M. percent of control of three experiments. Control condition was arbitrarily set as 100%. P,0.05 vs control; ‘P, 0.05 vs 1 mM oxaliplatin. doi:10.1371/journal.pone.0102758.g002 Figure 2. Expression and activity of catalase in astrocyte cell culture. Astrocytes (5?105 cells/well) were treated with the PPARc antagonist G3335 (30 mM) or with oxaliplatin (1 mM) in the absence or in the presence of the PPARc agonist rosiglitazone (10 mM). Expression and activity were measured after 48 h- (A and B, respectively) or 5 day-treatment (C and D, respectively). GAPDH normalization was performed for each sample. Values are expressed as the mean 6 S.E.M. percent of control of three experiments. Control condition was arbitrarily set as 100%. P,0.05 vs control; ‘P, 0.05 vs 1 mM oxaliplatin. doi:10.1371/journal.pone.0102758.g002 July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 3 3 Oxaliplatin Neuropathy and Peroxisome Impairment Oxaliplatin (mM) 0 0.1 0.3 1 3 10 30 100.064.5 98.065.3 91.862.4 92.663.2 90.963.4 88.162.3* 82.063.8* 10 mM 109.764.5 99.762.6 94.963.8 91.863.3 91.363.2 92.761.1* 75.464.8* e treated with increasing concentrations of oxaliplatin (1–300 mM) in the presence or in the absence of 10 mM rosiglitazone. Incubation was allowed for 5 days. Cell viability was measured by MTT assay. Control arbitrarily set as 100% and values are expressed as the mean 6 S.E.M. of three experiments. P,0.05 in comparison to control (oxaliplatin 0 mM). urnal.pone.0102758.t002 Oxaliplatin (mM) 0 0.1 0.3 1 3 10 30 control 100.064.5 98.065.3 91.862.4 92.663.2 90.963.4 88.162.3 82.063.8* rosiglitazone 10 mM 109.764.5 99.762.6 94.963.8 91.863.3 91.363.2 92.761.1* 75.464.8* Ht-29 cells were treated with increasing concentrations of oxaliplatin (1–300 mM) in the presence or in the absence of 10 mM rosiglitazone. Incubation was allowed for 5 days. Cell viability was measured by MTT assay. Control condition was arbitrarily set as 100% and values are expressed as the mean 6 S.E.M. Oxaliplatin Neuropathy and Peroxisome Impairment Four rats were housed per cage (size 26641 cm); animals were fed with standard laboratory diet and tap water ad libitum, and kept at 2361uC with a 12 hour light/dark cycle, light at 7 a.m. All animal manipulations were carried out according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive of 24 November 1986 (86/609/EEC). The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85-23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the experiments described was obtained from the Animal Subjects Review Board of the University of Florence. All efforts were made to minimize animal suffering and to reduce the number of animals used. Paw pressure test The nociceptive threshold in the rat was determined with an analgesimeter (Ugo Basile, Varese, Italy), according to the method described by Leighton et al. [43]. Briefly, a constantly increasing pressure was applied to a small area of the dorsal surface of the hind paw using a blunt conical probe by a mechanical device. Mechanical pressure was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. Rats scoring below 40 g or over 75 g during the test before drug administration were rejected (25%). For analgesia measures, mechanical pressure application was stopped at 120 g. Rats were randomly assigned to each experimental group and individually habituated to handling before testing. Negative controls were carried out by omitting the primary or the secondary antibodies. Staining was performed in a single session, to minimize artifactual differences in the staining. Numerous (.10) photomicrographs of cells were randomly taken using a digital photomicroscopy apparatus with a 406 objective. Each microscopic field corresponds to a test area of 38,700 mm2. On the digitized images peroxisomes were counted in every single cell and cell area was also measured Measurements were carried out using ImageJ 1.33 free-share image analysis software (ImageJ, NIH, Bethesda, Maryland, USA). The results were expressed as number of peroxisomes/mm2. On the digitized images, measure- ments of optical density of catalase-immunostained peroxisomes were carried out after determining the appropriate threshold to include only immune-reactive peroxisomes. The results were expressed as mean of optical density/number of peroxisomes. After determination of number and optical density of peroxisomes, Rota-Rod Test The Rota-rod apparatus (Ugo Basile, Varese, Italy) consisted of a base platform and a rotating rod with a diameter of 6 cm and a non-slippery surface. The rod was placed at a height of 25 cm from the base. The rod, 36 cm in length, was divided into 4 equal sections by 5 disks. Thus, up to 4 rats were tested simultaneously on the apparatus, with a rod-rotating speed of 10 r.p.m. The integrity of motor coordination was assessed on the basis of the time the animals kept their balance on the rotating rod up to a maximum of 10 min (600 s). After a maximum of 6 falls from the rod, the test was suspended and the time was recorded. Tissue collection On day 21, at the end of the behavioral test session, animals were sacrificed by decapitation. L4-L5 dorsal root ganglia (DRG) were dissected and frozen using liquid nitrogen. L4/L5 segments of the spinal cord were exposed from the lumbovertebral column via laminectomy and identified by tracing the dorsal roots from their respective DRG. After dissection, this lumbar portion was frozen using liquid nitrogen or fixed by immersion in 4% neutral buffered formalin. Blood was collected in heparin-treated tubes and plasma fraction was isolated by centrifugation. Catalase immunoreaction in astrocyte cell culture Cytological specimens were fixed in 4% paraformaldehyde for 10 minutes and then washed in PBS. For immunolabeling, slides were treated with 0.3% H2O2 (v/v) in water to quench endogenous peroxidase and then pre- incubated for 15 minutes in Ultra V Block (Thermo Scientific, Rancom Cheshire, UK). Successively, the slides were incubated with rabbit polyclonal anti- Catalase antiserum at final dilution of 1:100 (Novus Biological, Littelton, CO, USA). Immuno-reaction was revealed by biotin- conjugated goat anti-rabbit IgG to a final dilution of 1:200 (Dako, Glostrup, Denmark) followed by incubation with Streptavidin Peroxidase Complex (Thermo Scientific, Rancom Cheshire, UK). The morphology, size and cellular localization allowed us to identify the stained bodies as peroxisomes. Oxaliplatin Neuropathy and Peroxisome Impairment Oxaliplatin Neuropathy and Peroxisome Impairment Figure 3. Pain threshold measurements. A) Noxious stimulus, Paw-pressure test. Rats were daily intraperitoneally treated with 2.4 mg kg21 oxaliplatin (dissolved in 5% glucose). Rosiglitazone (3 and 10 mg kg21, suspended in CMC) was per os daily administered starting from the first day of oxaliplatin administration. B) Non-noxious stimulus, Cold plate test. The response to a thermal stimulus was evaluated by cold plate test measuring the latency (seconds) to pain-related behaviors (lifting or licking of the paw). Control animals were treated with vehicles. Behavioral measures were performed on day 7, 14 and 21, 24 h after the last treatment. Each value represents the mean of 10 rats per group, performed in 2 different experimental set. P,0.01 vs vehicle + vehicle (control); ‘P,0.01 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.g003 and the time (s) of the first sign was recorded. The cut-off time of the latency of paw lifting or licking was set at 60 s. and the time (s) of the first sign was recorded. The cut-off time of the latency of paw lifting or licking was set at 60 s. at the beginning of the experimental procedure were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least 1 week after their arrival. Four rats were housed per cage (size 26641 cm); animals were fed with standard laboratory diet and tap water ad libitum, and kept at 2361uC with a 12 hour light/dark cycle, light at 7 a.m. All animal manipulations were carried out according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive of 24 November 1986 (86/609/EEC). The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85-23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the experiments described was obtained from the Animal Subjects Review Board of the University of Florence. All efforts were made to minimize animal suffering and to reduce the number of animals used. at the beginning of the experimental procedure were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least 1 week after their arrival. Oxaliplatin model and pharmacological treatments p p g Oxaliplatin neuropathy was induced as described by Cavaletti et al. [42]. Rats were treated with 2.4 mg kg21 oxaliplatin, administered intraperitoneally (i.p.) for 5 consecutive days every week for 3 weeks (15 i.p. injections). Oxaliplatin was dissolved in 5% glucose solution. Rosiglitazone at the doses of 3 and 10 mg kg21 was suspended in 1% carboxymethylcellulose sodium salt (CMC) and administered per os (p.o.) daily starting from the first day of oxaliplatin administration up to day 20. Control animals received an equivalent volume of vehicles: i.p. glucose or p.o. CMC (vehicle). Behavioral, morphological and biochemical tests were performed on day 21, 24 hours after last treatments. On day 21, organic platinum plasmatic levels evaluated by Inductively Coupled Mass Spectrometry (ICP-MS) were 3.57360.271 mg/mL (corresponding to oxaliplatin 7.27460.552 mg/mL or 18.3 mM). July 2014 \| Volume 9 \| Issue 7 \| e102758 Animals of three experiments. P,0.05 in comparison to control (oxaliplatin 0 mM). doi:10.1371/journal.pone.0102758.t002 Oxaliplatin (mM) 0 1 3 10 30 100 300 control 100.062.4 99.761.1 95.161.1 89.161.4* 79.861.6* 38.663.7 17.261.3 rosiglitazone 10 mM 101.063.6 96.063.4 91.764.5 92.163.4 85.866.7* 44.964.3 16.962.5 Ht-29 cells were treated with increasing concentrations of oxaliplatin (1–300 mM) in the presence or in the absence of 10 mM rosiglitazone. Incubation was allowed for 48 h. Cell viability was measured by MTT assay. Control condition was arbitrarily set as 100% and values are expressed as the mean 6 S.E.M. of three experiments. P,0.05 and P,0.01 in comparison to control (oxaliplatin 0 mM). doi:10.1371/journal.pone.0102758.t001 NE \| www.plosone.org Oxaliplatin (mM) 0 1 3 10 30 100 300 control 100.062.4 99.761.1 95.161.1 89.161.4 79.861.6* 38.663.7 17.261.3 rosiglitazone 10 mM 101.063.6 96.063.4 91.764.5 92.163.4 85.866.7* 44.964.3 16.962.5 Ht-29 cells were treated with increasing concentrations of oxaliplatin (1–300 mM) in the presence or in the absence of 10 mM rosiglitazone. Incubation was allowed for 48 h. Cell viability was measured by MTT assay. Contro condition was arbitrarily set as 100% and values are expressed as the mean 6 S.E.M. of three experiments. P,0.05 and P,0.01 in comparison to control (oxaliplatin 0 mM). doi:10.1371/journal.pone.0102758.t001 Table 2. HT-29 cell viability, 5 days. Oxaliplatin (mM) 0 0.1 0.3 1 3 10 30 control 100.064.5 98.065.3 91.862.4 92.663.2 90.963.4 88.162.3 82.063.8* rosiglitazone 10 mM 109.764.5 99.762.6 94.963.8 91.863.3 91.363.2 92.761.1* 75.464.8* Ht-29 cells were treated with increasing concentrations of oxaliplatin (1–300 mM) in the presence or in the absence of 10 mM rosiglitazone. Incubation was allowed for 5 days. Cell viability was measured by MTT assay. Contro condition was arbitrarily set as 100% and values are expressed as the mean 6 S.E.M. of three experiments. P,0.05 in comparison to control (oxaliplatin 0 mM). doi:10.1371/journal.pone.0102758.t002 NE \| www.plosone.org 4 July 2014 \| July 2014 \| Volume 9 \| Issue 7 \| e102758 4 Oxaliplatin Neuropathy and Peroxisome Impairm Oxaliplatin Neuropathy and Peroxisome Impairment LOS ONE \| www.plosone.org 5 July 2014 \| Volume 9 \| Issue 7 \| e10275 July 2014 \| Volume 9 \| Issue 7 \| e102758 Oxaliplatin Neuropathy and Peroxisome Impairment Oxaliplatin Neuropathy and Peroxisome Impairment Figure 4. Motor coordination in oxaliplatin-treated rats. The integrity of the animals’ motor coordination was assessed using a rota-rod apparatus. Rats were placed on a rotating rod (10 rpm) for a maximum of 10 minutes (600 seconds). The number of falls (A) and the time spent in the balance (B) during 10 minutes were counted. Treatments (oxaliplatin 2.4 mg kg21 i.p. and rosiglitazone 3 and 10 mg kg21 p.o.) were performed daily. Motor coordination was evaluated on day 21, 24 h after the last treatment. Each value represents the mean of 10 rats per group, performed in 2 different experimental set. P,0.01 vs vehicle + vehicle (control); ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.g004 collected, subjected to a freeze–thaw cycle and centrifuged at 13,0006g for 10 min at 4uC. Nervous tissue (DRG and spinal cord) from treated animals was homogenized in the lysis buffer described before. The suspension was sonicated on ice using three 10 s bursts at high intensity with a 10 s cooling period between each burst and then centrifuged (13,0006g for 15 minutes at 4uC). Protein concentration was quantified by bicinchoninic acid assay. Forty mg of each sample were resolved with 10% SDS-PAGE before electrophoretic transfer onto nitrocellulose membranes (Biorad, Milan, Italy). Membranes were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 (PBST) and then probed overnight at 4uC with primary antibody specific versus catalase (1:1000; 60 kDa; Novus Biological, Littleton, CO, USA), GAPDH (1:1000; 38 kDa; Cell Signaling, Boston, MA, USA). Membranes were then incubated for 1 hour in PBST containing the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000; Cell Signalling, USA). ECL (Enhanced chemi- luminescence Pierce, Rockford, IL, USA) was used to visualize the peroxidase-coated bands. Densitometric analysis was performed using the ‘‘ImageJ’’ analysis software (ImageJ, NIH, Bethesda, Maryland, USA) and results were normalized to GAPDH immunoreactivity as internal control. Values are reported as percentages in comparison to control which was arbitrarily fixed at 100%. the slides were counterstained with hematoxylin to highlight the nuclei. Carbonylated protein evaluation Carbonylated proteins were evaluated in tissue homogenates as described previously [7]. Twenty mg of each spinal cord sample were denatured by 6% SDS and derivatized by 15-minute incubation with 2,4 dinitrophenyl hydrazine (DNPH; Sigma- Aldrich, Italy) at room temperature. Samples were separated on a 4–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel by electrophoresis and transferred onto nitrocellulose membranes (Biorad, Italy). The membranes were incubated overnight with primary antibody specific versus DNPH (1:5000; Sigma-Aldrich, Milan, Italy). Afterwards the procedure described for Western blotting analysis was followed. For each experiment the density of all bands showed in a lane was reported as mean. b-actin was used as loading control. Catalase activity Enzymatic activity was measured both in astrocyte culture and in nervous tissue. After incubation, cells were washed once with PBS and scraped with PBS on ice. Cells were then collected, subjected to a freeze–thaw cycle and centrifuged (13,0006g for 10 min at 4uC). DRG and spinal cord were homogenated in PBS. The suspension was sonicated on ice using three 10 s bursts at high intensity with a 10 s cooling period between each burst and then centrifuged (13,0006g for 15 minutes at 4uC). Catalase activity was measured in the supernatant by Amplex Red Catalase Assay Kit (Invitrogen, Monza, Italy) following the manufacturer’s instructions. Protein concentration was quantified by bicinchoninic acid assay (Sigma–Aldrich, Milan, Italy). Catalase activity for each sample was normalized to protein concentration. Control condi- tions in the absence of treatment were set as 100%. Lipid peroxidation Thiobarbituric acid reactive substances (TBARS) were quanti- fied in spinal cord tissue homogenate as described previously [7]. Tissue homogenate (1.5 mg) was added to 4 mL reaction mixture consisting of 36 mM thiobarbituric acid (Sigma-Aldrich, Milan, Italy) solubilized in 10% CH3COOH, 0.2% SDS, pH was adjusted to 4.0 with NaOH. The mixture was heated for 60 minutes at 100uC and the reaction was stopped by placing the vials in ice bath for 10 minutes. After centrifugation (at 1.600 g at 4uC for 10 minutes) the absorbance of the supernatant was measured at 532 nm (Perkin-Elmer spectrometer, Monza, Italy) and TBARS were quantified in mmoles/milligram of total protein using 1,1,3,3-tetramethoxypropane as standard. Protein homoge- nate concentration was measured by bicinchoninc acid (BCA; Sigma-Aldrich, Milan, Italy) assay. Cold Plate Test The animals were placed in a stainless box (12 cm620 cm610 cm) with a cold plate as floor. The temper- ature of the cold plate was kept constant at 4uC61uC. Pain-related behaviors (i.e. lifting and licking of the hind paw) were observed July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 6 Oxaliplatin Neuropathy and Peroxisome Impairment PLOS ONE \| www.plosone.org 7 July 2014 \| Volume 9 \| Issue 7 \| e102 PLOS ONE \| www.plosone.org 7 July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 7 July 2014 \| Volume 9 \| Issue 7 \| e10275 July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 7 Determination of tissue platinum concentration Plasma samples were pre-treated with nitric acid as described by [44] with minor modifications. Platinum levels were measured by Inductively Coupled Mass Spectrometry (ICP-MS) according to [45] in the ‘‘Laboratorio di Microanalisi’’ of the University of Florence. Glial Fibrillary Acid Protein (GFAP) immuno reaction Glial Fibrillary Acid Protein (GFAP) immuno reaction GFAP signal in immunostained sections was quantified using FIJI software (distributed by ImageJ, NIH, Bethesda, Maryland, USA) by automatic thresholding images with the aid of the ‘‘Moments’’ algorithm, which we found to provide the most consistent pattern recognition across all acquired images. Area fraction (%) occupied by the thresholded GFAP signal revealed a common trend between GFAP expression and astrocyte cell number. Five spinal cord sections were analyzed for each animal. Glial Fibrillary Acid Protein (GFAP) immuno reaction Formalin-fixed cryostat sections (20 mm) were incubated for 1 h in blocking solution (Bio-Optica; Milan, Italy) at room tempera- ture; and were then incubated for 24 h at 4uC in PBST containing rabbit primary antisera diluted 1:1000 and 5% normal donkey serum. The primary antibody was directed against glial fibrillary acidic protein (GFAP; 1:5000; Chemicon, Temecula, USA) for astrocyte staining. After rinsing in PBST, sections were incubated in donkey anti-rabbit IgG secondary antibody labeled with Alexa Fluor 488 (1:1000, Invitrogen, Monza, Italy) at room temperature for 1 h. Negative control sections (no exposure to the primary antisera) were processed concurrently with the other sections for all immunohistochemical studies. We obtained a single optical density value for the dorsal horns by averaging the two sides in each rat, and these values were compared to the homologous average values from the vehicle-treated animals. Images were acquired by a motorized Leica DM6000B microscope equipped with a DFC350FX camera (Leica, Mannheim, Germany). Microglia and astrocyte morphology was assessed by inspection of at least three fields (40X 0.75NA objective) in the dorsal horn and cerebral areas per section. Quantitative analysis of GFAP - positive cells was performed by collecting at least three indepen- dent fields through a 20X 0.5NA objective. GFAP-positive cells were counted using the ‘‘cell counter’’ plugin of ImageJ. The Oxaliplatin Neuropathy and Peroxisome Impairment Oxaliplatin Neuropathy and Peroxisome Impairment Figure 5. Expression and activity of catalase in the nervous tissue of oxaliplatin-treated animals. On day 21, dorsal root ganglia (DRG) and spinal cord were analyzed to measure both expression and activity of catalase. Densitometric analysis and representative Western blot of catalase expression in DRG (A) and spinal cord (C) are shown. GAPDH normalization was performed for each sample. Catalase enzymatic activity measurements in DRG (B) and spinal cord (D). Values are expressed as the mean 6 S.E.M. percent of control of 10 rats per group, performed in 2 different experimental set. Each value represents the mean of P,0.05 vs vehicle + vehicle; ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.g005 ue of oxaliplatin-treated animals. On day 21, dorsal root ganglia (DRG) catalase. Densitometric analysis and representative Western blot of catalase malization was performed for each sample Catalase enzymatic activity Figure 5. Expression and activity of catalase in the nervous tissue of oxaliplatin-treated animals. On day 21, dorsal root ganglia (DRG) and spinal cord were analyzed to measure both expression and activity of catalase. Densitometric analysis and representative Western blot of catalase expression in DRG (A) and spinal cord (C) are shown. GAPDH normalization was performed for each sample. Catalase enzymatic activity measurements in DRG (B) and spinal cord (D). Values are expressed as the mean 6 S.E.M. percent of control of 10 rats per group, performed in 2 different experimental set. Each value represents the mean of P,0.05 vs vehicle + vehicle; ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.g005 Western blotting analysis After incubation, astrocyte cell cultures were washed once with PBS and scraped on ice with lysis buffer containing 50 mM Tris- HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, Complete Protease Inhibitor (Roche, Milan, Italy). Cells were then Table 3. Lipid peroxidation in spinal cord. TBARS (mmol/mg protein) vehicle + vehicle 35.0567.25 oxaliplatin + vehicle 133.02623.45* oxaliplatin + rosiglitazone 3 mg kg21 29.6867.12‘ oxaliplatin + rosiglitazone 10 mg kg21 39.27693.45‘ On day 21, the lumbar tract of the spinal cord was explanted for the analysis of lipid peroxidation. Data were expressed as mean 6 SEM of Thiobarbituric Acid Reactive Substances (TBARS) levels (mmol/mg protein). Each value represents the mean of 10 rats per group, performed in 2 different experimental set. P,0.01 vs vehicle + vehicle (control); ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.t003 Table 3. Lipid peroxidation in spinal cord. On day 21, the lumbar tract of the spinal cord was explanted for the analysis of lipid peroxidation. Data were expressed as mean 6 SEM of Thiobarbituric Acid Reactive Substances (TBARS) levels (mmol/mg protein). Each value represents the mean of 10 rats per group, performed in 2 different experimental set. P,0.01 vs vehicle + vehicle (control); ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.t003 On day 21, the lumbar tract of the spinal cord was explanted for the analysis of lipid peroxidation. Data were expressed as mean 6 SEM of Thiobarbituric Acid Reactive Substances (TBARS) levels (mmol/mg protein). Each value represents the mean of 10 rats per group, performed in 2 different experimental set. P,0.01 vs vehicle + vehicle (control); ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.t003 July 2014 \| Volume 9 \| Issue 7 \| e102758 July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 8 Statistical analysis Results are expressed as mean 6 SEM and analysis of variance (ANOVA) was performed. A Bonferroni’s significant difference procedure was used as post hoc comparison. All assessments were made by researchers blinded to cell or rat treatments. Slides from control and experimental groups were labeled with numbers so that the person performing the image analysis was blinded as to the experimental group. In addition all images were captured and July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 9 Oxaliplatin Neuropathy and Peroxisome Impairment Figure 6. Levels of carbonylated proteins in the spinal cord of oxaliplatin-treated rats. At 21th day, the lumbar tract of the spina xplanted and analyzed to measure protein oxidation. Densitometric analysis (top panel) and representative Western blot (lower panel) are PLOS ONE \| www.plosone.org 10 July 2014 \| Volume 9 \| Issue 7 10 July 2014 \| Volume 9 \| Issue 7 \| e102758 Ex vivo evaluation Aimed to evaluate in vivo the relationship between oxaliplatin neurotoxicity and peroxisome, catalase expression and activity were measured in peripheral and central nervous tissue on day 21 of anticancer treatment. In DRG, catalase expression decreased up to 72.9610.7% in comparison to the control value (10068.7%, Figure 5A), the enzymatic activity was reduced up to 75.665.8% (Figure 5B). Rosiglitazone (3 mg kg21 and 10 mg kg21) prevented catalase impairment (Figure 5A and B). Figures 5C and 5D show the oxaliplatin-induced decrease of catalase expression (68.067.2%) and functionality (66.269.9%) in the spinal cord. The lower dosage of rosiglitazone (3 mg kg21) only, was able to prevent catalase alteration in spinal cord (Figure 5C and D). Moreover, both the doses of rosiglitazone limited the oxidative damage induced by oxaliplatin. As shown in Table 3, the lipid peroxidation promoted by the anticancer agent (up to 4 times as the basal value) was prevented by 3 and 10 mg kg21 rosiglitazone. Protein oxidation, evaluated as increase of the expression level of carbonylated protein was significantly prevented by rosiglitazone (Figure 6). Aimed to better understand the role of astrocyte cells in the modulation of neuropathic pain, we performed immunohis- tochemical analysis of GFAP-positive cells in the dorsal horn of the spinal cord (Figure 7). The oxaliplatin-dependent increase of cell number (about 25% increase in oxaliplatin + vehicle as compared to vehicle + vehicle), was prevented by 10 mg kg21 rosiglitazone (Figure 7). Similar alterations of catalase expression profile were measur- able by Western blot. Protein level decreased from the control value of 100% to 68.368.1% after 48 h incubation with 10 mM oxaliplatin (Figure 2A) and to 79.465.4% after 5 day incubation with 1 mM oxaliplatin (Figure 2C). Moreover, the chemothera- peutic agent impaired the enzymatic activity of catalase. After 48 h, 10 mM oxaliplatin reduced catalase activity from 100% (control) to 62.365.1% (Figure 2B); five day incubation with 1 mM oxaliplatin reduced activity to 62.065.5% (Figure 2D). Effects evoked by oxaliplatin in 5 day incubation were mimicked by the PPAR-c antagonist G3335 (30 mM). Catalase expression de- creased to 64.467.2% (Figure 2C) and activity up to 62.265.3% (Figure 2D). The PPAR-c agonist rosiglitazone (10 mM) prevented both oxaliplatin- (Figure 2D) and G3335-dependent (data not shown) alterations. Neither G3335 nor rosiglitazone were effective after 48 h of treatment. Ex vivo evaluation Aimed at evaluating the potential interaction between rosiglitazone treatment and the therapeutic property of oxaliplatin, we measured the viability of the human colon cancer cell line HT-29. Tables 1 and 2 show the lack of influence by the PPAR-c agonist on the concentration-dependent (0.1–100 mM) oxaliplatin lethal effect after 48 h and 5 day incubation. Discussion Neuropathy is a dose limiting side effect of many anticancer agents, including oxaliplatin. In the clinical practice, the common human dosage of oxaliplatin is 85 mg/m2 and cumulative doses higher than 1000 mg/m2 [4,5] causes chronic neuropathy in approximately 50% of patients. The human plasmatic concentra- tion of inorganic platinum after a single i.v. injection of 85 mg/m2 is on average about 3 mg/mL and only limited accumulation is observed in plasma after repeated cycles (five consecutive cycles at 85 or 130 mg/m2 every 3 weeks) [46,47]. Oxaliplatin Neuropathy and Peroxisome Impairment actin normalization was performed for each sample. Values are expressed as the mean 6 S.E.M. percent of control of 10 rats per group, performed in 2 different experimental set. Each value represents the mean of P,0.05 vs vehicle + vehicle; ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.g006 actin normalization was performed for each sample. Values are expressed as the mean 6 S.E.M. percent of control of 10 rats per group, performed in 2 different experimental set. Each value represents the mean of P,0.05 vs vehicle + vehicle; ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.g006 analyzed by an investigator other than the one who performed measurements to avoid possible bias. Data were analyzed using the ‘‘Origin 8.1’’ software (OriginLab, Northampton, MA, USA). by rosiglitazone (Figure 3A). In Figure 3B the withdrawal threshold to non-noxious thermal stimulus was shown. Cold plate test highlighted a decreased pain threshold starting from the 2nd week of oxaliplatin treatment (9.660.8 s, oxaliplatin + vehicle group, in comparison to vehicle + vehicle group, 19.360.3 s). On day 14 pain threshold was increased by 49% and 84% in 3 and 10 mg kg21 rosiglitazone-treated animals, respectively. Both rosiglitazone dosages were effective on day 21 increasing pain threshold by 39% and 49%, respectively (Figure 3B). On day 21, motor coordination was evaluated by Rota rod test measuring the walking time and the number of falls in 600 s. In comparison with control rats (number of falls 1.360.3, time 600 s) oxaliplatin treated animals fell down 5.660.4 times (Figure 4A) and maintained the balance for 162.0638.5 seconds (Figure 4B) and. Three mg kg21 rosiglitazone reduced the number of falls to 2.860.6 (Figure 4A) and improved the time of walking to 490645 seconds (Figure 4B). The higher dosage of rosiglitazone was ineffective. analyzed by an investigator other than the one who performed measurements to avoid possible bias. Data were analyzed using the ‘‘Origin 8.1’’ software (OriginLab, Northampton, MA, USA). Primary rat astrocytes In astrocyte cell culture, peroxisomes were highlighted as catalase-positive organelles. After 5 day incubation, 1 mM oxaliplatin increased the number of peroxisomes by 54% (Figure 1). The PPARc agonist rosiglitazone (10 mM) fully prevented the increase whereas 10 mM rosiglitazone per se (without oxaliplatin) did not modify peroxisome number. On the contrary the PPARc antagonist G3335 (30 mM) was able to increase peroxisomes to the same extent of oxaliplatin (45%; Figure 1). G3335 effect was rosiglitazone-inhibitable (data not shown). The incubation for 48 h with 10 mM oxaliplatin induced similar effects (data not shown). Aimed to investigate the expression level of catalase in peroxisomes, we measured the ratio between the optical density value of catalase immunostaining and the number of peroxisomes. Oxaliplatin (1 mM, 5 day incubation) reduced the ratio by about 60% in comparison to the control value (Figure 1). G3335 (30 mM) decreased catalase expression similarly to oxaliplatin while 10 mM rosiglitazone significantly prevented alterations (Figure 1). After 48 h incubation, 10 mM oxaliplatin and 30 mM G3335 reduced the ratio by 26% and 49% (data not shown). July 2014 \| Volume 9 \| Issue 7 \| e102758 ure 6. Levels of carbonylated proteins in the spinal cord of oxaliplatin-treated rats. At 21th day, the lumbar tract o anted and analyzed to measure protein oxidation Densitometric analysis (top panel) and representative Western blot (lower Figure 6. Levels of carbonylated proteins in the spinal cord of oxaliplatin-treated rats. At 21th day, the lumbar tract of the spinal cord was explanted and analyzed to measure protein oxidation. Densitometric analysis (top panel) and representative Western blot (lower panel) are shown. B- PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 10 Oxaliplatin Neuropathy and Peroxisome Impairment Oxaliplatin Neuropathy and Peroxisome Impairment On the contrary, i P ki ’ d l h i ff i d d b Among the debated biomolecular mechanisms of nervous damage induced by platinum derivatives [50], we previously indicated the oxidative stress as exploitable pathological target for the treatment of chemotherapy-induced neuropathy. In our model, a relationship between oxaliplatin-induced neuropathic pain and oxidative damage was shown [7]. The cellular redox balance is mainly regulated by two organelles, mitochondria and peroxisomes, and the oxidative homeostasis is due to their cooperation [51]. Mitochondrion was extensively studied and it is considered a pivotal target of platinum neurotoxicity since Zheng et al. [10] described an increase in the incidence of swollen and vacuolated mitochondria in peripheral nerve axons in animals treated with oxaliplatin. Moreover, we have recently confirmed that oxaliplatin promotes a significant cytosolic release of cytochrome C in astrocyte cell culture, indicating a mitochondrial suffering [52]. On the contrary, the peroxisomal compartment was poor analyzed and its relevance in cellular redox metabolism has been underestimated for a long time [22]. Peroxisomes are single- membrane bound organelles with a protein-rich matrix and play a key role in both the production and scavenging of ROS in the cell [15]. Catalase is the most important antioxidant defense enzyme in mammalian peroxisomes breaking down hydrogen peroxide to water and oxygen [26,53,54]. Catalase alterations are highlighted in many neurodegenerative conditions correlate to oxidative damage. Brain of aged patients showed a reduced catalase functionality [55], similarly catalase activity was affected in a rat model of Parkinson’s disease [56]. Catalase impairment was also described in neuropathic conditions since decreased catalase efficiency was described in sciatic nerve of rats affected by diabetic neuropathy [57] as well as in brain regions of animals with delayed neuropathy induced by organophosphate [58]. On the other hand, the oxidative unbalance of these painful conditions has never been associated with peroxisome alterations. The present results highlight the relevance of peroxisomes in oxaliplatin-dependent neurotoxicity. In astrocyte culture, glial cells implied in the development and maintenance of chronic pain [59], [60] and sensitive to the platinum drug toxicity [8], oxaliplatin increases the number of peroxisomes. Peroxisomes are able to respond to physiological changes in cellular environment adapting their number, morphology, enzyme content and metabolic functions [61]. In particular, alterations of peroxisome number were described during carcinogenesis and liver cirrhosis, suggest- ing proliferative mechanisms as well as peroxisome division [61]. Oxaliplatin Neuropathy and Peroxisome Impairment Scale bar 50 mm. In the lower panel quantitative analysis of cellular density is shown. Each value represents the mean 6 S.E.M. of 10 rats per group, performed in 2 different experimental sets. P,0.05 vs vehicle + vehicle; ‘P,0.05 vs oxaliplatin + vehicle. doi:10 1371/journal pone 0102758 g007 Scale bar 50 mm. In the lower panel quantitative analysis of cellular density is shown. Each value represents the mean 6 S.E.M. of 10 rats per group, performed in 2 different experimental sets. *P,0.05 vs vehicle + vehicle; ‘P,0.05 vs oxaliplatin + vehicle. doi:10.1371/journal.pone.0102758.g007 highlighted in DRGs and spinal cord. These data suggest an impairment of peroxisome that may participate to oxaliplatin- induced redox unbalance previously observed in astrocyte culture as well as in the nervous tissue of neuropathic animals [7,8]. conversion of animal doses to the Human Equivalent Dose [48,49]). The daily repeated administration of 2.4 mg kg21 performed in the animal model allows to obtain a cumulative dose of 36 mg kg21 corresponding to 1332 mg/m2. This dosage mimics the clinical cumulative oxaliplatin dose causing chronic neuropathy. Moreover, in our condition the inorganic platinum plasmatic levels after 21 days of treatment is 3.57360.217 mg/mL in line to human plasma concentration. conversion of animal doses to the Human Equivalent Dose [48,49]). The daily repeated administration of 2.4 mg kg21 performed in the animal model allows to obtain a cumulative dose of 36 mg kg21 corresponding to 1332 mg/m2. This dosage mimics the clinical cumulative oxaliplatin dose causing chronic neuropathy. Moreover, in our condition the inorganic platinum plasmatic levels after 21 days of treatment is 3.57360.217 mg/mL in line to human plasma concentration. Oxaliplatin-induced alteration of catalase, in terms of activity and expression, is comparable to that evoked by the pharmaco- logical blockade of PPARc (by the selective and reversible PPARc antagonist G3335 [41]). PPARs belong to a nuclear receptor superfamily actively involved in immunoregulation. Membrane lipid composition, cell proliferation, sensitivity to apoptosis, energy homeostasis, and various inflammatory transcription factors are regulated by the trans-repression capabilities of these receptors [64]. The c subtype of PPARs is expressed both in neurons [65] and glia cells [66] and PPARc stimulation protects neuronal and axonal damage induced by oxidative stimuli [67]. This property has been associated with a concomitant increase in the enzymatic activity of catalase [67] accordingly to the evidence of a direct modulation of this enzyme by PPARc [25]. Oxaliplatin Neuropathy and Peroxisome Impairment The similarity of oxaliplatin- and G3335-mediated effects on astrocyte catalase and peroxisome number suggests a common dysregulation of these organelles. Since oxaliplatin impairs catalase in 48 h whereas G3335 needs 5 days, we can hypothesize a direct effect of oxaliplatin on the peroxisome machinery. On the other hand, 5 days incubation with the selective PPARc agonist rosiglitazone, reduces the enzymatic failure promoted by both oxaliplatin and G3335 and normalizes the peroxisome number. Accordingly, the repeated administration of rosiglitazone improves catalase effi- ciency in the nervous tissue of oxaliplatin-treated rats and prevents spinal oxidative alterations reducing the lipid peroxidation and carbonylated protein levels. The maintenance of the defensive properties of catalase, and the consequent redox balance improvement, are concomitant with the control of pain exerted by the PPARc agonist. A relationship between pain and catalase impairment is suggested. Rosiglitazone reduces oxaliplatin-depen- dent alterations of the pain threshold when both noxious or non- noxious stimuli are used. The anti-neuropathic effect is dose- and time-dependent till day 14. On day 21, the effect of 3 and 10 mg kg21 is similar in the Cold plate test. On the same day, the low dose treated animals (3 mg kg21) show an improvement in motor coordination and a significant restoration of catalase expression and activity in the central nervous system, whereas the beneficial effect of the higher dose (10 mg kg21) disappears. These evidences suggest the need of a mild PPARc stimulation to obtain a protective antineuropathic effect. Interestingly, the 10 mg kg21 dosage prevents the increase of astrocyte number in the spinal cord, on the contrary the lower dose is ineffective. Glia cells contribute to the persistence of pain [68] as well as to several omeostatic functions above all neuroprotection [69]. The block of glial-related signals impairs functional recovery after nerve injury [70], suggesting that tout court glial inhibition may relieve pain but hinders the rescue mechanisms that protect nervous tissue. Accordingly, the present data suggest that the lower dose of rosiglitazone (which is unable to decrease astrocyte cell number) yields the better balance between neuroprotective and anti- hyperalgesic effects. On the other hand, the effect of rosiglitazone on glia cells is not univocally depending by pathological condition and by CNS area. In rats, 0.1 mg kg21 rosiglitazone (i.p.) was able to decrease the cognitive impairment after status epilepticus and to inhibit astrocyte activation in the striatum [71]. Behavioural measurements Seven days after the beginning of oxaliplatin treatment (2.4 mg kg21 i.p., daily) pain sensitivity towards noxious stimulus (Paw pressure test) was altered. The weight tolerated on the posterior paw significantly decreased from the control value of 73.360.8 g to 49.061.5 g (Figure 3A). Rosiglitazone, 3 and 10 mg kg21, per os administered daily, limited hypersensitivity increasing the tolerated weight (60.062.1 g and 61.962.3, respectively; Figure 3A). The progression of oxaliplatin-dependent neuropathic state on day 14 and 21 (Figure 3A) was reduced dose dependently The model used for the present research is consistent with the clinical practice. 2.4 mg kg21 oxaliplatin corresponds to the common human dosage (considering the Km factor 37 for the July 2014 \| Volume 9 \| Issue 7 \| e102758 11 PLOS ONE \| www.plosone.org Oxaliplatin Neuropathy and Peroxisome Impairment Figure 7. Glial profile in spinal cord scored with GFAP-positive cells in the dorsal horn of the lumbar tract. Transverse sections of s cord imaged with 20X objective of A) vehicle + vehicle, B) oxaliplatin + vehicle, C) and D) oxaliplatin + rosiglitazone 3 and 10 mg kg21, respect PLOS ONE \| www plosone org 12 July 2014 \| Volume 9 \| Issue 7 \| e10 Figure 7. Glial profile in spinal cord scored with GFAP-positive cells in the dorsal horn of the lumbar tract. Transverse sections of ure 7 G Figure 7. Glial profile in spinal cord scored with GFAP-positive cells in the dorsal horn of the lumbar tract. Transverse sections of spinal cord imaged with 20X objective of A) vehicle + vehicle, B) oxaliplatin + vehicle, C) and D) oxaliplatin + rosiglitazone 3 and 10 mg kg21, respectively. PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e102758 12 Oxaliplatin Neuropathy and Peroxisome Impairment July 2014 \| Volume 9 \| Issue 7 \| e102758 References 23. Issemann I, Green S (1990) Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347: 645–650. 1. Andre´ T, Boni C, Mounedji-Boudiaf L, Navarro M, Tabernero J, et al. (2004) Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med 350: 2343–2351. 24. Crosby MB, Zhang J, Nowling TM, Svenson JL, Nicol CJ, et al. (2006) Inflammatory modulation of PPAR gamma expression and activity. Clin Immunol 118: 276–283. g J 2. Kannarkat G, Lasher EE, Schiff D (2007) Neurologic complications of chemotherapy agents. Curr Opin Neurol 20: 719–725. 3. Wolf S, Barton D, Kottschade L, Grothey A, Loprinzi C (2008) Chemotherapy induced peripheral neuropathy: prevention and treatment strategies. Eur J Cancer 44: 1507–1515. 25. Girnun GD, Domann FE, Moore SA, Robbins ME (2002) Identification of a functional peroxisome proliferator-activated receptor response element in the rat catalase promoter. Mol Endocrinol 16: 2793–2801. 4. De Gramont A, Figer A, Seymour M, Homerin M, Hmissi A, et al. (2000) Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J Clin Oncol 18: 2938–2947. 26. Putnam CD, Arvai AS, Bourne Y, Tailner JA (2000) Active and inhibited catalase structures: ligand and NAPH binding and catalytic mechanism J Mol Biol 296: 295–309. 5. Souglakos J, Mavroudis D, Kakolyris S, Kourousis CH, Vardakis N, et al. (2002) Triplet combination with irinotecan plus oxaliplatin plus continuous-infusion fluorouracil and leucovorin as first-line treatment in metastatic colorectal cancer: a multicenter phase II trial. J Clin Oncol 20: 2651–2657. 27. Powers SK, Jackson MJ (2008) Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. Physiol. Rev 88: 1243– 1276. 28. Diab A, Deng C, Smith JD, Hussain RZ, Phanavanh B, et al. (2002) Peroxisome proliferator-activated receptor-gamma agonist 15-deoxy-Delta(12,14)-prosta- glandin J(2) ameliorates experimental autoimmune encephalomyelitis. J Immunol 168: 2508–2515. 6. Cavaletti G, Marmiroli P (2010) Chemotherapy-induced peripheral neurotox- icity. Nat Rev Neurol 6: 657–666. 7. Di Cesare Mannelli L, Zanardelli M, Failli P, Ghelardini C (2012) Oxaliplatin- induced neuropathy: oxidative stress as pathological mechanism. Protective effect of silibinin. J Pain 13: 276–284. 29. Feinstein DL, Galea E, Gavrilyuk V, Brosnan CF, Whitacre CC, et al. (2002) Peroxisome proliferator-activated receptor-gamma agonists prevent experimen- tal autoimmune encephalomyelitis. Ann Neurol 51: 694–702. 8. References Napolitano M, Costa L, Palermo R, Giovenco A, Vacca A, et al. (2011) Protective effect of pioglitazone, a PPARgamma ligand, in a 3 nitropropionic p gy 15. Schrader M, Fahimi HD (2006) Peroxisomes and oxidative stress. Biochim Biophys Acta 1763: 1755–1766. acid model of Huntington’s disease. Brain Research Bulletin 85: 23 p y 16. Powes JM, Mosers HW (1998) Peroxisomal disorders: genotype, phenotype, major neuropathologic lesions, and pathogenesis. Brain Pathol 8: 101–120. 37. Benedusi V, Martorana F, Brambilla L, Maggi A, Rossi D (2012) The Peroxisome Proliferator-activated Receptor gamma (PPARgamma) Controls Natural Protective Mechanisms against Lipid Peroxidation in Amyotrophic Lateral Sclerosis. The Journal Of Biological Chemistry 287: 35899–35911. 17. Bottelbergs A, Verheijden S, Van Veldhoven PP, Just W, Devos R, et al. (2012) Peroxisome deficiency but not the defect in ether lipid synthesis causes activation of the innate immune system and axonal loss in the central nervous system. J Neuroinflammation 9: 61. 38. Lourenco MV, Ledo JH (2013) Targeting Alzheimer’s pathology through PPARc signaling: modulation of microglial function. J Neurosci 33: 5083–5084. 18. Trompier D, Vejux A, Zarrouk A, Gondcaille C, Geillon F, et al. (2014) Brain peroxisomes. Biochimie 98: 102–110. 39. McCarty KD, de Vellis J (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85: 890–902. p 19. D’amico A, Bertini E (2013) Metabolic neuropathies and myopathies. Handb Clin Neurol 113: 1437–1455. 40. Perdomo MC, Santos JE, Badinga L (2011) Trans-10, cis-12 conjugated linoleic acid and the PPAR-c agonist rosiglitazone attenuate lipopolysaccharide-induced TNF-a production by bovine immune cells. Domest Anim Endocrinol 41: 118– 25. 20. Cimini A, Moreno S, D’Amelio M, Cristiano L, D’Angelo B, et al. (2009) Early biochemical and morphological modifications in the brain of a transgenic mouse model of Alzheimer’s disease: a role for peroxisomes. J Alzheimers Dis 18: 935– 952. 41. Ye F, Zhang ZS, Luo HB, Shen JH, Chen KX, et al. (2006) The dipeptide H- Trp-Glu-OH shows highly antagonistic activity against PPARgamma: bioassay with molecular modeling simulation. Chembiochem 7: 74–82. 21. Kou J, Kovacs GG, Ho¨ftberger R, Kulik W, Brodde A, et al. (2011) Peroxisomal alterations in Alzheimer’s disease. Acta Neuropathol 122: 271–283. 42. Cavaletti G, Tredici G, Petruccioli MG, Donde` E, Tredici P, et al. (2001) Effects of different schedules of oxaliplatin treatment on the peripheral nervous system of the rat. Eur J Cancer 37: 2457–2463. 22. Acknowledgments Finally, it is important to highlight the absence of interaction between the PPAR-c agonist and the lethal effect exerted by oxaliplatin on the human colon cancer cells HT-29. Moreover, thiazolidinediones reduce the growth of different tumors, arresting cancer cell proliferation by affecting cell cycle checkpoints or inhibiting growth factors [77]. Preclinical and clinical studies have demonstrated the antitumoral effect of rosiglitazone alone or in combination [78]. This research was funded by the Italian Ministry of Instruction, University and Research (MIUR) and by the University of Florence. The authors declare no conflict of interest. Oxaliplatin Neuropathy and Peroxisome Impairment In our condition, the increase in peroxisome number is insufficient to provide the physiological level of catalase functionality. Both activity and expression of this antioxidant enzyme are reduced in astrocytes by oxaliplatin treatment. Astrocytes are generally less susceptible to oxidative injury than neurons and provide for their health and functionality [62,63]. The dysregulation of astrocyte antioxidant machinery can influence neuron functionality and evoke nervous circuit alterations. Accordingly, in the rat model of oxaliplatin-induced neuropathy a similar alteration of catalase is July 2014 \| Volume 9 \| Issue 7 \| e102758 13 PLOS ONE \| www.plosone.org Oxaliplatin Neuropathy and Peroxisome Impairment 3 mg kg21 rosiglitazone (i.p.) is concomitant with an increase in GFAP expression in the hippocampus [72]. Noteworthy, the capability of rosiglitazone to penetrate the blood brain barrier is debated [73–75], though central effects have been demonstrated [76]. these phenomena and controls pain. The optimal profile shown by the lower dosage suggests the mild stimulation of PPARc as possible approach to the oxaliplatin neuropathy. Author Contributions Conceived and designed the experiments: CG PF LDCM. Performed the Conceived and designed the experiments: CG PF LDCM. Performed the experiments: MZ LM LC PF. Analyzed the data: MZ. Wrote the paper: MZ LDCM CG PF. experiments: MZ LM LC PF. Analyzed the data: MZ. Wrote the paper: MZ LDCM CG PF. In summary, oxaliplatin-dependent neurotoxicity alters perox- isome functionality. The PPARc agonist rosiglitazone prevents References Di Cesare Mannelli L, Zanardelli M, Failli P, Ghelardini C (2013a) Oxaliplatin- induced oxidative stress in nervous system-derived cellular models: Could it correlate with in vivo neuropathy?. Free Radic Biol Med 61C: 143–150. 30. Raikwar HP, Muthian G, Rajasingh J, Johnson CN, Bright JJ (2006) PPARgamma antagonists reverse the inhibition of neural antigen-specific Th1 response and experimental allergic encephalomyelitis by Ciglitazone and 15- deoxy-Delta12,14-prostaglandin J2. J Neuroimmunol 178: 76–86. 9. Di Cesare Mannelli L, Pacini A, Matera C, Zanardelli M, Mello T, et al. (2013b) Involvement of a7 nAChR subtype in rat oxaliplatin-induced neuropathy: Effects of selective activation. Neuropharmacology 79C: 37–48. deoxy-Delta12,14-prostaglandin J2. J Neuroimmunol 178: 76– 31. Heneka MT, Landreth GE, Hu¨ll M (2007) Drug insight: effects mediated by peroxisome proliferator-activated receptor-gamma in CNS disorders. Nat Cli Pract Neurol 3: 496–504. p gy 10. Zheng H, Xiao WH, Bennett GJ (2011) Functional deficits in peripheral nerve mitochondria in rats with paclitaxel- and oxaliplatin-evoked painful peripheral neuropathy. Exp Neurol 232: 154–161. 32. Bernardo A, Minghetti L (2008) Regulation of Glial Cell Functions by PPAR gamma. Natural and Synthetic Agonists. PPAR Res 2008: 864140. 11. De Duve C, Baudhuin P (1966) Peroxisomes (microbodies and related particles). Physiol Rev 46: 323–357. 33. Landreth G, Jiang Q, Mandrekar S, Heneka M (2008) PPARgamma agonists as therapeutics for the treatment of Alzheimer’s disease. Neurotherapeutics 5: 481– 489. 12. Elliott BM, Dodd NJ, Elcombe CR (1986) Increased hydroxyl radical production in liver peroxisomal fractions from rats treated with peroxisome proliferators. Carcinogenesis 7: 795–799. 34. Lee YJ, Han SB, Nam SY, Oh KW, Hong JT (2010) Inflammation and Alzheimer’s disease. Arch Pharm Res 33: 1539–1556. 13. Zwacka RM, Reuter A, Pfaff E, Moll J, Gorgas K, et al. (1994) The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species EMBO J 13: 5129–5134. 35. Swanson CR, Bondarenko VJV, Brunner K, Simmons HA, Ziegler TE, et al. (2011) The PPAR-c agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys Journal of Neuroinflammation 8: 91. yg p 14. Stolz DB, Zamora R, Vodovotz Y, Loughran PA, Billiar TR, et al. (2002) Peroxisomal localization of inducible nitric oxide synthase in hepatocytes. Hepatology 36: 81–93. 36. Napolitano M, Costa L, Palermo R, Giovenco A, Vacca A, et al. (2011) Protective effect of pioglitazone, a PPARgamma ligand, in a 3 nitropropionic acid model of Huntington’s disease. Brain Research Bulletin 85: 231–237. 36. Oxaliplatin Neuropathy and Peroxisome Impairment Gray E, Ginty M, Kemp K, Scolding N, Wilkings A (2012) The PPAR- c agonist pioglitazone protects cortical neurons from inflammatory mediators via improvement in peroxisomal function. J Neuroinflammation 9: 63. p 49. Reagan-Shaw S, Nihal M, Ahamad N (2008) Dose translation from animal to human studies revisited. FASEB J 22: 659–661. 68. Scholz J, Woolf CJ (2007) The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 10: 1361–1368. 50. Grisold W, Cavaletti G, Windebank AJ (2012) Peripheral neuropathies from chemotherapeutics and targeted agents: diagnosis, treatment, and prevention. Neuro Oncol Suppl 4: iv 45–54. 69. Milligan ED, Watkins LR (2009) Pathological and protective roles of glia in chronic pain. Nat Rev Neurosci 10: 23–36. pp 51. Wang B, Veldhoven PP, Brees C, Rubio C, Nordgren M, et al. (2013) Mitochondria are targets for peroxisome-derived oxidative stress in cultured mammalian cells. Free Radic Biol Med 65: 882–894. 70. Boivin A, Pineau I, Barrette B, Filali M, Vallie`res N, et al. (2007) Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. J Neurosci 27: 12565–12576. 52. Zanardelli M, Di Cesare Mannelli L, Micheli L, Cinci L, Baptista De Souza D, et al. (2013) oxaliplatin-dependent apoptosis: different apoptotic process in primary astrocytes vs colorectal cancer cells. Proceeding of 36uCongress of the Italian Society of Pharmacology, Turin, 23–26 October. Available: http:// congresso.sifweb.org/abs/444.pdf. 71. Hong S, Xin Y, HaiQin W, GuiLian Z, Ru Z, et al. (2012) The PPARc agonist rosiglitazone prevents cognitive impairment by inhibiting astrocyte activation and oxidative stress following pilocarpine-induced status epilepticus. Neurol Sci 33: 559–566. 53. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, et al. (2002) Intracellular Compartments and Protein Sorting. In: Garland Science, editor. Molecular biology of the cell, 4th edition. New York. 72. Lee EY, Lee JE, Park JH, Shin IC, Koh HC (2012) Rosiglitazone, a PPAR-c agonist, protects against striatal dopaminergic neurodegeneration induced by 6- OHDA lesions in the substantia nigra of rats. Toxicol Lett 213: 332–344. 54. Powers SK, Lennon SL (1999) Analysis of cellular responses to free radicals: focus on exercise and skeletal muscle. Proc Nutr Soc 58: 1025–1033. 73. Festuccia WT, Oztezcan S, Laplante M, Berthiaume M, Michel C, et al. (2008) Peroxisome proliferato-activated receptor-gamma-mediated positive energy balance in the rat is associated with reduced sympathetic drive to adipose tissues and thyroid status. Endocrinology 149: 2121–2130. 55. Oxaliplatin Neuropathy and Peroxisome Impairment 43. Leighton GE, Rodriguez RE, Hill RG, Hughes J (1988) kappa-Opioid agonists produce antinociception after i.v. and i.c.v. but not intrathecal administration in the rat. Br J Pharmacol 93: 553–560. 61. Ribeiro D, Castro I, Fahimi HD, Schrader M (2012) Peroxisome morphology in pathology. Histol Histopathol 27: 661–676. 62. Chen Y, Chan PH., Swanson RA (2001) Astrocytes overexpressing Cu, Zn superoxide dismutase have increased resistance to oxidative injury. Glia 33: 343– 347. J 44. Goulle´ JP, Mahieu L, Castermant J, Neveu N, Bonneau L, et al. (2005) Metal and metalloid multi-elementary ICP MS validation in whole blood, plasma, urine and hair. Reference values. Forensic Sci Int 153: 39–44. 63. Choi JH, Kim DH, Yun IJ, Chang JH, Chun BG, et al. (2007) Zaprinast inhibits hydrogen peroxide-induced lysosomal destabilization and cell death in astrocytes. Eur J Pharmacol 571: 106–115. 45. Morrison JG, White P, McDougall S, Firth JW, Woolfrey SG, et al. (2000) Validation of a highly sensitive ICP-MS method for the determination of platinum in biofluids: application to clinicalpharmacokinetic studies with oxaliplatin. J Pharm Biomed Anal 24: 1–10. 64. Hunter RL, Dragicevic N, Seifert K, Choi DY, Liu M, et al. (2007) Inflammation induces mitochondrial dysfunction and dopaminergic neurode- generation in the nigrostriatal system. J Neurochem 100: 1375–1386. p J 46. Graham MA, Lockwood GF, Greenslade D, Brienza S, Bayssas M, et al. (2000) Clinical pharmacokinetics of oxaliplatin: a critical review. Clin Cancer Res 6: 1205–1218. generation in the nigrostriatal system. J Neurochem 100: 1375–138 65. Sarruf DA, Yu F, Nguyen HT, Williams DL, Printz RL, et al. (2009) Expression of peroxisome proliferator-activated receptor-gamma in key neuronal subsets regulating glucose metabolism and energy homeostasis. Endocrinology 150: 707–712. 47. Han CH, Khwaounjoo P, Kilfoyle DH, Hill A, McKeage MJ (2013) Phase I drug-interaction study of effects of calcium and magnesium infusions on oxaliplatin pharmacokinetics and acute neurotoxicity in colorectal cancer patients. BMC Cancer 13: 495. 66. Carniglia L, Durand D, Caruso C, Lasaga M (2013) Effect of NDP-a-MSH on PPAR-c and -b expression and anti-inflammatory cytokine release in rat astrocytes and microglia. PLoS One 8: e57313. 48. Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper HE (1996) Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep 50: 219–244. 67. References Fransen M, Nordgren M, Wang B, Apanasets O (2012) Role of peroxisomes in ROS/RNS-metabolism: implications for human disease. Biochim Biophys Acta. 1822: 1363–1373. July 2014 \| Volume 9 \| Issue 7 \| e102758 July 2014 \| Volume 9 \| Issue 7 \| e102758 14 PLOS ONE \| www.plosone.org Oxaliplatin Neuropathy and Peroxisome Impairment Venkateshappa C, Harish G, Mahadevan A, Srinivas Bharath MM, Shankar SK (2012) Elevated oxidative stress and decreased antioxidant function in the human hippocampus and frontal cortex with increasing age: implications for neurodegeneration in Alzheimer’s disease. Neurochem Res 37: 1601–1614. 74. Strum JC, Shehee R, Virley D, Richardson J, Mattie M, et al. (2007) Rosiglitazone induces mitochondrial biogenesis in mouse brain. J Alzheimers Dis 11: 45–51. 56. Khan MM, Ahmad A, Ishrat T, Khan MB, Hoda MN, et al. (2010) Resveratrol attenuates 6-hydroxydopamine-induced oxidative damage and dopamine depletion in rat model of Parkinson’s disease. Brain Res 1328: 139–151. 75. Sheu WH, Chuang HC, Cheng SM, Lee MR, Chou CC, et al. (2011) Microdialysis combined blood sampling technique for the determination of rosiglitazone and glucose in brain and blood of gerbils subjected to cerebral ischemia. J Pharm Biomed Anal 54: 759–764. 57. Kamboj SS, Vasishta RK, Sandir R (2010) N-acetylcysteine inhibits hypergly- cemia-induced oxidative stress and apoptosis markers in diabetic neuropathy. J Neurochem 112: 77–91. 76. Lu M, Sarruf DA, Talukdar S, Sharma S, Li P, et al. (2011) Brain PPAR-c promotes obesity and is required for the insulin-sensitizing effect of thiazolidi- nediones. Nat Med 17: 618–622. 58. Masoud A, Sandhir R (2012) Increased oxidative stress is associated with the development of organophosphate-induced delayed neuropathy. Hum Exp Toxicol 31: 1214–1227. 77. Blanquicett C, Roman J, Hart CM (2008) Thiazolidinediones as anti-cancer agents. Cancer Ther 6(A): 25–34. 59. Gao YJ, Ji RR (2010) Targeting astrocyte signaling for chronic pain. Neurotherapeutics 7: 482–493. 78. Joshi H, Pal T, Ramaa CS (2014) A new dawn for the use of thiazolidinediones in cancer therapy. Expert Opin Investig Drugs 23: 501–510. 60. Salvemini D, Neumann W (2010) Targeting peroxynitrite driven nitroxidative stress with synzymes: A novel therapeutic approach in chronic pain management. Life Sci 86: 604–614. 60. Salvemini D, Neumann W (2010) Targeting peroxynitrite driven nitroxidative stress with synzymes: A novel therapeutic approach in chronic pain management. Life Sci 86: 604–614. July 2014 \| Volume 9 \| Issue 7 \| e102758 PLOS ONE \| www.plosone.org 15
https://openalex.org/W2054613070	https://zenodo.org/records/2472052/files/article.pdf	German	null	Die Fehler der Uhrschraube am 30‐cm‐Refraktor der Leipziger Sternwarte	Astronomische Nachrichten	1,911	public-domain	3,655	Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Stern aktor der Leipziger Sternwarte. Von F. Hnyn. Bewegung gebracht und nun in moglichst rascher Folge bei laufendem Uhrwerk Einstellungen auf den Stern ausgefihrt wurden. Das Uhrwerk des Leipziger Refraktors hat die bekannte RcpsoMsche Konstruktion rnit Federpendelregulator. Die Be- wegung wird voni Uhrwerk durch einen Schliissel und ein Paar gleicher Winkelriider auf eine Schnecke ubertragen, die in ein Schneckenrad rnit 36 Zahnen eingreift. Dieses Rad sitzt fest auf der Spindel der Uhrschraube, die den Uhrkreis von 54 cm Durchinesser bewegt. Der Kreis besitzt 1200 Zahne, die Uhrschraube macht in 72 Sek., die Schnecke und ebenso der lange Schlussel in 2 Sek. eine Umdrehung. Der Moment der Einstellung wurde registriert, zudem von Zeit zu Zeit der Moment, wo ein an der Uhrschraube angebrachter Index die Nullstellung passierte. Man erhiilt so, wie bei jeder Schraubenuntersuchung, die zur Trommel- ablesung gehorige Verschiebung der Mutter. Als Trommel ist hier das Schneckenrad gedacht, dessen 36 ZHhne also einer Trommelteilung in 36 partes entsprechen. Ein posi- tiver Fehler bedeutet, daD das Fernrohr vorausgeeilt ist. Im Juni I 9 I I wurde diese Untersuchung wiederholt ; die Fehler fanden sich in guter Ubereinstimmung rnit denen vom Jahre 1903. Das Mittel beider Bestimmungen befindet sich unter I der folgenden Tabelle. In den ersten Jahren ,nach Indienststellung des Re- fraktors wurde der Gang des Uhrwerks ofter untersucht. Zu dem Zwecke befindet sich an einem der letzten rasch laufen- den Riider (eine Umdrehung in I Z ? ~ ) ein elektrischer Kon- takt, der rnit Hilfe des Chronographen eine Kontrolle des Ganges gestattet. Diese Untersuchungen des Ganges, des Ein- flusses von Gewichtsllnderungen, Abhagigkeit von der Schwin- gungsamplitude des Pendels etc. gaben die erfreuliche Ge- wiDheit, daD der Isochronismus des Regulators sehr befriedi- gend ist. Zahlenangaben hieruber sollen einer spiiteren Mit- teilung vorbehalten bleiben. Ftir die Beobachtungen der ersten Jahre, soweit. sie iiberhaupt rnit Uhrwerk angestellt wurden, konnte die Bewegung des Fernronrs als vollig exakt angesehen werden. Als ich im Jahre 1903 zum erstenmale Distanzen von der Gr6k des Mondradius bestimmen muOte, trat die Notwendigkeit ein, die Ubertragung der Bewegung vom Uhrwerk auf das Fernrohr einer genaueren Untersuchung zu unterziehen, urn die Fehler in Rechnung ziehen zu konnen. Diese Untersuchungen sind veroffentlicht in der Abhandlung : Selenographische Koordinaten, 11. Teil, Abh. der Kgl. SHchs. Gesellschafi der Wissenschaften, 29. Band, S. 33 u. ff. 4556 347 Fur die noch ausstehende Feststellung der Beziehung zwischen der Helligkeit und Farbe der Sterne kommt, solange eine exakte MeDmethode nicht gefunden ist, nur die Schiit- zung nach der Abkiihlungsskala in Frage. Sicher scheint zu sein, dab rnit der Abnahme der Helligkeit eine Vertiehng der Farbe eintritt, die zwischen den Sternen erster bis vierter Grebe im Durchschnitt in allen Spektralklassen reichkh eine Stufe der zehnteiligen Abkiihlungsskala betriigt '). Alle Farben- schiitzungen sollten fiir Untersuchungen dieser Art deshalb bis auf OCI genau ausgef&rt werden, .und alle Beobachter sollten sich mtiglichst der Abkuhlungsskala bedienen. Die Hagcnsche Einteilung bietet vor der Osth#schen den Vorteil der symmetrischen Anordnung ; der Beobachter braucht nur die den Zahlen 3 und 6 zukommenden Farben genau seinem Gedslchtnis einzupriigen und sich zu erinnern, daD die Haupt- farben eine einfache arithmetische Progression mit der Diffe- renz 3 vorstellen. In praktischer Beziehung bedeutet also die Hagmche neunteilige Skala einen Fortschritt, durch welchen die Farbenschiitzungen erleichtert werden. O i E d : R 1 'it I I 0 P FY. Kt%gcr. Aarhus, 19 I I Dezember 7. ') Niiheres hiertiber siehe in Nr. 3 der Publikationen der Specola Vaticana. ') Niiheres hiertiber siehe in Nr. 3 der Publikationen der Specola Vaticana. raktor der Leipziger Sternwarte. Von F. Hnyn. Bewegung gebracht und nun in moglichst rascher Folge bei laufendem Uhrwerk Einstellungen auf den Stern ausgefihrt wurden. Die Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Sternwarte. Von F. Hnyn. B b h d i li h h F l b i 347 O i E d 4556 : R 1 'it I I 0 P Fur die noch ausstehende Feststellung zwischen der Helligkeit und Farbe der Sterne k eine exakte MeDmethode nicht gefunden ist, zung nach der Abkiihlungsskala in Frage. Sic sein, dab rnit der Abnahme der Helligkeit e der Farbe eintritt, die zwischen den Sternen er Grebe im Durchschnitt in allen Spektralklassen Stufe der zehnteiligen Abkiihlungsskala betriigt ') schiitzungen sollten fiir Untersuchungen diese bis auf OCI genau ausgef&rt werden, .und a sollten sich mtiglichst der Abkuhlungsskala b Hagcnsche Einteilung bietet vor der Osth#sche der symmetrischen Anordnung ; der Beobacht die den Zahlen 3 und 6 zukommenden Farben Gedslchtnis einzupriigen und sich zu erinnern, d farben eine einfache arithmetische Progression renz 3 vorstellen. In praktischer Beziehung die Hagmche neunteilige Skala einen For welchen die Farbenschiitzungen erleichtert we Aarhus, 19 I I Dezember 7. FY. Kt ') Niiheres hiertiber siehe in Nr. 3 der Publikationen der Specola Vaticana. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Sternwarte. Vo Das Uhrwerk des Leipziger Refraktors hat die bekannte Msche Konstruktion rnit Federpendelregulator. Die Be- Bewegung gebracht und nun in moglichst ras laufendem Uhrwerk Einstellungen auf den St 347 O i E d 4556 : R 1 'it I I 0 P Fur die noch ausstehende Feststellung zwischen der Helligkeit und Farbe der Sterne k eine exakte MeDmethode nicht gefunden ist, zung nach der Abkiihlungsskala in Frage. Sic sein, dab rnit der Abnahme der Helligkeit e der Farbe eintritt, die zwischen den Sternen er Grebe im Durchschnitt in allen Spektralklassen Stufe der zehnteiligen Abkiihlungsskala betriigt ') schiitzungen sollten fiir Untersuchungen diese bis auf OCI genau ausgef&rt werden, .und a sollten sich mtiglichst der Abkuhlungsskala b Hagcnsche Einteilung bietet vor der Osth#sche der symmetrischen Anordnung ; der Beobacht die den Zahlen 3 und 6 zukommenden Farben Gedslchtnis einzupriigen und sich zu erinnern, d farben eine einfache arithmetische Progression renz 3 vorstellen. In praktischer Beziehung die Hagmche neunteilige Skala einen For welchen die Farbenschiitzungen erleichtert we Aarhus, 19 I I Dezember 7. FY. Kt ') Niiheres hiertiber siehe in Nr. 3 der Publikationen der Specola Vaticana. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Sternwarte. Vo Das Uhrwerk des Leipziger Refraktors hat die bekannte Msche Konstruktion rnit Federpendelregulator. Die Be- Bewegung gebracht und nun in moglichst ras laufendem Uhrwerk Einstellungen auf den St 47 O i E d 4556 : R 1 'it I I 0 P 34 Fur die noch ausstehende Feststellung der Be zwischen der Helligkeit und Farbe der Sterne kommt, eine exakte MeDmethode nicht gefunden ist, nur die zung nach der Abkiihlungsskala in Frage. Sicher sc sein, dab rnit der Abnahme der Helligkeit eine Ve der Farbe eintritt, die zwischen den Sternen erster bi Grebe im Durchschnitt in allen Spektralklassen reich Stufe der zehnteiligen Abkiihlungsskala betriigt '). Alle schiitzungen sollten fiir Untersuchungen dieser Art bis auf OCI genau ausgef&rt werden, .und alle Be sollten sich mtiglichst der Abkuhlungsskala bediene Hagcnsche Einteilung bietet vor der Osth#schen den 348 4556 ') Niiheres hiertiber siehe in Nr. 3 der Publikationen der Specola Vaticana. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Stern Um kurz den Gang der Untersuchung hier wiederzugeben, genugt es zu sagen, daO das Fernrohr auf einen Aquatorstern ge- richtet, die Mikrometerschraube in die Richtung der tiiglichen Eine Umdrehung der Schraube bewegt das Fernrohr urn 1080'; man sieht also, daD bei einer Amplitude des periodischen Fehlers von 174 nach den Erfahrungen rnit guten Mikrometerschrauben dieser Fehler als sehr klein be- zeichnet werden mu& Da nun die Absicht besteht, den Re- fraktor in Zukunft zu scharfen Untersuchungen photographi- scher Natur zu verwenden, muDte versucht werden, die hier- bei doch sehr sttirenden periodischen Fehler der Schraube zu beseitigen. Es lag die Vermutung nahe, daO bei dem eigentum- lichen Verlaufe der Fehlerkurve die Ursache teilweise in dem Schneckenrade zu suchen sei; denn das Rad hatte den aukrordentlich kleinen Durchmesser von I 8 mm, seine a h n e ebenso wie die Schnecke waren gllnzlich ausgeschliffen. Ich machte den Versuch, rnit einer improvisierten Einrichtung die Teilungsfehler des Rades zu bestimmen. Die gefundenen Werte finden sich in der Spalte I1 der Tabelle; sie sind infolge der unvollkommenen Vomchtung etwas unsicher aus- 45 56 349 3 5 0 sekunden, d. h. so gegeben, wie sie im Fernrohr erscheinen. Zieht man 111 von I ab, so muB man die periodischen Fehler erhalten, die auf Rechnung der Schraube nebst Mutt& zu setzen sind, siehe Spalte IV. gefallen, geben aber ein richtiges Bild von ihrem Verlaufe. Diese Fehler sind so zu verstehen, daB ein positiver Fehler dann vorhanden ist, wenn eine Umdrehung der Schnecke das Rad nicht um genau IOO, sondern um etwas mehr be- wegt. Unter 111 sind die Fehler der Spalte I1 in Bogen- wegt. Unter 111 sind die Fehler der Spalte I1 in Bogen- Trom- inel _. _. _I - 0 I 2 3 4 5 6 7 8 9 I 0 11 I 2 '3 14 ' 5 16 17 18 19 2 0 2 1 2 2 23 24 25 26 27 28 29 30 3' 32 33 34 35 - 0137 -0.33 - 0.30 -0.23 -0.23 -0.27 - 0.30 -0.31 - 0 . 2 0 - 0.40 --0.47 - 0.40 -0.31 -0.33 - 0.30 -0.23 - 0.07 +0.23 +041 + 0.63 +073 +0.80 +0.87 +0.90 +0.87 +0.63 +0.50 +0.23 -0.13 -0.13 -0.33 - 0.40 - 0.43 - 0.3 I -0.10 0.00 - I' + I + I -- 4 -. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Stern 7 - 9 0 I - - 1 1 - I 2 - I 2 - 1 1 - 9 - 8 - 5 - 3 + ? + 4 + 5 + I + I + I + 5 + 3 + 3 + 3 + 3 0 or0 0.0 0.0 0.0 0.0 -0.2 - 0.3 -- 0.4 - 0.5 - 0.6 - 0.6 -0.5 - 0.4 - 0.4 -0.2 -0.1 0.0 +O.I +0.2 +0.2 +0.3 +0.2 +0.2 +0.2 +0.2 +0.2 +om3 +0.3 + 4 ' +0.2 + 4 +0.2 + 4 +0.2 + 3 +O.I + I 0.0 I 0.0 I 0.0 I 0.0 - - - - or4 - 0.3 - 0.3 - 0.2 - 0.2 0.0 0.0 +O.I +O.I +0.2 +O.I. +O.I +O.I 0.0 -0.1 - 0.1 -0.1 -0.2 0.0 +0.3 +0.3 -1-0.5 +0.7 +0.7 +0.7 +0.4 +0.4 + 0.3 0.0 - 0.2 - 0.3 - 0.3 - 0.4 -.0.4 - 0.4 - 0.2 V VI v11 VIIL . - .- - ... .. . . . - 0737 - 0.3 3 -0.33 -0.20 -0.10 -0.03 0.00 0.00 0.00 0.00 -0.07 -0.13 -0.10 - 0 . 2 0 - 0.20 -0.23 -0.13 - 0.03 + O . I 7 '0.33 +0.50 +0.60 +0.60 +0.57 + 0.60 +0.53 +0.37 +0.27 +0.17 -0.13 -0.27 - 0.3 7 -0.43 -9.43 -0.31 0.00 + 7!4 + 6.6 + 6.6 + 4.0 + 2.0 + 0.6 0.0 0.0 0.0 0.0 + rJ4 + 2.0 + 2.6 + 4.0 + 4.0 + 4.6 + 2.6 + 0.6 - 3.4 - 6.6 - 10.0 - 12.0 - 12.0 -11.4 - 10.6 - 12.0 - 7.4 - 5.4 - 3.4 + 2.6 + 5.4 + 8.6 + 8.6 0.0 1.4 + 7.4 Der Gedanke lag nun nahe, auf die Uhrschraube ein eues Rad zu setzen und diesem absichtlich solche Fehler u geben, daB sie die Fehler der Schraube zum Verschwinden ringen muaten. Die nun zu beschreibenden Anderungen n der Uhrschraube sind in der Werkstatt der Stemwarte on deren Mechanikus, Herrn Bmst hh, ausgefiihrt worden. s mag hierbei gleich e m h t werden, daB er Schnecke und ad mit einem gut schlieknden Staubschutz versehen hat, ne sehr notwendige Einrichtung, da sonst Staub und Sand, e in dies rasch laufende Getriebe hineinfallen, den Eingriff ald verderben. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Stern Es ist zu verwundern, daB man, wllhrend onst jedes Mikrometerwerk sorgaltig eingeschlossen wird, + 7!3 + 6.4 + 6.5 + 4.5 + 2.1 + 1.9 + 0.4 0.2 0.2 - - - 0.5 + 0.7 + 1.5 + 0.8 + 3.1 + 3.6 + 4.1 + 1.8 0.0 -- 3.4 - 5-3 - 8.9 - 10.6 - 11.5 - 10.9 - I 1.4 - 9.5 - 6.4 - 4.5 - 2.8 + 0.8 + 2.8 + 5.5 + 8.2 + 8.4 + 1.1 1.4 +or36 +0.32 +0.32 +0.22 +o.r3 +0.09 +0.02 - 0.0 I - 0.0 I - 0.02 +0.03 +0.04 +0.18 +0.20 +0.09 0.00 +0.07 +o.15 -0.17 -0.26 - 0.44 -0.53 -0.51 - 0.54 -0.51 -0.47 - 0.3 2 - 0 . 2 2 -0.14 +0.04 +o.14 +0.27 +041 +0.41 +0.42 +0.38 - of0 1 - 0.0 I - 0.0 I + 0.0 2 +0.03 +0.06 +0.02 - 0.0 I - 0.0 I - 0.02 - 0.04 - 0.03 - 0.09 - 0.05 - 0.03 - 0'.04 - 0.03 +0.07 +0.06 +0.07 +0.03 - 0.02 0.00 +0.03 +0.03 +0.06 +0.05 + 0.0 5 +0.03 + 0.04 +O.OI 0.00 0.00 - 0.02 - 0.0 I +o.o1 + 0103 0.00 0.00 +0.03 + 0.03 0.00 - 0.03 0.00 0.00 0.00 - 0.03 - 0.03 - 0.07 -0.13 -0.13 -0.13 -0.10 - 0 . 1 0 -0.03 +O.Ol +0.10 +O.IO +0.07 +O.Ol 0.00 +0.10 +o.ro +O.Ol +O.Ol +0.07 +0.03 0.00 - 0.03 - 0.03 - 0.03 0.00 +or0 I 0.00 0.00 +0.02 +0.03 +o.o:s 0.00 c.00 0.00 - 0.0 I - 0.03 - 0.03 - 0.08 - 0.09 - 0.08 - 0.08 - 0.07 - 0.06 +0.07 + 0.08 +0.08 +0.05 +0.01 +0.05 +0.08 + 0.07 +0.06 +0.05 +0.05 +0.02 0.00 - 0.0 I -0.02 - 0.02 0.00 -0.01 diesen wichtigen Instrumententeil stets ungeschtitzt 1HB hider gestattete der Raum nicht, das neue Rad gr6l3er a 32 mm im Durchmesser zu machen. Schon dieser Durch messer hat zur Folge, daB das Femrohr in der unteren Lag etwas an Bewegungsfreiheit einbtiBt. Es bedeutet dies alle dings kaum einen Verlust, machte aber einen Anschlag no wendig, damit die Klemm- und Feinbewegungsstiicke nich gegen das neue Rad stokn. Zunkhst galt es die periodischen Fehler scharf zu be stimmen. Dazu muate die Schraube mit einem fehlerfreie Rad versehen werden. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Stern Wenn auch die Lagerung zwischen Spitzen an sich gut ist, wenn die Spitzen gut justiert und wirklich rund sind, so unterliegt sie bekanntlich einer raschen Abnutzung, die Spitzen werden unrund und miissen nachgestellt werden, eine Un- veriinderlichkeit der periodischen Fehler kann hiernach nicht erwartet werden. Rad aufgesteckt und festgeklemmt wurde. Ein fehlerfreies Rad zu schneiden gelang nun freilich vorerst nicht, da der Teilkreis der Drehbank nicht fehlerfrei war. Die Teilung des neuen Rades wurde nun genau untersucht und ebenso an mehreren Abenden das System Rad-Schraube. Hierbei wurden vier um 90° voneinander entfernte Stellen des Kreises benutzt. um zu sehen, ob die Fehler an allen Stellen des Uhrkreises die gleichen waren. Eine Verschiedenheit in diesem Sinne konnte nicht nachgewiesen werden. 'Die aus diesen Beobachtungen folgenden Werte, verbessert um die Teilungsfehler des Rades, stehen in Spalte V. Sollen diese Schraubenfehler durch ein Rad kompensiert werden, so muO dieses die Teilungsfehler unter VI haben. Es wurde nun ein anderes, genau gleich groRes Rad angefertigt und ihm die gewunschte Teilung gegeben. Es bietet dies keine Schwierigkeiten, zumal in einer Prazisions- werkstatt, wo die notigen Einrichtungen vorhanden sind. Not- wendig ist aber, daD erst die periodischen Fehler der Schraube recht scharf bestimmt werden. Als namlich das neue Rad angefertigt wurde, waren sie noch nicht genau genug bekannt, wie die darauf folgende Untersuchung an Sternen ergab; die Folge war, daB die ZSLhne korrigiert werden muOten, wollte man nicht ein ganz neues Rad schneiden. Um zu sehen, ob eine solche Korrektur besondere Schwierigkeiten bietet, habe ich die Retouche selbst rnit dern Gewindebohrer aus- gefhhrt; die Korrektionen betrugen bis 5'. Das Rad besitzt nun die unter VII gegebenen Teilungsfehler; in Spalte VIII steht ihr scheinbarer Wert. Die Teilung. laBt sich rnit aller Schgrfe untersuchen, wenn man auf dem Rade einen Kreis befestigt und beobachtet, welche Winkelbewegung einer Um- drehung der Schnecke entspricht. Da periodische Schraubenfehler sich nie ganz oder nur sehr schwer vermeiden lassen und der Erbauer eines Re- fraktors wohl kaum in der Lage sein wird sie zu bestimmen, so enipfiehlt es sich, die Uhrschraube zungchst rnit einem wirklich fehlerfreien Rade zu versehen. Hat dann der Be- obachter die Schraubenfehler scharf bestimmt, so wird nun ein in den Abmessungen vtillig gleiches Rad hergestellt, dessen Zahnliicken an ihrem berechneten Orte erst mtiglichst gut vorgefribt und dann mit dem Gewindebohrer nachge- schnitten werden. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Stern Das alte Rad wurde abgedreht bi auf einen schwach konischen Zapfen, auf den nun das neu diesen wichtigen Instrumententeil stets ungeschtitzt 1HBt. hider gestattete der Raum nicht, das neue Rad gr6l3er als 32 mm im Durchmesser zu machen. Schon dieser Durch- messer hat zur Folge, daB das Femrohr in der unteren Lage etwas an Bewegungsfreiheit einbtiBt. Es bedeutet dies aller- dings kaum einen Verlust, machte aber einen Anschlag not- wendig, damit die Klemm- und Feinbewegungsstiicke nicht gegen das neue Rad stokn. Der Gedanke lag nun nahe, auf die Uhrschraube ein neues Rad zu setzen und diesem absichtlich solche Fehler zu geben, daB sie die Fehler der Schraube zum Verschwinden bringen muaten. Die nun zu beschreibenden Anderungen an der Uhrschraube sind in der Werkstatt der Stemwarte von deren Mechanikus, Herrn Bmst hh, ausgefiihrt worden. Es mag hierbei gleich e m h t werden, daB er Schnecke und Rad mit einem gut schlieknden Staubschutz versehen hat, eine sehr notwendige Einrichtung, da sonst Staub und Sand, die in dies rasch laufende Getriebe hineinfallen, den Eingriff bald verderben. Es ist zu verwundern, daB man, wllhrend sonst jedes Mikrometerwerk sorgaltig eingeschlossen wird, g g Zunkhst galt es die periodischen Fehler scharf zu be- stimmen. Dazu muate die Schraube mit einem fehlerfreien Rad versehen werden. Das alte Rad wurde abgedreht bis auf einen schwach konischen Zapfen, auf den nun das neue 25' 4556 35 2 35 1 sein. Nun konnte noch als Nachteil der Methode bezeichnet werden, daB sie versagen muO, wenn die Fehler des Rades oder der Schraube sich verindern. Meine Beobachtungen haben ergeben, daO selbst unter den ungiinstigsten Verhillt- nissen die Summe beider Fehler in 8 Jahren, in denen das Uhrwerk sehr vie1 benutzt worden ist, sich nicht wesentlich geiindert hat. Eine Anderung der Teilungsfeh\er des neuen Rades ist um so weniger zu erwarten, da ein zu starkes und ungleichmHOiges Ausschleifen einerseits durch den neuen Staubschutz und andererseits durch die nunmehr gleichformige Geschwindigkeit des Fernrohrs verhindert wird. Eine Ande rung der periodischen Fehler der Uhrschraube ist bei so sicherer und exakter Lagerung, wie sie das Leipziger Instru- ment zeigt, ganz unbegrundet. Die Lagerung besteht wie bei vertikalen Achsen aus Konus, Zylinder und ebenem Widerlager. Uhrschrauben allerdings, die z. B. zwischen Spitzen gefiihrt werdeo, wurden diese Gewihr nicht leisten. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Stern Beim Befestigen des Rades mu13 naturlich Sorge getragen werden, daO es so aufgesteckt wird, daO in der Tat eine Kompensation der Fehler eintritt. Wurde man z. B. unser neues Kad um einen Zahn oder 10' auf der Schraubenspindel drehen, so wurde man durch Summation von V und VIII nicht die Werte 1X erhalten, sondern stellen- weise wesentlich andere. Addiert man VIII zu V, so erhailt man die Fehler unter IX, die nunmehr noch das System Schneckenrad-Uhrschraube- Uhrkreis besitzt. Kontrollbeobachtungen am Himmel ergaben die Werte unter X, deren Widerspruche gegen IX hauptlch- lich in der Unsicherheit der Spalte X begriindet sind. Die GroOen IX besitzen mindestens das doppelte Gewicht ; sieht man aber davon ab in Anbetracht, daO die Werte X direkt bestimmt sind, und bildet einfach das Mittel XI, so erhalt man ein Bild, welche Verbesserung das neue Rad gegen das von Repsold (I) bedeutet. Das gesteckte Ziel ist erreicht, die periodischen Fehler sind so verkleinert, daO ein Stern nicht mehr als 011 von der Mittellage abweicht. Der Vorteil der Methode liegt meines Erachtens erstens in dem Umstande, daO sie anwendbar bleibt, wenn die Fehlerkurve keine Sinuskurve ist, sondern irgend eine Gestalt besitzt, und ferner darin, daO die Fehler der Schrauhe 2.B. in unserem Falle durch Zahnfehler von 70-facher G r o b kompensiert werden; in diesem Umstande Iiegt die GewHhr fir langjithrige Brauchbarkeit der getroffenen Einrichtung. g Das geschilderte Verfahren ist naturlich nur anwendbar, wenn die periodischen Fehler der Schraube klein sind. Wurde man dem Schneckenrade grtiUere Teilungsfehler geben, so wurde der Eingriff der Schnecke nicht mehr einwandfrei F. Havn. LeiDzin. I Q I I Dez. 2 I. LeiDzin. I Q I I Dez. 2 I. Beobachtungen von Kometen am Boguslawskischen Mikrometer rnit einem Positionskreis des 0.16 m Merz-Refraktors der Universitlts-Sternwarte zu Warschau. Von S. Tschwny. 191 I IM.Z.Warsch.1 da I Ad I Vgl. I a app. I logpd I d app. I IogpAl Red. ad 1. app. 1 * -. . ._.._._ __ . . . . . . - - .- - . .. .- . . _ - ... . _.__ __ ___-____ Koniet 1911 c (Brooks). 19 30 25.83 j 8.832 1 +53 5 0 36.0 9.399n I +r.63 +11.7 I , 2 1 9 ~ 30m25?44 ! 8.833 ' + 5 3 O 50' 4016 [ 9.399a + 1t62 + 1117 1 I Sept. Fehler der Uhrschraube am SO-cm-Refraktor der Leipziger Stern 2 / 9h 12'40s + 1m31f12 + 0' 12r7 2 I 9 1 2 37 I - 0 4.53 I - 3 58.1 Beobachtungen von Kometen am Boguslawskischen Mikrometer rnit einem Positionskreis des 0.16 m Merz-Refraktors der Universitlts-Sternwarte zu Warschau. Von S. Tschwny. 191 I IM.Z.Warsch.1 da I Ad I Vgl. I a app. I logpd I d app. I IogpAl Red. ad 1. app. 1 * -. . ._.._._ __ . . . . . . - - .- - . .. .- . . _ - ... . _.__ __ ___-____ Koniet 1911 c (Brooks). 19 30 25.83 j 8.832 1 +53 5 0 36.0 9.399n I +r.63 +11.7 I , 2 1 9 ~ 30m25?44 ! 8.833 ' + 5 3 O 50' 4016 [ 9.399a + 1t62 + 1117 1 I Sept. 2 / 9h 12'40s + 1m31f12 + 0' 12r7 2 I 9 1 2 37 I - 0 4.53 I - 3 58.1
https://openalex.org/W4224440824	https://thrombosisjournal.biomedcentral.com/track/pdf/10.1186/s12959-022-00382-2	English	null	Prevalence and risk factors of preoperative venous thromboembolism in patients with malignant musculoskeletal tumors: an analysis based on D-dimer screening and imaging	Thrombosis journal	2,022	cc-by	5,521	Prevalence and risk factors of preoperative venous thromboembolism in patients with malignant musculoskeletal tumors: an analysis based on D‑dimer screening and imaging Kenta Hayashida1, Yusuke Kawabata1, Keiju Saito1, Shintaro Fujita1, Hyonmin Choe1, Ikuma Kato2, Masanobu Takeyama1 and Yutaka Inaba1 Kenta Hayashida1, Yusuke Kawabata1, Keiju Saito1, Shintaro Fujita1, Hyonmin Choe1, Ikuma Kato2, Masanobu Takeyama1 and Yutaka Inaba1 Kenta Hayashida1, Yusuke Kawabata1, Keiju Saito1, Shintaro Fujita1, Hyonmin Choe1, Ikuma Kato2, Masanobu Takeyama1 and Yutaka Inaba1 Hayashida et al. Thrombosis Journal (2022) 20:22 https://doi.org/10.1186/s12959-022-00382-2 Abstract Background: Venous thromboembolism (VTE) is a major complication in patients with malignant tumors and ortho- pedic disorders. Although it is known that patients undergoing surgery for malignant musculoskeletal tumor are at an increased risk of thromboembolic events, only few studies have investigated this risk in detail. Therefore, the aim of this study was to determine the prevalence and risk factors for preoperative VTE in malignant musculoskeletal tumors patients. rospectively reviewed the medical records of 270 patients who underwent surgical procedures, Methods: We retrospectively reviewed the medical records of 270 patients who underwent surgical procedures, including biopsy for malignant musculoskeletal tumor, have undergone measurements of preoperative D-dimer lev- els, and were subsequently screened for VTE by lower extremity venous ultrasonography and/or contrast-enhanced computed tomography scans. Statistical analyses were performed to examine the prevalence and risk factors for VTE. Receiver operating characteristic (ROC) analysis was performed to verify the D-dimer cutoff value for the diagnosis of VTE. Results: Overall, 199 patients (103 with primary soft tissue sarcomas, 38 with primary bone sarcomas, 46 with meta- static tumors, and 12 with hematologic malignancies) were included. D-dimer levels were high in 79 patients; VTE was detected in 19 patients (9.5%). Multivariate analysis indicated that age ≥ 60 years (P = 0.021) and tumor location in the lower limbs (P = 0.048) were independent risk factors for VTE. ROC analysis showed that the D-dimer cutoff value for the diagnosis of VTE was 1.53 µg/mL; the sensitivity and specificity were 89.5% and 79.4%, respectively. Conclusions: Our study indicated that age and tumor location in the lower limbs were independent risk factors for preoperative VTE in malignant musculoskeletal tumors patients. D-dimer levels were not associated with VTE in the multivariate analysis, likely because they are affected by a wide variety of conditions, such as malignancy and aging. D-dimer is useful for exclusion diagnosis because of its high sensitivity, but patients with high age and tumor location Correspondence: yusuke0807kawabata@yahoo.co.jp; md09061181766@gmail.com 1 Department of Orthopaedic Surgery, Yokohama City University, 3‑9 Fukuura, Kanazawa‑ku, Yokohama 236‑0004, Japan Full list of author information is available at the end of the article Background Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is a major complication in orthopedic surgery. VTE is also known to be associated with malignancy, which increases the risk of VTE by 2- to sevenfold [1–3]. Several mecha- nisms may be involved in the promotion of thromboem- bolic events in cancer patients [4]. Because the risk of VTE is reported to be particularly high during the first few months after the diagnosis of malignancy [3], it is important to assess affected patients regarding this con- dition before initiating treatment. Because such limitations may cause an underestima- tion of the prevalence of VTE, only limited evidence is available for understanding preoperative VTE and associated risks for malignant musculoskeletal tumors patients. Therefore, we investigated the prevalence and risk factors for VTE in these patients and hypothesized that a combination of measuring D-dimer levels and imaging-based examination may be the best screening strategy for VTE. D-dimer, the breakdown product of stabilized fibrin, is frequently used to screen for VTE as there is well- established evidence that levels below 0.5 µg/mL have an exceedingly high negative predictive value for the exclu- sion of PE [5–7]. Although lower extremity venography is the mainstay for the diagnosis of DVT, it has recently been replaced by lower extremity venous ultrasonogra- phy and contrast-enhanced computed tomography (CT) [8–10]. The combination of clinical symptoms, D-dimer concentrations, and contrast-enhanced CT has made it possible to diagnose 97.9% of VTE cases [11, 12].h © 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, visithttp://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. Hayashida et al. Thrombosis Journal (2022) 20:22 Page 2 of 8 Trial registration: Our study was approved by the institutional review board. The registration number is B200600056. The registration date was July 13, 2020. Trial registration: Our study was approved by the institutional review board. The registration numb The registration date was July 13, 2020. Keywords: Preoperative venous thromboembolism, VTE, Deep vein thrombosis, DVT, Malignant tumor, Malignant musculoskeletal tumor, D-dimer Keywords: Preoperative venous thromboembolism, VTE, Deep vein thrombosis, DVT, Malignant musculoskele tumor, Malignant musculoskeletal tumor, D-dimer 22% [13–21]. A myriad of factors, such as age, size and location of the tumor, chemotherapy, Ewing sarcoma family of tumors, metastases, pathological fracture, and type of surgery, have been suggested to be related to the occurrence of VTE [13–17, 20–23]. However, most pre- vious studies analyzed postoperative, rather than pre- operative, patients who underwent orthopedic surgery [13, 15–18, 22]. In addition, routine screening for VTE was not considered; therefore, the reported rate of VTE represented only those that were clinically symptomatic [15, 17]. Methods After obtaining approval from our institutional review board, we retrospectively reviewed the medical records of patients treated at our institution between January The prevalence of perioperative VTE in malignant musculoskeletal tumors patients ranges from 2.7% to Fig. 1 Flow chart depicting the venous thromboembolism assessment strategy. Patients were evaluated using D-dimer screening, lower extremity venous ultrasonography, and contrast-enhanced computed tomography. A total of 23 patients were not included based on the exclusion criteria. Additional extensive resections for unplanned resections or positive margins and non-orthopedic surgery patients were included. *Only cases with D-dimer > 1.0 μg/ml and not yet evaluated imaging studies were indicated. a CT: computed tomography; b VTE: venous thromboembolism; c DOACs: direct oral anticoagulant Fig. 1 Flow chart depicting the venous thromboembolism assessment strategy. Patients were evaluated using D-dimer screening, lower extremity venous ultrasonography, and contrast-enhanced computed tomography. A total of 23 patients were not included based on the exclusion criteria. Additional extensive resections for unplanned resections or positive margins and non-orthopedic surgery patients were included. *Only cases with D-dimer > 1.0 μg/ml and not yet evaluated imaging studies were indicated. a CT: computed tomography; b VTE: venous thromboembolism; c DOACs: direct oral anticoagulant Fig. 1 Flow chart depicting the venous thromboembolism assessment strategy. Patients were evaluated using D-dimer screening, lower extremity venous ultrasonography, and contrast-enhanced computed tomography. A total of 23 patients were not included based on the exclusion criteria. Additional extensive resections for unplanned resections or positive margins and non-orthopedic surgery patients were included. **Only cases with D-dimer > 1.0 μg/ml and not yet evaluated imaging studies were indicated. a CT: computed tomography; b VTE: venous thromboembolism; c DOACs: direct oral anticoagulant Hayashida et al. Thrombosis Journal (2022) 20:22 Page 3 of 8 in patients with metastases was 10.9%. The tumors com- prised 103 primary soft tissue sarcomas, 38 primary bone sarcomas, 46 metastatic tumors, and 12 hematologic malignancies. The pathological diagnoses of the patients are summarized in Table 2. Proximal VTE and PE were detected in 3 (2.5%) and 2 (1.7%) patients, respectively. Most patients had asymptomatic VTE, but 3/19 patients (15.8%) had local edema or pain. The clinical profiles of the patients with VTE are summarized in Table 3. The patients comprised 10 men and 9 women with a mean age of 59.7 years (range, 19–91 years). Methods The tumor was located in the lower limbs in 17 patients, accounting for 89.5% of the patients with VTE. The mean D-dimer level was 2.64 µg/mL (range, 1.05–11.72 µg/mL). Among the patients with VTE, 9 had soft tissue sarcomas and 3 had bone sarcomas. The cases of soft tissue sarcoma included 4 cases of myxofibrosarcomas, 3 undifferenti- ated pleomorphic sarcomas (UPSs), and 2 leiomyosarco- mas. The tumor was located on the surface of the fascia in 4 patients and deeper than the fascia in 5 patients. The cases of bone sarcoma included 2 UPSs of the bone and 1 osteosarcoma. Seven patients had bone metastases and hematologic malignancies. ROC analysis showed that the cutoff value of D-dimer for the diagnosis of VTE was 1.53 µg/mL using by Youden’s index; the area under the curve was 0.857 (95% CI, 0.804—0.911), and the sensitiv- ity and specificity were 90.0% and 79.6%, respectively.h 2014 and June 2020. Out of 270 malignant musculoskel- etal tumors patients who underwent orthopedic surgical procedures, including open biopsy, and were assessed for preoperative VTE according to a flowchart developed by our institution, a total of 199 patients were included (Fig. 1). VTE was screened by measuring preopera- tive D-dimer levels and subsequently performing lower extremity venous ultrasonography and/or contrast- enhanced CT. D-dimer levels were measured by LPIA D-dimer (Mitsubishi Chemical Medience, Tokyo, Japan); owing to the sensitivity of this assay, levels < 0.50 µg/mL were considered 0.50 µg/mL. Ultrasonography and CT scans were performed if the D-dimer level was ≥ 1.0 µg/ mL. In this study, DVT in the lower extremities that involved the popliteal vein or those above was defined as the proximal type; DVT involving the area below the popliteal vein was defined as the distal type.hi i The clinical profiles of patients with VTE were retro- spectively assessed. The anatomical location of the tumor was defined as the lower limb, including the pelvis, hip, thigh, knee, lower leg, ankle, and foot. Tumor location was also assessed based on the depth of the tumor. A soft tissue tumor located on the surface of the fascia was defined as superficial. Soft tissue tumors deeper than the fascia and bone tumors were defined as deep.h i The Mann–Whitney test was used for continuous vari- ables, and the chi-squared test or Fisher’s exact test were used for categorical variables. Logistic regression analysis was used for the multivariate analysis. Results We excluded patients if they met any of the follow- ing criteria: benign condition; history of VTE; treat- ment with direct oral anticoagulants or heparin before the assessment of VTE; reoperation within 3 months; absence of preoperative D-dimer measurements; and absence of lower extremity venous ultrasonography or contrast-enhanced CT examinations in cases with D-dimer > 1.0 μg/ml. Baseline information was sum- marized in Table 1. The patients comprised 113 men and 86 women, with a mean age of 60.9 years (range, 14–91 years). The tumor location was the lower limb in 133 patients. The D-dimer concentration was ≥ 1.0 µg/ mL in 79 (39.7%) patients. The overall prevalence of pre- operative VTE detected by ultrasonography and con- trast-enhanced CT scans was 9.5% (19 patients). VTE was detected in 8.7% and 7.9% of patients with soft tissue and bone sarcomas, respectively. The prevalence of VTE Methods Receiver operating characteristic (ROC) analysis was performed to verify the cutoff value of D-dimer for the diagnosis of VTE. Statis- tical significance was set at P < 0.05. All statistical analy- ses were performed using R (R Foundation for Statistical Computing, Vienna, Austria). i The risk factors for VTE were analyzed using univariate analysis; we found that age ≥ 60 years (P = 0.011), tumor location in the lower limbs (P = 0.038), and D-dimer lev- els (P < 0.001) were associated with the prevalence of VTE (Table 1). In contrast, sex, body mass index, size, origin, performance status, pathological fracture, and hema- tologic status, including hemoglobin, white blood cell, platelet, and C-reactive protein levels, were not identified as significant risk factors. Multivariate analysis indicated that age ≥ 60 years (P = 0.021), and tumor location in the lower limbs (P = 0.048) were independent risk factors for VTE. The D-dimer level was not significant in the multi- variate analysis (P = 0.51). Discussion We investigated the prevalence of preoperative VTE in malignant musculoskeletal tumors patients. The most important finding in this study was that more affected patients could potentially have VTE than that reported by previous studies. In this study, VTE was detected in 19 of the 199 included patients (9.5%); the prevalence was therefore higher than the rate of perioperative VTE reported in previous studies [13–18]. Several condi- tions in our study that differed from those in previous studies may have influenced the results. First, routine screening for VTE was performed. We performed DVT Hayashida et al. Thrombosis Journal (2022) 20:22 Page 4 of 8 Table 1 Demographic and clinical variables for patients Table 1 Demographic and clinical variables for patients a Fisher’s exact test Variable Number of patients Univariate analysis Multivariate analysis VTE (-) VTE ( +) Odds ratio 95% confidence interval p-value Odds ratio 95% confidence interval p-value Number of patients (n = 199) 180 19 (9.5%) Diagnosis Soft tissue sarcoma 94 9 (8.7%) Bone sarcoma 35 3 (7.9%) Metastasis 41 5 (10.9%) Hematologic malig- nancy 10 2 (16.7%) Sex Male 103 10 Female 77 9 0.81 0.28–2.31 0.85a Age (years) 59.7 ± 1.4 70.8 ± 3.7 < 60 74 2 ≧ 60 106 17 5.89 1.33–54.1 0.011a 5.80 1.32–26.5 0.021c BMI (cm/kg) 22.46 21.39 0.24b Size (mm) 80.4 65.8 0.79b Location Lower limb 116 17 Other 64 2 4.66 1.05–42.9 0.038a 4.56 1.01–20.4 0.048c Origin Bone 80 10 Soft 100 9 0.72 0.25–2.08 0.63a Subcutaneous ~ fascia 36 4 Fascia ~ bone 144 15 1.14 0.26–3.90 0.77a D-dimer 0.65 2.64 > 0.001b 1.01 0.96–1.08 0.51c Performance Status 0–2 154 16 3–4 26 3 1.11 0.19–4.29 0.74a Pathological fracture 23 3 1.28 0.22–4.99 0.71a Hematologic status Hemoglobin (g/dL) 12.9 13 0.88b White blood cell (/μL) 7010 6080 0.38b Platelet (× 104/μL) 28 26.2 0.99b C related protein (mg/L) 1.78 0.56 0.80b osteoarthritis, previous report suggested that perform- ing MDCT before and after total knee arthroplasty may be useful to clarify the incidence of VTE and to develop appropriate strategies for treatment and prevention [26]. Therefore, for the management of preoperative malig- nant musculoskeletal tumors patients, it is crucial to recognize that more patients have asymptomatic VTE than previously thought. Second, our study included patients with metastases and hematologic malignancies. The rate of DVT in surgical oncology patients receiv- ing no prophylaxis was reported to be 35.2% [27]. Discussion Thrombosis Journal (2022) 20:22 Page 5 of 8 Table 2 Diagnosis of all patients in this study Tumor origin Pathological diagnosis Number of patients Primary Bone Chondrosarcoma 15 Osteosarcoma 13 UPS 5 Ewing sarcoma 2 Chordoma 2 Malignant giant cell tumor of bone 1 Subtotal 38 Soft tissue Myxofibrosarcoma 27 UPS 24 Myxoid liposarcoma 10 MPNST 8 Synovial sarcoma 7 Dedifferentiated liposarcoma 6 Leiomyosarcoma 7 Extraskeltal myxoid chondrosarcoma 5 Dermatofibrosarcoma protuberans 3 Pleomorphic liposarcoma 2 Rhabdomyosarcoma 2 Others 2 Subtotal 103 Metastasis Renal cell cancer 16 Lung cancer 11 Prostatic cancer 5 Breast cancer 3 Esophageal cancer 2 Colorectal cancer 2 Bladder cancer 2 Thyroid cancer 1 Gastric cancer 1 Liver cancer 1 Pancreatic cancer 1 Uterine cancer 1 Subtotal 46 Hematologic malignancy Lymphoma 9 Multiple myeloma 3 Subtotal 12 Total 199 Pathological diagnosis UPS Undifferentiated pleomorphic sarcoma, MPNST Malignant peripheral nerve sheath tumor of patients Primary Bone Chondrosarcoma 15 Osteosarcoma 13 UPS 5 Ewing sarcoma 2 Chordoma 2 Malignant giant cell tumor of bone 1 Subtotal 38 Soft tissue Myxofibrosarcoma 27 UPS 24 Myxoid liposarcoma 10 MPNST 8 Synovial sarcoma 7 Dedifferentiated liposarcoma 6 Leiomyosarcoma 7 Extraskeltal myxoid chondrosarcoma 5 Dermatofibrosarcoma protuberans 3 Pleomorphic liposarcoma 2 Rhabdomyosarcoma 2 Others 2 Subtotal 103 Metastasis Renal cell cancer 16 Lung cancer 11 Prostatic cancer 5 Breast cancer 3 Esophageal cancer 2 Colorectal cancer 2 Bladder cancer 2 Thyroid cancer 1 Gastric cancer 1 Liver cancer 1 Pancreatic cancer 1 Uterine cancer 1 Subtotal 46 Hematologic malignancy Lymphoma 9 Multiple myeloma 3 Subtotal 12 Total 199 Metastasis Hematologic malignancy Hematologic malignancy Hematologic malignancy UPS Undifferentiated pleomorphic sarcoma, MPNST Malignant peripheral nerve sheath tumor Several risk factors associated with an increased sus- ceptibility of preoperative VTE in malignant musculo- skeletal tumors patients have been identified. Our study showed that increasing age and tumor location are inde- pendent risk factors. Age has been previously suggested to be associated with an increased risk of VTE [13, 20]. Kim et al. reported an odds ratio of 5.84 in patients older than 60 years [13]. Our results showed a similar trend, and only two patients with VTE were younger than 60 years of age. Patients with bone or soft tissue sarco- mas located in the hip or thigh have been suggested to have an increased risk of VTE [17]. Yamaguchi et al. Discussion The cancer type affects the prevalence of VTE in adolescent and young adult patients; therefore, oncology patients, except for those with sarcomas, may have a different total prevalence of VTE. assessment in combination with routine D-dimer meas- urement, lower extremity ultrasonography, and contrast- enhanced CT scans. This screening strategy allowed for the diagnosis of patients with asymptomatic VTE. Symp- tomatic VTE in this study was 3/199 patients (1.5%). The necessity of anticoagulation therapy for these patients is controversial; however, Galanaud et al. reported that patients with cancer-related isolated distal DVT had a prognosis similar to that of patients with cancer-related isolated proximal DVT and a dramatically poorer prog- nosis than those with isolated distal DVT without can- cer [24]. In addition, Gary et al. reported Asymptomatic deep vein thrombosis and superficial vein thrombo- sis in ambulatory cancer patients are associated with poor survival despite anticoagulation therapy [25]. In Hayashida et al. Discussion patients with tumors in the lower limbs are suggested to be at an increased risk of VTE occurrence. In this study, 17 of 19 VTE patients had tumors in the lower limbs (89.5%), and the two VTE patients with tumors in the upper extremities were both older than 90 years of age. y g Whilst the level of D-dimer showed an association with preoperative VTE in the univariate analysis, this was not confirmed in the multivariate analysis. This was likely due to the influence of increased age on D-dimer levels, as they need to be corrected by age, and the value of this fibrin degradation product itself should be evalu- ated carefully [5, 7]. In addition, the D-dimer cutoff value in cancer patients was reported to be higher than that commonly used in clinical practice [28]. The cutoff value of D-dimer for the diagnosis of VTE was found to be 1.53 µg/mL in the present study, which was almost the same as that used for other cancer types [29, 30]. The appropriate consideration of D-dimer limits the overuse and added cost of ultrasonography without a negative impact [31]. If D-dimer exceeds a level of 1.53 µg/mL, additional assessment of VTE by lower extremity ultra- sonography and contrast-enhanced CT should be per- formed. In particular, patients older than 60 years and with tumor location in the lower limbs are a high-risk group and should be considered for imaging evaluation such as ultrasonography regardless of D-dimer levels. Discussion demonstrated a high prevalence of VTE (22%) in patients after resection of musculoskeletal tumors of the lower limb [20]. Since surgery involving the pelvis has been associated with the development of proximal DVT [22], Hayashida et al. Thrombosis Journal (2022) 20:22 Page 6 of 8 Table 3 Characteristic of patients with venous thromboembolism Table 3 Characteristic of patients with venous thromboembolism BMI Body mass index, PS Performance status, VTE Venous thromboembolism, UPS Undifferentiated pleomorphic sarcoma Age Sex BMI (cm/kg) Location Depth Diagnosis Pathological fracture PS D-Dimer (μg/mL) Location of VTE 1 76 Male 22.3 Thigh Superficial Myxofibrosarcoma - 0 1.05 Distal 2 66 Male 18.7 Femur Bone Lung cancer - 2 5.69 Distal 3 66 Male 29.1 Thigh Superficial UPS - 0 1.58 Distal 4 86 Female 22.2 Thigh Deep Leiomyosarcoma - 1 3.69 Proximal 5 79 Male 29.2 Lower leg Superficial UPS - 1 2.16 Distal 6 77 Female 17.0 Patella Bone Malignant lymphoma - 2 2.33 PE, distal 7 58 Male 19.8 Femur Bone Lung cancer + 3 11.72 PE, distal 8 73 Female 16.4 Femur Bone UPS + 4 2.43 Distal 9 64 Female 18.0 Tibia Bone Uterine cancer - 1 4.65 Distal 10 91 Male 20.4 Forearm Superficial Myxofibrosarcoma - 1 2.32 Distal 11 78 Female 20.2 Pelvis Bone UPS - 1 4.23 Proximal 12 67 Female 17.9 Buttocks Deep UPS - 1 2.71 Distal 13 76 Female 25.0 Lower leg Deep Leiomyosarcoma - 1 1.21 Distal 14 19 Male 22.2 Tibia Bone Osteosarcoma - 1 1.53 Distal 15 91 Female 20.4 Elbow Deep Myxofibrosarcoma - 1 2.49 Distal 16 60 Female 23.2 Femur Bone Breast cancer + 4 9.28 Distal 17 75 Male 24.5 Pelvis Bone Malignant lymphoma - 1 1.76 Distal 18 85 Male 15.4 Sacrum Bone Lung cancer - 1 2.85 Distal 19 79 Male 24.8 Lower leg Deep Myxofibrosarcoma - 1 1.91 Proximal Our study has several limitations. First, it was ret- rospective in nature and had a relatively small sample size. Some predictive variables may not have had suf- ficient statistical power; therefore, some important variables may have been ignored. Second, patients with metastases and hematologic malignancies were included. Regarding the evaluation of VTE risk in bone and soft tissue sarcoma patients, these malignancies should be evaluated in separate categories with suffi- cient sample sizes. Finally, lower extremity ultrasonog- raphy and contrast-enhanced CT for VTE evaluation were not unified. Declarations 13. Kim SM, Park JM, Shin SH, Seo SW. Risk factors for post-operative venous thromboembolism in patients with a malignancy of the lower limb. Bone Joint J. 2013;95-B(4):558–62. Availability of data and materials Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. 12. van Belle A, Büller HR, Huisman MV, Huisman PM, Kaasjager K, Kamphu- isen PW, et al. Effectiveness of managing suspected pulmonary embolism using an algorithm combining clinical probability, D-dimer testing, and computed tomography. JAMA. 2006;295(2):172–9. Abbreviations 4. Donnellan E, Khorana AA. Cancer and venous thromboembolic disease: a review. Oncologist. 2017;22(2):199–207. CT: Computed tomography; DOAC: Direct oral anticoagulant; DVT: Deep vein thrombosis; PE: Pulmonary embolism; ROC: Receiver operating characteristic; VTE: Venous thromboembolism. 5. Righini M, Perrier A, Moerloose PD, Bounameaux H. D-Dimer for venous thromboembolism diagnosis: 20 years later. J Thromb Haemost. 2008;6(7):1059–71. 6. Nisio MD, Squizzato A, Rutjes AWS, Büller HR, Zwinderman PMM Bossuyt. Diagnostic accuracy of D-dimer test for exclusion of venous thromboem- bolism: a systematic review. J Thromb Haemost. 2007;5(2):296–304. Ethics approval and consent to participate This research has been approved by the IRB of the Yokohama City University Hospital. The approval number is B200600056. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants or, if participants are under 16, from a parent and/or legal guardian. 14. Morii T, Mochizuki K, Tajima T, Aoyagi T, Satomi K. Venous thromboem- bolism in the management of patients with musculoskeletal tumor. J Orthop Sci. 2010;15(6):810–5. 15. Damron TA, Wardak Z, Glodny B, Grant W. Risk of venous thromboem- bolism in bone and soft-tissue sarcoma patients undergoing surgical intervention: a report from prior to the initiation of SCIP measures. J Surg Oncol. 2011;103(7):643–7. Funding The authors declare that they have no funding. 11. Wells PS, Anderson DR, Rodger M, Ginsberg JS, Kearon C, Gent M, et al. Derivation of a simple clinical model to categorize patients’ probability of pulmonary embolism: increasing the model’s utility with the SimpliRED D-dimer. Thromb Haemost. 2000;83(3):416–20. Authors’ contributions K.H. was a major contributor in writing the manuscript. Y.K. have made design of the work. K.S. analyzed the patient data. S.F. interpreted the patient data and prepared Table 1 and 2. I.K. was a major contributor in pathological assess- ment. H.C. and M.T. have made substantial contributions to the conception. Y.I. have approved the submitted version. All authors read and approved the final manuscript. 10 Duwe KM, Shiau M, Budorick NE, Austin JH, Berkmen YM. Evaluation of the lower extremity veins in patients with suspected pulmonary embolism: a retrospective comparison of helical CT venography and sonography. 2000 ARRS Executive Council Award I. American Roentgen Ray Society. AJR Am J Roentgenol. 2000;175(6):1525–31. Acknowledgements Not applicable. 9. Bernardi E, Camporese G, Büller HR, Siragusa S, Imberti D, Berchio A, et al. Serial 2-point ultrasonography plus D-dimer vs whole-leg color- coded Doppler ultrasonography for diagnosing suspected sympto- matic deep vein thrombosis: a randomized controlled trial. JAMA. 2008;300(14):1653–9. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 17. Mitchell SY, Lingard EA, Kesteven P, McCaskie AW, Gerrand CH. Venous thromboembolism in patients with primary bone or soft-tissue sarcomas. J Bone Joint Surg Am. 2007;89(11):2433–9. Consent for publication Consent for publication was obtained from all participants or, if participants are under 16, from a parent and/or legal guardian. 16. Kaiser CL, Freehan MK, Driscoll DA, Schwab JH, Bernstein KDA, Lozano- Calderon SA. Predictors of venous thromboembolism in patients with primary sarcoma of bone. Surg Oncol. 2017;26(4):506–10. Received: 19 November 2021 Accepted: 17 April 2022 20. Yamaguchi T, Matsumine A, Niimi R, Nakamura T, Asanuma K, Hasegawa M, et al. Deep-vein thrombosis after resection of musculoskeletal tumours of the lower limb. Bone Joint J. 2013;95-B(9):1280–4. 21. Li M, Guo Q, Hu W. Incidence, risk factors, and outcomes of venous thromboembolism after oncologic surgery: a systematic review and meta-analysis. Thromb Res. 2019;173:48–56. Conclusions Our study indicated that age and tumor location in the lower limbs were independent risk factors for preopera- tive VTE in malignant musculoskeletal tumors patients. D-dimer levels were not identified as independent risk factors in the multivariate analysis because it is affected by a wide variety of conditions, including malignancy and aging. D-dimer is useful for exclusion diagnosis because of its high sensitivity, but patients older than 60 years and with tumor location in the lower limbs are a high-risk group and should be considered for imag- ing evaluation such as ultrasonography regardless of D-dimer levels. Hayashida et al. Thrombosis Journal (2022) 20:22 Page 7 of 8 Author details 1 1 Department of Orthopaedic Surgery, Yokohama City University, 3‑9 Fukuura, Kanazawa‑ku, Yokohama 236‑0004, Japan. 2 Department of Molecular Pathol- ogy, Yokohama City University, Yokohama, Japan. 18. Singh VA, Yong LM, Vijayananthan A. Is DVT prophylaxis necessary after oncology lower limb surgery? A pilot study. Springerplus. 2016;5(1):943.i 19. Tuy B, Bhate C, Beebe K, Patterson F, Benevenia J. IVC filters may prevent fatal pulmonary embolism in musculoskeletal tumor surgery. Clin Orthop Relat Res. 2009;467(1):239–45. Received: 19 November 2021 Accepted: 17 April 2022 Received: 19 November 2021 Accepted: 17 April 2022 Additional file 1. 8. Kearon C, Akl EA, Comerota AJ, Prandoni P, Bounameaux H, Goldhaber SZ, et al. Antithrombotic therapy for VTE disease: antithrombotic therapy and prevention of thrombosis, 9th ed: American college of chest physi- cians evidence-based clinical practice guidelines. Chest. 2012;141(2 Suppl):e419S-e496S. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12959-022-00382-2. 7. Nybo M, Hvas AM. Age-adjusted D-dimer cut-off in the diagnostic strategy for deep vein thrombosis: a systematic review. Scand J Clin Lab Invest. 2017;77(8):568–73. References A systematic review of deep venous thrombosis prophylaxis in cancer patients: implications for improving quality. Ann Surg Oncol. 2007;14(2):929–36. y 28. Chen C, Li G, Liu YD, Gu YJ. A new D-dimer cutoff value to improve the exclusion of deep vein thrombosis in cancer patients. Asian Pac J Cancer Prev. 2014;15(4):1655–8. 28. Chen C, Li G, Liu YD, Gu YJ. A new D-dimer cutoff value to improve the exclusion of deep vein thrombosis in cancer patients. Asian Pac J Cancer Prev. 2014;15(4):1655–8. 29. Kawaguchi R, Furukawa N, Kobayashi H. Cut-off value of D-dimer for pre- diction of deep venous thrombosis before treatment in ovarian cancer. J Gynecol Oncol. 2012;23(2):98–102. 29. Kawaguchi R, Furukawa N, Kobayashi H. Cut-off value of D-dimer for pre- diction of deep venous thrombosis before treatment in ovarian cancer. J Gynecol Oncol. 2012;23(2):98–102. y 30. Osaki T, Saito H, Fukumoto Y, Kono Y, Murakami Y, Shishido Y, et al. Risk and incidence of perioperative deep vein thrombosis in patients under- going gastric cancer surgery. Surg Today. 2018;48(5):525–33. 31. Mousa AY, Broce M, Gill G, Kali M, Yacoub M, AbuRahma AF. Appropriate use of D-dimer testing can minimize over-utilization of venous duplex ultrasound in a contemporary high-volume hospital. Ann Vasc Surg. 2015;29(2):311–7. References 1. Stein PD, Beemath A, Meyers FA, Skaf E, Sanchez J, Olson RE. Incidence of venous thromboembolism in patients hospitalized with cancer. Am J Med. 2006;119(1):60–8. 1. Stein PD, Beemath A, Meyers FA, Skaf E, Sanchez J, Olson RE. Incidence of venous thromboembolism in patients hospitalized with cancer. Am J Med. 2006;119(1):60–8. 2. Heit JA, Silverstein MD, Mohr DN, Petterson TM, O’Fallon WM, Mel- ton LJ 3rd. Risk factors for deep vein thrombosis and pulmonary embolism: a population-based case-control study. Arch Intern Med. 2000;160(6):809–15. 3. Blom JW, Doggen CJM, Osanto S, Rosendaal FR. Malignancies, Prothrombotic Mutations, and the Risk of Venous Thrombosis. JAMA. 2005;293(6):715–22. 1. Stein PD, Beemath A, Meyers FA, Skaf E, Sanchez J, Olson RE. Incidence of venous thromboembolism in patients hospitalized with cancer. Am J Med. 2006;119(1):60–8. y 22. Nathan SS, Simmons KA, Lin PP, Hann LE, Morris CD, Athanasian EA, et al. Proximal deep vein thrombosis after hip replacement for oncologic indications. J Bone Joint Surg Am. 2006;88(5):1066–70. 2. Heit JA, Silverstein MD, Mohr DN, Petterson TM, O’Fallon WM, Mel- ton LJ 3rd. Risk factors for deep vein thrombosis and pulmonary embolism: a population-based case-control study. Arch Intern Med. 2000;160(6):809–15. indications. J Bone Joint Surg Am. 2006;88(5):1066–70. 23. Ogura K, Yasunaga H, Horiguchi H, Ohe K, Kawano H. Incidence and risk factors for pulmonary embolism after primary musculoskeletal tumor surgery. Clin Orthop Relat Res. 2013;471(10):3310–6. 3. Blom JW, Doggen CJM, Osanto S, Rosendaal FR. Malignancies, Prothrombotic Mutations, and the Risk of Venous Thrombosis. JAMA. 2005;293(6):715–22. 24. Galanaud JP, Sevestre MA, Pernod G, Genty C, Richelet S, Kahn SR, et al. Long-term outcomes of cancer-related isolated distal deep vein throm- bosis: the OPTIMEV study. J Thromb Haemost. 2017;15(5):907–16. Page 8 of 8 Hayashida et al. Thrombosis Journal (2022) 20:22 Hayashida et al. Thrombosis Journal (2022) 20:22 25. Gary T, Belaj K, Steidl K, Pichler M, Eisner F, Stöger H, et al. Asymp- tomatic deep vein thrombosis and superficial vein thrombosis in ambulatory cancer patients: impact on short-term survival. Br J Cancer. 2012;107(8):1244–8. 26. Watanabe H, Sekiya H, Kariya Y, Hoshino Y, Sugimoto H, Hayasaka S. The incidence of venous thromboembolism before and after total knee arthroplasty using 16-row multidetector computed tomography. J Arthroplasty. 2011;26(8):1488–93. 27. Leonardi MJ, McGory ML, Ko CY. A systematic review of deep venous thrombosis prophylaxis in cancer patients: implications for improving quality. Ann Surg Oncol. 2007;14(2):929–36. 27. Leonardi MJ, McGory ML, Ko CY. Publisher’s Note S Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from:
https://openalex.org/W3047523109	https://zenodo.org/record/5112653/files/Z-BB-Jinvest-Vol2-Issue1-pp23-27.pdf	English	null	Data Fabrication and Data Falsification in the paper entitled "Regulation of DARPP-32 dephosphorylation at PKA and Cdk5-sites by NMDA and AMPA receptors", authored by Nishi, A., Bibb, J.A., Matsuyama, S., Hamada, M., Higushi, H., Nairn, A.C. and Greengard. P., and published in the Journal of Neurochemistry. [J. Neurochem. (2002) Vol. 5, pp832-841].	null	2,020	cc-by	1,633	JOURNAL OF INVESTIGATIVE CRITIQUES OF PUBLISHED SCIENTIFIC ARTICLES VOLUME TWO, ISSUE ONE (JANUARY, 2020), REPORT ONE (pp 23-27): REPORT ONE (pp 23-27): Data Fabrication and Data Falsification in the paper entitled "Regulation of DARPP-32 dephosphorylation at PKA and Cdk5-sites by NMDA and AMPA receptors", authored by Nishi, A., Bibb, J.A., Matsuyama, S., Hamada, M., Higushi, H., Nairn, A.C. and Greengard. P., and published in the Journal of Neurochemistry. [J. Neurochem. (2002) Vol. 5, pp832-841]. Author: H.Y. Lim Tung, PH.D., Peptide and Protein Chemistry Research Laboratory, Nacbraht Biomedical Research Institute, 3164 21st Street, Suite 117, Astoria (NYC), NY 11106, USA. E mail: hyltung2010@nacbrahtbiomedresins.net Author: H.Y. Lim Tung, PH.D., Peptide and Protein Chemistry Research Laboratory, Nacbraht Biomedical Research Institute, 3164 21st Street, Suite 117, Astoria (NYC), NY 11106, USA. E mail: hyltung2010@nacbrahtbiomedresins.net Author: H.Y. Lim Tung, PH.D., Peptide and Protein Chemistry Research Laboratory, Nacbraht Biomedical Research Institute, 3164 21st Street, Suite 117, Astoria (NYC), NY 11106, USA. E mail: hyltung2010@nacbrahtbiomedresins.net Abstract. The paper entitled "Regulation of DARPP-32 dephosphorylation at PKA and Cdk5-sites by NMDA and AMPA receptors", authored by Nishi, A., Bibb, J.A., Matsuyama, S., Hamada, M., Higushi, H., Nairn, A.C. and Greengard. P., and published in the Journal of Neurochemistry. [J. Neurochem. (2002) Vol. 5, pp832-841] describes the study the effects of ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices and purported to show that activation of NMDA and AMPA receptors caused the decrease of phosphorylations of threonine 34 and threonine 75 DARPP-32 mediated by Ca2+-dependent activation of calcineurin and protein phosphatase-2A respectively. However, no supporting scientific results were provided. Figures 1A, 1B, 1C and 1D are duplicates of each other. With exactly the same figures depicting exactly the same immunublots, the authors somehow came up with different bar charts that supposedly quantify the extent of DARPP-32 phosphorylation levels under 23 different conditions. Figures 3A and 3C are also duplicates of each other. It is not clear how the reviewer(s) and the Editor of the Journal of Neurochemistry could have missed the glaring evidence of Data Fabrication and Data Falsification. This is an Investigative Critique of the paper entitled "Regulation of DARPP-32 dephosphorylation at PKA and Cdk5-sites by NMDA and AMPA receptors", authored by Nishi, A., Bibb, J.A., Matsuyama, S., Hamada, M., Higushi, H., Nairn, A.C. and Greengard. P., and published in the Journal of Neurochemistry. [J. Neurochem. (2002) Vol. 5, pp832-841]. JOURNAL OF INVESTIGATIVE CRITIQUES OF PUBLISHED SCIENTIFIC ARTICLES VOLUME TWO, ISSUE ONE (JANUARY, 2020), REPORT ONE (pp 23-27): According to the authors of the paper, they investigated the effects of ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices and claimed to have shown that activation of NMDA and AMPA receptors caused the decrease of phosphorylations of threonone 34 and threonine 75 DARPP-32 mediated by Ca2+-dependent activation of calcineurin and protein phosphatase-2A respectively. Various data were presented including Figure 1 which showed (i) Immunoblots of Phospho-Thr34 in neostriatal slices following no treatment, treatment with AMPA, treatment with NMDA plus MK801, and treatment with MK801 only (Figure 1a), (ii) Immunoblots of Phospho-Thr34 in neostriatal slices following no treatment, treatment with AMPA, treatment with NMDA plus CNQX, and treatment with CNQX alone (Figure 1b), (iii) Immunoblots of Phospho-Thr75 in neostriatal slices following no treatment, treatment with AMPA, treatment with NMDA plus MK801, and treatment with MK801 only (Figure 1c), (ii) Immunoblots of Phospho-Thr34 in neostriatal slices following no treatment, treatment with AMPA, treatment with NMDA plus CNQX, and treatment with CNQX alone (Figure 1d). Careful analysis of Figure 1(a), 1(b), 1(c) and 1(d) revealed that they are all the same exact duplicates of each other except that Figure (1b), 1(c) and (1(d) have been manipulated unscientifically to look differentially lighter than Figure 1(a). Figure 1(a), 1(b), 1(c) and 1(d) are clones of each other. Although, Figure 1(a), 1(b), 1(c) and 1(d) were generated from the same results and Immunoblots, somehow, the authors of the 24 paper entitled "Regulation of DARPP-32 dephosphorylation at PKA and Cdk5-sites by NMDA and AMPA receptors" and published in the Journal of Neurochemistry. [J. Neurochem. (2002) Vol. 5, pp832-841] were able to magically come up with different statistical analyses showing that the four different figures were distinct experiments giving different results. The authors state in the results section the following: (i) "treatment of slices with NMDA (100 µM) for 5 min decreased the level of phospho- Thr34 DARPP-32 to 26.4 ± 7.9% of control" (Fig. 1a), (ii) "Pretreatment of slices with an NMDA receptor antagonist, MK801 (100 µM), for 10 min did not affect the basal level of phospho-Thr34 DARPP-32. How-ever, the effect of NMDA on DARPP-32 Thr34 phosphory-lation was abolished by MK801", (iii) Treatment of slices with AMPA (50 µM) for 5 min decreased the level of phospho-Thr34 DARPP-32 to 66.0 ± 7.6% of control (Fig. JOURNAL OF INVESTIGATIVE CRITIQUES OF PUBLISHED SCIENTIFIC ARTICLES VOLUME TWO, ISSUE ONE (JANUARY, 2020), REPORT ONE (pp 23-27): 1b), (iv) Pretreatment of slices with an AMPA receptor antagonist, CNQX (20 µM), for 10 min increased the basal level ofphospho-Thr34 DARPP-32, possibly by antagonizing the effects of endogenously released glutamate. The effect of AMPA on DARPP-32 Thr34 phosphorylation was antagon-ized by CNQX. How with the same Immunoblot (Figure 1(a) and Figure 1(b), the authors were able to come up with different statistical analysis of 26.4 ± 7.9% of control for Figure 1(a) and 66.0 ± 7.6% of control for Fig. 1b is beyond normal scientific comprehension and in the realm of science fiction. Figure 1(c) and Figure 1(d) are also exact replicas of Figure 1(a) except that they have been unscientifically manipulated to appear lighter. Yet somehow, the authors of the paper were able to come up with statistical analysis of of 22.1 ± 2.2% of control for Figure 1(c) and 47 ± 5.6% of control. Figure 2(a), Lanes for control and NMDA and Figure 2(c), Lanes for control and KCl looked suspiciously similar. Figure 3(a) and 3(c) are exact duplicates except that Figure 3(c) has unscientifically manipulated to look lighter. Despite the fact that Figure 3(a) and Figure 3(c) are exact duplicates, they are stated to depict different treatment with NMDA (Figure 1a) and KCl (Figure 1c), In conclusion, the paper entitled "Regulation of DARPP-32 dephosphorylation at PKA and Cdk5-sites by NMDA and AMPA receptors", authored by Nishi, A., Bibb, J.A., 25 25 Matsuyama, S., Hamada, M., Higushi, H., Nairn, A.C. and Greengard. P., and published in the Journal of Neurochemistry. [J. Neurochem. (2002) Vol. 5, pp832-841] must be retracted because it contains data that was fabricated and falsified. How such blatant cheating managed to pass through the "peer review" system of the Journal of Neurochemistry is unfathomable. Is it because the paper came from a so called "Star Laboratory" and "Star Principal Scientific Researcher". Pursuant to the Office of Research Integrity of the United States Department of Health and Human Services, Scientific Misconduct is defined as Data Fabrication, Data Falsification and Plagiarism in proposing, performing or reviewing research, or in reporting research results [1]. The group of authors that published this paper have been shown to have published several papers with Fabricated Data and Falsified Data [2-5]. JOURNAL OF INVESTIGATIVE CRITIQUES OF PUBLISHED SCIENTIFIC ARTICLES VOLUME TWO, ISSUE ONE (JANUARY, 2020), REPORT ONE (pp 23-27): While Scientific Misconduct is not a criminal act per se in the United States of America, the use of the fruits of Scientific Misconduct to defraud a person or entity, like obtaining a research grant from the NIH or DOD with the Fabricated Data and Falsified Data as Preliminary or Published Results in support of the grant application is a criminal act that can be prosecuted. Many Scientific Researchers have been prosecuted, found guilty, imprisoned and fined [6] References References References 1. Https//ori.hhs.gov. 2. Tung, H.Y.L. (2019) J. Invest. Cri. Pub. Sci. Articles, Vol. 1, pp5-19. Scientific Misconduct, Scientific Fraud and Dishonest Scientific Report. 2. Tung, H.Y.L. (2019) J. Invest. Cri. Pub. Sci. Articles, Vol. 1, pp5-19. Scientific Misconduct, Scientific Fraud and Dishonest Scientific Report. 3. Tung, H.Y.L. (2019) J. Invest. Cri. Pub. Sci. Articles, Vol. 1, pp21-36 Evidence of Data Fabrication, Data Falsification, Dishonest Scientific Report, Theft of Idea and Theft of Data in the Scientific Article "Regulation of Protein Phosphatase-1I by Cdc25C-associated kinase 1 (C-TAK1) and PFTAIRE Protein Kinase" [J. Biol. Chem (2014) Vol 289, pp23893-23900.] 4. Tung, H.Y.L. (2019) J. Invest. Cri. Pub. Sci. Articles, Vol. 1, pp37-43. Showing Data Fabrication and Data Falsification in the Scientific Article "Synthetic Peptide Analogs of DARPP-32 (Mr 32,000 Dopamine- and cAMP- regulated Phosphoprotein), an Inhibitor of Protein Phosphatase-1" authored by “Hugh C. Hemmings, Jr., Angus C. Nairn, James I. Elliot, and Paul Greengard”, 4. Tung, H.Y.L. (2019) J. Invest. Cri. Pub. Sci. Articles, Vol. 1, pp37-43. Showing Data Fabrication and Data Falsification in the Scientific Article "Synthetic Peptide Analogs of DARPP-32 (Mr 32,000 Dopamine- and cAMP- regulated Phosphoprotein), an Inhibitor of Protein Phosphatase-1" authored by “Hugh C. Hemmings, Jr., Angus C. Nairn, James I. Elliot, and Paul Greengard”, Tung, H.Y.L. (2019) J. Invest. Cri. Pub. Sci. Articles, Vol. 1, pp37-43. 26 and published in the Journal of Biological Chemistry [J. Biol. Chem. (1990) Vol. 265, pp20369-20376]. 5. Tung, H.Y.L. (2020) J. Invest. Cri. Pub. Sci. Articles, Vol. 2, pp28-31. Anomalies, contradictory data and dishonest scientific report in the paper entitled "Phosphorylation of DARPP-32, a dopamine and cAMP regulated phosphoprotein by casein kinase II", authored by Girault, J.-A., Hemmings, H.C.Jr., Williams, K.R., Nairn, A. and Greengard, P. (1989) and published in the Journal of Biological Chemistry [J. Biol. Chem. (1989) Vol. 264, pp21748-21759]. 5. 6. Tung, H.Y.L. (2019) In the Matter of Scientific Misconduct and Fraud,1st Edition, Cactoa Scientific Publishers, Inc. Astoria (NYC), New York, USA. 6. Tung, H.Y.L. (2019) In the Matter of Scientific Misconduct and Fraud,1st Edition, Cactoa Scientific Publishers, Inc. Astoria (NYC), New York, USA. 6. 27 27
https://openalex.org/W1997128949	https://europepmc.org/articles/pmc3879602?pdf=render	English	null	Genome Sequence of the Boron-Tolerant and -Requiring Bacterium Bacillus boroniphilus	Genome announcements	2,014	cc-by	1,083	uence of the Boron-Tolerant and -Requiring Bacterium i hil Bekir Çöl,a Zeynep Özkeserli,b Dibyendu Kumar,c Hilal Özdag˘,b Yes¸im D. Alakoçb Mug˘la SK University, Science Faculty, Department of Biology, Kötekli, Mug˘la, Turkeya; Ankara University, Biotechnology Institute, Cen Turkeyb; Waksman Institute of Microbiology, Piscataway, New Jersey, USAc Mug˘la SK University, Science Faculty, Department of Biology, Kötekli, Mug˘la, Turkeya; Ankara University, Biotechnology Institute, Central Laboratory, Tandog˘an, Ankara, Turkeyb; Waksman Institute of Microbiology, Piscataway, New Jersey, USAc Bacillus boroniphilus is a highly boron-tolerant bacterium that also requires this element for its growth. The complete genome sequence of B. boroniphilus was determined by a combination of shotgun sequencing and paired-end sequencing using 454 py- rosequencing technology. A total of 84,872,624 reads from shotgun sequencing and a total of 194,092,510 reads from paired-end sequencing were assembled using Newbler 2.3. The estimated size of the draft genome is 5.2 Mb. Received 5 October 2013 Accepted 2 December 2013 Published 2 January 2014 Citation Çöl B, Özkeserli Z, Kumar D, Özdag˘ H, Alakoç YD. 2014. Genome sequence of the boron-tolerant and -requiring bacterium Bacillus boroniphilus. Genome Announc. 2(1):e00935-13. doi:10.1128/genomeA.00935-13. Copyright © 2014 Çöl et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Yes¸im D. Alakoç, ydalakoc@gmail.com. Bacillus boroniphilus is a highly boron-tolerant bacterium that also requires this element for its growth. The complete genome sequence of B. boroniphilus was determined by a combination of shotgun sequencing and paired-end sequencing using 454 py- rosequencing technology. A total of 84,872,624 reads from shotgun sequencing and a total of 194,092,510 reads from paired-end sequencing were assembled using Newbler 2.3. The estimated size of the draft genome is 5.2 Mb. Received 5 October 2013 Accepted 2 December 2013 Published 2 January 2014 Citation Çöl B, Özkeserli Z, Kumar D, Özdag˘ H, Alakoç YD. 2014. Genome sequence of the boron-tolerant and -requiring bacterium Bacillus boroniphilus. Genome Announc. 2(1):e00935-13. doi:10.1128/genomeA.00935-13. B oron is an essential micronutrient for plants, required primarily for maintaining the integrity of cell walls (1). As an ultratrace element, it is also necessary for the optimal health of some animals (2). It was initially reported that B is essential for some cyanobacteria (3). Moreover, it is known to be toxic for living cells when present above a certain threshold (4). uence of the Boron-Tolerant and -Requiring Bacterium i hil However, in 2007, a B-tolerant and -requiring bacterium was isolated from a naturally high-B- containingareainKütahya,Turkey.Theso-calledBacillusboroniphi- lus requires B for its growth and also can tolerate up to 450 mM boric acid (5). This feature makes it an excellent candidate for being a model organism to investigate the boron-bacterium relationship us- ing molecular and biochemical approaches. toxiﬁcation (9), and stress response genes with no category (33) are particularly striking. Furthermore, it was found that a rela- tively signiﬁcant number of the annotated genes were classiﬁed into the “poorly characterized” COG groups, which indicates that there is still much to study on the genome of this organism. There- fore, the genome of B. boroniphilus will likely shed light on the studies regarding boron biology, stress phenotypes, and beyond. B Nucleotide sequence accession number. This genome se- quence has been deposited at GenBank with the accession no. AWXY00000000. ACKNOWLEDGMENTS This study was supported by a Boron Research Center of Turkey (BO- REN) grant (no. Ç0238). This study was supported by a Boron Research Center of Turkey (BO- REN) grant (no. Ç0238). This study was supported by a Boron Research Center of Turkey (BO- REN) grant (no. Ç0238). We received the B. boroniphilus DSM 17376 from the German Collection of Microorganisms and Cell Culture (DZMZ). The bacte- rium was grown with Difco sporulation medium (DSM) 92 with 50mMboricacid(30°Cat200rpm).ThegenomicDNAwasisolated with the Promega Wizard Genomic DNA puriﬁcation kit. The com- plete genome sequence of B. boroniphilus was determined by a com- bination of a shotgun sequencing and a paired-end sequencing run using 454 pyrosequencing technology on a GS FLX Titanium plat- form. We thank Iftikhar Ahmed for helpful discussions regarding B. bo- roniphilus. REFERENCES 1. Warington K. 1923. The effect of boric acid and borax on the broad bean and certain other plants. Ann. Bot. 37:629–672. 1. Warington K. 1923. The effect of boric acid and borax on the broad bean and certain other plants. Ann. Bot. 37:629–672. 2. Rowe RI, Eckhert CD. 1999. Boron is required for zebraﬁsh embryogen- esis. J. Exp. Biol. 202(Pt 12):1649–1654. 3. Mateo P, Bonilla I, Fernández-Valiente E, Sanchez-Maeso E. 1986. Es- sentiality of boron for dinitrogen ﬁxation in Anabaena sp. Pcc 7119. Plant Physiol. 81:430–433. http://dx.doi.org/10.1104/pp.81.2.430. A total of 84,872,624 reads from shotgun sequencing and a total of 194,092,510 reads from paired-end sequencing were as- sembled using Newbler 2.3, generating three large and 10 small scaffolds. The annotation was performed using both the NCBI- PGAAP and RAST annotation servers (6, 7). 4. Nable RO, Banuelos GS, Paull JG. 1997. Boron toxicity. Plant Soil 193: 181–198. http://dx.doi.org/10.1023/A:1004272227886. 5. Ahmed I, Yokota A, Fujiwara T. 2007. A novel highly boron tolerant bacterium, Bacillus boroniphilus sp. nov., isolated from soil, that requires boron for its growth. Extremophiles 11:217–224. http://dx.doi.org/10.100 7/s00792-006-0027-0. The draft genome of B. boroniphilus contains 4,650,916 bases in 13 scaffolds, excluding gaps. The genome comprises 4,538 pre- dicted coding sequences, 61 tRNAs, and 4 rRNAs genes. The GC content of the genome was found to be 41.35%. The predicted coding sequences were found to be in three COG categories (in- formation storage and processing, metabolism, and cellular pro- cesses and signaling). RAST annotation results revealed 94 activi- ties that belong to the group of stress response genes, which might be involved in providing the extremophile strain of B. boroniphilus to tolerate or even perhaps require B. Among these genes, those for osmotic stress (4), oxidative stress (40), heat shock (15), de- 6. Angiuoli SV, Gussman A, Klimke W, Cochrane G, Field D, Garrity G, Kodira CD, Kyrpides N, Madupu R, Markowitz V, Tatusova T, Thomson N, White O. 2008. Toward an online repository of Standard Operating Procedures (SOPs) for (meta)genomic annotation. OMICS 12: 137–141. http://dx.doi.org/10.1089/omi.2008.0017. 7. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. January/February 2014 Volume 2 Issue 1 e00935-13 REFERENCES BMC Genomics 9:75. http://dx.doi.org/10.1186/1471-2164-9-75. Genome Announcements genomea.asm.org 1
W3036263868.txt	https://downloads.hindawi.com/journals/ijac/2020/2187646.pdf	en		Development of the High Sensitivity and Selectivity Method for the Determination of Histamine in Fish and Fish Sauce from Vietnam by UPLC-MS/MS	International journal of analytical chemistry	2,020	cc-by	5,959	Hindawi International Journal of Analytical Chemistry Volume 2020, Article ID 2187646, 9 pages https://doi.org/10.1155/2020/2187646 Research Article Development of the High Sensitivity and Selectivity Method for the Determination of Histamine in Fish and Fish Sauce from Vietnam by UPLC-MS/MS Quang Hieu Tran ,1 Thanh Tan Nguyen,2 and Kim Phuong Pham2 1 Chemistry Division, Basic Sciences Department, Saigon Technology University, 180 Cao Lo, Ward 4, District 8, Ho Chi Minh City 700000, Vietnam 2 Saigon STC Hi-Tech Analytical Center, Ho Chi Minh City 700000, Vietnam Correspondence should be addressed to Quang Hieu Tran; hieu.tranquang@stu.edu.vn Received 20 February 2020; Revised 19 April 2020; Accepted 29 May 2020; Published 17 June 2020 Academic Editor: Kevin Honeychurch Copyright © 2020 Quang Hieu Tran 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. A selective, sensitive, and rapid method by using ultraperformance liquid chromatography-tandem mass spectrometry (UPLCMS/MS) for the determination of histamine in ﬁsh and ﬁsh sauce was developed. The optimal conditions of liquid chromatographic separation and mass spectroscopy of histamine have also been investigated. The linear ranges of the method were 20.0 ÷ 1000 ng/mL, and the corresponding correlation coeﬃcient was 0.9993. Mean recoveries of the analyte at three spike levels (low, medium, and high) were within the range of 98.5% ÷ 102.5% (n � 7). The limit of detection (LOD) and limit of quantiﬁcation (LOQ) values were 3.83 and 11.50 ng/mL for the ﬁsh sauce sample and 4.71 and 14.12 ng/mL for the ﬁsh sample, respectively. The inﬂuence of the matrix eﬀect on the accuracy, repeatability, and recovery of the method was negligible. The recommended method was applied to determine the content of this substance in 21 ﬁsh sauce samples and 4 kinds of ﬁsh samples, which were collected from Ho Chi Minh City, Vietnam, in 2019. 1. Introduction For a long time, the content of histamine in ﬁsh and ﬁshery products as well as its poisoning has attracted a lot of attention from scientists around the world. This compound is formed during the breakdown of the amino acid histidine which follows two ways. The major route of catabolism of histidine passes through its conversion to glutamic acid, which begins with the degradation of histidine to urocanic acid by the action of the enzyme histidase. The glutamate product is converted to alpha-ketoglutarate, which is an intermediate in the citric acid cycle (Krebs cycle). The second way is decarboxylation (loss of COO-) for the action of the enzyme histidine decarboxylase to form histamine [1, 2]. Scombroid syndrome/histamine poisoning occurs worldwide, and the number of cases is increasing, in spite of the improved knowledge on seafood safety [3]. The symptoms of histamine poisoning generally resemble the symptoms encountered with IgE-mediated food allergies. The symptoms include nausea, vomiting, diarrhea, an oral burning sensation or peppery taste, hives, itching, red rash, and hypotension [4]. The onset of the symptoms usually occurs within a few minutes after the ingestion of the implicated food, and the duration of symptoms ranges from a few hours to 24 h [1]. Due to the toxicity of histamine, the content of this substance has been strictly controlled. According to Codex 302-2011, histamine is considered to be a hazard in ﬁsh sauce, and the contents of this compound must not exceed 400 mg/kg [5, 6]. The Commission Regulation (EC), no. 1019/2013, recommended that the level of histamine must not be exceeded than 400 mg/kg in ﬁsh sauce and 200–400 mg/kg in the ﬁshery products [7]. The European Union and the United States Food and Drug Administration (FDA) have ﬁxed 100 2 and 50 mg/kg for the maximum level of histamine in ﬁsh and ﬁshery products, respectively [8, 9]. Fish sauce, a fundamental ingredient used in many Southeast Asian dishes, a dipping condiment, is gradually gaining popularity worldwide [10]. Fish sauce is a clear brown liquid produced by spontaneous fermentation of diverse ﬁsh such as anchovies, sardines, and menhaden [11]. These ﬁshes typically possess high levels of free histidine and other amino acids. The quantity of histamine produced in traditional ﬁsh sauce is related to the free histidine content in the raw materials. During fermentation, protein hydrolysis is caused by endogenous proteinases in the ﬁsh muscle and digestive tract as well as proteinases produced by halophilic bacteria [2, 12]. The species most commonly used for ﬁsh sauce production is Indian anchovy (Stolephorus spp.). Anchovies are normally caught and kept on board. Producers with good manufacturing practices would mix ﬁsh with salt after the catch, which retards histamine formation. However, some keep ﬁsh without salt for up to 8 hours before landing and transport to a factory in an open container without a proper cooling system [13]. Due to the nature of raw materials and the production methods for traditional ﬁsh sauce, high levels of histamine are found in many samples [14, 15]. There are more than 2,800 ﬁsh sauce production facilities in Vietnam, which has been producing more than 200 million liters per year, worth over VND 4,800 billion. However, Vietnam’s ﬁsh sauce exports account for only about 3–5% of the production. The leading cause of the current low export volume is that there are no well-established brands of Vietnamese ﬁsh sauce overseas. Besides, another important issue is that traditional Vietnamese ﬁsh sauce often encounters technical barriers with some international quality standards, such as histamine level [16]. Currently, histamine has been detected by many methods, such as electrophoretic [17], thin layer chromatography [18], nuclear magnetic resonance (NMR) [19], photoluminescence CdTe quantum dots [20], colorimetry [21, 22], enhanced Raman spectroscopy (SERS) [23], electrochemical [24], nanogold particles (AuNPs) [25], HLPC-UV [14, 25, 26], LCMS/MS [26], UHPLC-Q/TOF-MS [11], or UHPLC-MS/ MS [27]. Presently, the standard procedure for histamine analysis by UPLC-MS/MS has not been oﬃcially announced in Vietnam. Therefore, in this work, we have developed the analysis method to shorten the analysis time and increase the sensitivity and selectivity based on UPLCMS/MS. Thereby, we hope to provide a novel analytical method for testing the presence of histamine in ﬁsh sauce and ﬁshery products in Vietnam. 2. Materials and Methods 2.1. Chemicals and Reagents. All reagents were of analytical grade. The histamine reference standard was purchased from Merck (Darmstadt, Germany); acetonitrile (ACN) and formic acid (FA) were supplied from Sigma-Aldrich (Germany). Methanol was of HPLC grade and acquired from J. T. Baker (Phillipsburg, USA). International Journal of Analytical Chemistry 2.2. Instrumentation. The method development and validation was performed on an ultrahigh performance liquid chromatography (UPLC) system including the column oven and thermostat autosampler (Ultimate 3000, Thermo Fisher Scientiﬁc, Bremen, Germany) in combination with the Waters. TQD tandem quadrupole mass spectrometer, operated in the electrospray positive ion mode with selected reaction monitoring (SRM), was used for data acquisition. 2.3. Standard Solutions. The stock standard solution of 1.0 mg/mL of histamine was prepared in ACN at a concentration of 1.0 mg/mL each. A working solution was prepared immediately before use by diluting 0.1 mL of the stock standard solution to 10 mL ACN to obtain a solution having a known concentration of 0.01 mg/mL. All stock solutions were kept refrigerated (2–8°C) when not in use. 2.4. Sample Preparation 2.4.1. Fish Samples. Fish samples were collected from various commercial ﬁsh markets in Ho Chi Minh City. Each sample was placed individually in a plastic bag, kept in an icebox, and carried to the laboratory of Saigon STC Hi-Tech Analytical center, Ho Chi Minh City. For the examination, a representative ﬁsh sample (50 g) was chopped into small slices and ﬁnely ground with a blender to homogenize it before extraction. Then, 5.0 ± 0.1 g of the ﬁsh sample was transferred into a 50 mL centrifuge tube, added precisely 25 mL of MeOH, vortexed for 2.0 mins, and sonicated in an ultrasonic bath for 20 mins. The solution was transferred into a 50 mL volumetric ﬂask, and volume was makeup with MeOH. Then, this solution was ﬁltrated through a 0.45 μm Whatman membrane. The solution of the sample has been diluted in DI water (the dilution factor depends on the amount of histamine in the sample) and was ﬁltered through a 0.22 μm ﬁlter into a 1.5 mL amber LC vial. The volume of 5.0 μL of the sample solution was injected into the UPLCMS/MS system via the autosampler at the optimum experimental conditions. 2.4.2. Fish Sauce Samples. Fish sauce samples, including 10 industrial ﬁsh sauce (IFS) and 10 traditional ﬁsh sauce (TFS), were collected from the local markets and supermarkets in Ho Chi Minh City, Vietnam. The procedure for sample preparation is as follows: 1.0 mL of ﬁsh sauce was drawn accurately into the 50 mL volumetric ﬂask, dissolved, and made up to the mark with MeOH. Then, 0.5 mL of this solution was transferred to a 10 mL volumetric ﬂask, dissolved and made up to volume with DI water, and ﬁltered through a 0.45 µm ﬁlter. The solution of the sample was diluted in DI water (the dilution factor depends on the amount of histamine in the sample) and was ﬁltered through a 0.22 µm ﬁlter into a 1.5 mL amber LC vial. The analysis was performed on UPLC-MS/MS, as described in Section 2.4.1. International Journal of Analytical Chemistry 2.5. For Liquid Chromatographic Conditions. For liquidchromatographic separation, the HILIC Silica 3 µm, 2.1 × 50 mm, Atlantis column was applied for the separation of target analytes at the temperature of 40°C. The binary mobile phases were ACN 100 mM + HCOOH (A) and DI + 0.5% HCOOH (B). The mobile phase program for the loading pumps is shown in Table 1. The ﬂow rate was constantly kept at 0.3 mL/ min during the whole chromatographic analysis process. Both samples and standard solutions were kept at 10°C in the sample tray. A 5.0 µL of the standard or samples was injected into the UPLC-MS/MS system via an autosampler. The needle and the sample loop in the autosampler were washed triplicate, using the mixture of ACN and deionized water (1 :1, v : v). 3 Table 1: The mobile phase gradient program. Time (min) 0 4.2 4.3 9.5 Flow rate (mL/min) 0.3 0.3 0.3 0.3 ME(%) � B × 100, A A (%) 90 40 90 90 B (%) 10 60 10 10 (1) where A is the chromatographic peak area of the standard in neat solution, B is the peak area of the standard spiked into sample solution after extraction by methanol. 2.6. MS Conditions. For ﬁnding the optimal conditions of MS, selected reaction monitoring (SRM) spectra obtained in the positive ion mode were applied to recognize the speciﬁed analyte. The ion source parameters were optimized as follows: ionizing source: H-ESI (+), spray voltage: 3500 V, vaporizer temperature: 300°C, ion transfer tube temperature: 300°C, sheath gas: 40 arb, aux gas: 5 arb, and CID gas: 2 mTorr. TraceFinder 3.3 software was used for calculating the peak areas and peak area ratios. 2.7.4. Recovery and Precision. For examining recovery and precision, the experiment was carried out with two kinds of samples (ﬁsh and ﬁsh sauce). A series of the example was spiked histamine samples at 214, 428, and 856 mg/L. The solution was diluted 20 times in DI water. The analysis was performed on UPLC-MS/MS, as described in Section 2.4.1. At each level, the test was measured seven times for calculating SD, R%, and RSD%. 2.7. Method Validation 3. Results and Discussion 2.7.1. Linearity. For evaluating the linearity, a series of reference standard solutions of histamine were prepared in the range of concentrations of 20.0, 50.0, 150.0, 200.0, 500.0, and 1000.0 (ng/mL). At each level, the standard solutions were measured with triplicate. Based on the plotting of the peak area ratio to the internal standard versus the spiked concentration, the calibration curve equation was calculated. 2.7.2. Determination of LOD and LOQ. Limit of detection (LOD) and limit of quantiﬁcation (LOQ) are two important performance features in method validation. LOD and LOQ, strictly related to the magnitude of noises in the measurement system, could be determined in diﬀerent ways. In this work, these values were calculated by the formula LOD � 3SD and LOQ � 3LOD according to [28, 29]. 2.7.3. Matrix Eﬀect (ME). The evaluation of the matrix eﬀect was conducted by using an experimental model of Matuszewski et al. [30]. The matrix eﬀect during validation of the developed method was examined by measurement of the analytical signal of histamine in the postextraction spiked solution and that of the histamine standard in a neat solution. The experiment was carried out with two groups of pooled samples. The ﬁrst group was prepared by mixing ﬁve ﬁsh sauce samples, and the second group was formed by mixing four ﬁsh samples (sardines, anchovy, menhaden, and mackerel). The sample preparation was performed in Section 2.4.1. The ﬁnal solution (after sample extraction) was spiked in three levels of 50, 200, and 1000 ppb of standard histamine. The eﬀect of the sample matrix was determined by the following equation: 3.1. Chromatographic and MS Conditions 3.1.1. Chromatographic Conditions. The conditions for UPLC chromatography have been established with the HILIC Silica 3 µm, 2.1 × 50 mm, Atlantis column, mobile phases A (MeOH 5 mM HCOONH4 + 0.1% FA) and B (H2O 5 mM HCOONH4 + 0.1% FA), and 5.0 µL injection volume. The chromatogram of the standard solutions under selected conditions indicates the retention time of 3.69 mins for the neat solvent and 3.68 for both ﬁsh and ﬁsh sauce sample solutions. Figure 1 indicates that there is no signiﬁcant diﬀerence in the chromatograph of the standard histamine neat solvent and in the ﬁsh sauce sample solution at the same concentration. The retention time and the intensity of the analyte in both samples are related. This phenomenon implies that the eﬀect of the sample background on the retention time is not notable. 3.1.2. MS Parameters. Table 2 and Figure 2 describe the mass spectrometry parameters of histamine and its fragmented ions. The precursor ion shows 112.2 of m/z; the product ions are 68 of m/z, 83 of m/z, and 95 of m/z, respectively. The collision energy for the ions is 23.0 V (m/z 68), 16.0 V (m/z 83), and 15.0 V (m/z 95), respectively. The m/z 95 ion is selected for the quantitation analysis. This result is quite similar to previous research studies [14, 24]. 3.2. Method Validation 3.2.1. Linearity. As present in Section 3.1, the matrix eﬀects on two samples (ﬁsh and ﬁsh sauce) are not signiﬁcant on the retention time and the intensity of the method. For International Journal of Analytical Chemistry Relative intensity 4 100 90 80 70 60 50 40 30 20 10 0 RT: 3.69 histamine 0.17 0.67 0.5 1.11 1.51 1.73 1.97 2.18 1.0 1.5 2.0 2.72 2.5 3.22 3.45 3.0 3.5 4.78 4.0 4.5 5.0 RT (min) 5.34 5.5 5.76 6.13 6.0 6.57 6.5 7.13 7.27 7.54 8.00 8.30 8.60 8.88 9.20 7.0 8.0 7.5 8.5 9.0 9.5 Relative intensity (a) RT: 3.68 histamine 100 90 80 70 60 50 40 30 20 10 0 0.52 0.70 0.98 0.5 1.0 1.45 1.5 1.91 2.0 2.43 2.79 2.5 3.46 3.0 3.5 4.65 4.97 5.26 5.63 5.82 4.0 4.5 5.0 RT (min) 5.5 6.0 6.43 6.5 6.91 7.19 7.0 7.73 7.97 8.22 8.55 8.87 7.5 8.0 8.5 9.0 9.47 9.5 Relative intensity (b) 100 90 80 70 60 50 40 30 20 10 0 RT: 3.68 histamine 0.14 0.68 1.01 0.5 1.0 1.64 1.88 1.5 2.0 2.43 2.78 2.5 3.29 3.0 4.81 5.04 5.46 5.79 3.5 4.0 4.5 5.0 RT (min) 5.5 6.0 6.31 6.56 6.89 7.20 6.5 7.0 7.71 7.99 8.31 8.78 9.06 9.27 7.5 8.0 8.5 9.0 9.5 (c) Figure 1: Representative UPLC-MS/MS chromatograms of histamine at 50.0 ng/mL of concentration: (a) in ACN blank, (b) in ﬁsh sauce sample solution, and (c) in ﬁsh sample solution. Table 2: Mass parameters of histamine and its products in the SRM mode. Compound name Histamine Histamine Histamine ∗ Polarity Positive Positive Positive Precursor (m/z) 112.2 112.2 112.2 Product (m/z) 68.222 83.169 95.111∗ Collision energy (V) 23.0 16.0 15.0 Quantitative ion. simplifying the process, we have built the calibration curve in the pure solvent. The calibration curve in Figure 3 is linear over the range of 20.0 to 1000.0 ng/mL. The calibration equation obtained from the proposed method was y � 35536x + 692240. The least-squares regression exhibited an excellent correlation coeﬃcient of 0.9993. The relative standard deviation of each point (n � 3) was less than 3.0%. 3.2.2. LOD and LOQ. As can be seen in Table 3, LOD and LOQ of the validated method for ﬁsh sauce samples and ﬁsh samples are 3.83 and 11.52 and 4.71 and 14.12 ng/mL, respectively. Therefore, the developed method was suitable for direct analysis of this substance in ﬁsh and ﬁsh sauce samples according to the EC [7] and the USFDA [8]. International Journal of Analytical Chemistry 5 Multiple collision energy product scan for m/z = 112.20 m/z = 95.11 Maximum intensity 600000 500000 400000 300000 200000 m/z = 68.22 100000 20 30 40 50 60 m/z 70 m/z = 83.10 80 90 100 Figure 2: The maximum intensity of product scan for histamine in the SRM mode. 40000000 2 35000000 y = 35536x + 692240, R = 0.9993 30000000 Area 25000000 20000000 15000000 10000000 5000000 0 0 200 400 600 800 Histamine concentration (ng/mL) 1000 Figure 3: The graph of peak area vs. histamine concentration. 3.2.3. Selectivity. Endogenous sources of interference were not observed at the retention time of the analyte in Figure 1. This phenomenon indicates that the eﬀect of the sample background on the retention time is not signiﬁcant as compared with the sample blank. These values suggest that there is a minimum eﬀect of the sample background on ion suppression, ion enhancement, and the retention time of analytes. These data also prove that the selectivity of the method for the determination of histamine in real samples is excellent. 3.2.4. Matrix Eﬀect. As can be seen, in Table 4, at three sample backgrounds with diﬀerent fat contents, ME values in the range from 98.23% to 104.01% were in an acceptable range [30] (from 80 to 120%). The relative standard deviations at diﬀerent concentrations are suitable values. These values show that the inﬂuence of the sample background on the selectivity and the recovery of measurement is negligible. This phenomenon could be explained by the excellent separation ability of the HILIC Silica column and the high sensitivity of the MS/MS probe. Compared with other methods for the determination of histamine with complex extraction, this method signiﬁcantly shortens the analysis time, as well as reduces the analysis cost and the amount of solvent released after the sampling process. 3.2.5. Recovery and Precision. In this test, the levels of 214, 428, and 856 mg/L, approximately to the level of 0.5, 1.0, and 2.0 times compared with the EC threshold prescribed for histamine standards in ﬁsh sauce, were chosen to spike. Similarly, the levels of 25, 50, and 100 mg/kg equivalent to 0.5, 1.0, and 2.0 times with the EC regulation threshold for histamine standards in ﬁsh samples and ﬁsh products were selected for the determination of recovery, accuracy, and repeatability. As presented in Table 5, the recovery of the histamine detection method at three concentrations lies from 100.86% to 116.43% (for the ﬁsh sauce sample) and from 95.14% to 107.21% (for the ﬁsh sample). These values are consistent with the requirements of the AOAC [31] and similar to previous research studies [26, 32, 33]. Thus, the method in this work has a high recovery eﬃciency and can be applied to real sample analysis. The intraday repeatability was within the acceptable range, ranging from 1.61 to 2.52% of RSD, whereas interday repeatability ranges from 2.34 to 10.21% of RSD. Similar RSD values are found in the ﬁsh sample background. The observed RSD values for the precision study indicate that this method is suﬃciently precise for routine analysis. For illustration, the sensitivity of the developed method by UPLC-MS/MS, a comparison with the previously published methods for histamine determination is given in Table 6. The developed method provides higher sensitivity in comparison with published methods. The sensitivity of this method is similar to the selectivity of the cITP-CZE-COND method. 3.3. Application to Real Samples. The proposed method has been applied to analyze 21 ﬁsh sauce samples. As shown in Table 7 and Figure 4, the group of industrial ﬁsh sauce (IFS 00 to IFS 10) contains histamine with the content from 8.0 to 80.9 mg/L. In general, the histamine content in 11 types of industrial ﬁsh sauce analyzed was the equally low allowable threshold of the FDA [8] and below of the EC [7]. The cause may be due to the closed industrial ﬁsh sauce production process and the raw ﬁsh that has been salted or frozen immediately after catching. Therefore, the conversion rate from histidine to histamine is less. Also, it is possible that, in industrial products, the content of pure ﬁsh sauce is low, and 6 International Journal of Analytical Chemistry Table 3: The detection limit and quantitation limit of the UPLC-MS/MS method. Replica (n � 7) 1 2 3 4 5 6 7 Mean SD LOD � 3 SD (ng/mL) LOQ � 3 LOD (ng/mL) Fish sauce samples Spiked (ng/mL) Found (ng/mL) 20.00 19.21 20.00 21.32 20.00 21.20 20.00 18.70 20.00 20.15 20.00 21.10 20.00 22.31 20.57 1.28 3.83 11.50 Fish samples Spiked (ng/mL) Found (ng/mL) 20.00 18.12 20.00 22.13 20.00 21.40 20.00 18.36 20.00 21.14 20.00 21.20 20.00 20.90 20.46 1.57 4.71 14.12 Table 4: ME (%) of the method at diﬀerent matrix samples; the standard amount of histamine was spiked in three levels of 50, 200, and 1000 ppb after sample preparation. Samples Concentration of histamine in ﬁnal sample solution (ng/mL) Pooled ﬁsh sauce 531.4 Pooled ﬁsh 55.8 Spiked (ng/mL) (n � 3) 50 200 1000 50 200 1000 ME (%) 100.87 104.01 101.34 101.42 102.07 98.23 RSD (%) 2.31 1.67 2.24 2.44 0.74 1.21 Table 5: Recovery, intraday repeatability (day 1), and interday repeatability assay (day 3, day 5, and day 7) measured as %RSD of spiked ﬁsh sauce and ﬁsh samples (1.0 mL of ﬁsh sauce or 1.0 g of the ﬁsh sample, 50 mL of sample volume, and 20 times of dilution factor). Samples Concentration of histamine in the sample (ng/mL) Fish sauce, TFS 18 385.6 Mackerel ﬁsh 64.5 Spiked concentration (mg/L) Recovery, R (%) 214 428 856 25 50 100 109.21 100.86 116.43 105.32 95.14 107.21 Repeatability (intraday), RSD (%) Day 1 Day 3 Day 5 Day 7 1.83 2.34 3.51 6.71 2.55 3.22 8.34 4.29 1.81 2.44 6.65 10.21 2.35 3.12 3.59 6.71 3.49 3.67 6.15 6.91 2.92 3.13 7.14 11.52 Table 6: The comparison of the developed UPLC-MS/MS method with previously published methods for the histamine analysis. Methods UHPLC-HR-MS cITP-CZE-COND IRP-HPLC Colorimetric UPLC-FLD HPLC-UV UHPLC-MS/MS LOD (ng/mL) 100 4.0 1000 5000 5.5 130 3.38 LOQ (ng/mL) 300 12.0 3000 the manufacturer has added spices and ﬂavorings to create industrial ﬁsh sauce. Therefore, the histamine content may be much lower than that of traditional ﬁsh sauce. Meanwhile, in the traditional ﬁsh sauce group (TFS 10 to TFS 20), the histamine content ranges from 385.6 to 1436.4 mg/L. These values are higher than the FDA’s allowable threshold. The histamine content in traditional ﬁsh sauce in Vietnam is 15.6 450 11.5 Recovery (%) — 91 ± 9 86.00 >91 — 91 ÷ 115 100.1 ÷ 116 References [34] [17] [35] [21] [34] [15] This work quite similar to other countries in the region, such as Thailand [12, 14], Malaysia [15], and China [11]. In fact, in the past, people in Asian countries have used traditional ﬁsh sauce with high levels of histamine content. However, histamine poisoning from ﬁsh sauce is very rare, because the amount of ﬁsh sauce in the daily meal is not much. However, for traditional ﬁsh sauce to penetrate the market of International Journal of Analytical Chemistry 7 Table 7: The concentration of histamine in ﬁsh sauce samples. IFS00 IFS 01 IFS 02 IFS 03 IFS 04 IFS 05 IFS 06 IFS 07 IFS 08 IFS 09 IFS 10 TFS 11 TFS 12 TFS 13 TFS 14 TFS 15 TFS 16 TFS 17 TFS 18 TFS 19 TFS 20 Compared method (HPLC-PDA)1 Mean ± SD (mg/L) Not found 16.2 ± 1.2 13.5 ± 1.6 7.2 ± 0.6 48.3 ± 3.1 35.2 ± 1.8 24.3 ± 1.9 28.1 ± 2.3 66.3 ± 2.9 38.6 ± 2.7 87.4 ± 6.0 432.0 ± 31.3 761.4 ± 46.2 391.6 ± 31.7 589.3 ± 34.2 432.4 ± 26.8 1093.4 ± 72.6 410.4 ± 16.9 374.3 ± 22.7 1501.3 ± 79.6 424.8 ± 21.2 Developed method (UPLC-MS/MS) Mean ± SD (mg/L) Not found 15.1 ± 1.1 12.4 ± 0.8 8.0 ± 0.5 45.2 ± 2.2 32.0 ± 1.3 21.5 ± 1.2 31.2 ± 1.4 63.1 ± 3.2 35.2 ± 2.4 80.9 ± 5.0 458.0 ± 18.3 773.4 ± 50.5 410.3 ± 30.5 561.5 ± 41.6 463.6 ± 33.2 1130.1 ± 60.9 397.3 ± 17.6 385.6 ± 25.4 1436.4 ± 86.5 418.6 ± 18.3 Note. 1Compared method was carried out by using the Waters 2996 HPLC-PDA instrument with column C18 (Zorbax Eclipse XDB-C18, 5 mm × 4.6 mm × 250 mm, Agilent) and followed the standard protocol of the Ministry of Science and Technology of Vietnam [22] and AOAC [36]. Each value is expressed as mean ± SD (n � 3). 1600 Concentration of histamine in fish sauce (mg/L) 1400 1200 1000 800 600 400 200 IFS 01 IFS 02 IFS 03 IFS 04 IFS 05 IFS 06 IFS 07 IFS 08 IFS 09 IFS 10 TFS 11 TFS 12 TFS 13 TFS 14 TFS 15 TFS 16 TFS 17 TFS 18 TFS 19 TFS 20 0 Figure 4: Histamine concentration of ﬁsh sauce; values are expressed as the mean ± SD (n � 3). Error bars represent the standard deviation of three replicates. Table 8: The concentration of histamine in ﬁsh samples. Frozen Frozen Frozen Frozen sardines (Sardinella tawilis) anchovy (Engraulidae) menhaden (Brevoortia tyrannus) mackerel (Rastrelliger kanagurta) Developed method (UPLC-MS/MS) (mg/kg) 16.4 ± 0.8 13.5 ± 0.9 6.3 ± 0.7 64.5 ± 1.6 Compared method (HPLC-PDA) (mg/kg) 18.3 ± 1.2 12.1 ± 0.8 7.5 ± 0.6 58.5 ± 3.2 Note. Each value is expressed as mean ± SD (n � 3). European countries or the US, manufacturers, scientists, and managers must ﬁnd an explanation for the histamine content. Four raw ﬁsh samples commonly used to produce ﬁsh sauce have been analyzed. Table 8 indicates that the histamine content in these samples ranges from a fairly wide 8 range, with the highest in mackerel, reaching 65.4 mg/kg. The histamine content depends on the type and freshness of ﬁsh. The more the ﬁsh becomes spoiled, the higher the content of histamine will be [37]. The histamine content of ﬁsh samples analyzed was quite similar to the results of the previous report in Vietnam [38] and other countries [20, 26]. We have also applied the AOAC standard procedure with HPLC-FDA equipment to determine the level of histamine in ﬁsh sauce and ﬁsh samples. The results illustrated in Tables 7 and 8 also showed that both methods perform harmonious results. 4. Conclusions In this work, we have developed and evaluated a new analytical method that has excellent sensitivity, selectivity, and recovery. The method described here shows the promise of highly selective and sensitive quantiﬁcation of histamine in ﬁsh sauce and ﬁsh samples. The HPLC-FDA method has also been used for comparative analysis, showing that the proposed method has similar results but with shorter time and more straightforward sample processing. The study of real samples has also shown that the histamine content in traditional ﬁsh sauce, produced by individual establishments, is much higher than that of the EC and FDA standards. Hopefully, we have contributed a new analytical technique to assess and control histamine content in ﬁsh, ﬁshery products, food, and pharmaceuticals. Data Availability The data used to support the ﬁndings of this study are available from the corresponding author upon request. The MS optimization, some sample chromatographs, recovery performance calculations, and calibration curves are presented in Supplementary Information (available here). Conflicts of Interest The authors declare that there are no conﬂicts of interest regarding the publication of this paper. Authors’ Contributions All authors have contributed equally to this work. Acknowledgments This project was funded by the Saigon Technology University under Grant no. DTCS-02, 2019. Supplementary Materials S1: compound optimization report. S2: UPLC-MS/MS chromatograms of histamine in some of ﬁsh sauce and ﬁsh samples. S3: data for determination of R% and repeatability (ﬁsh sauce). S4: compound calibration report. (Supplementary Materials) International Journal of Analytical Chemistry References [1] S. L. Taylor, J. E. Stratton, and J. A. Nordlee, “Histamine poisoning (scombroid ﬁsh poisoning): an allergy-like intoxication,” Journal of Toxicology: Clinical Toxicology, vol. 27, no. 4-5, pp. 225–240, 1989. [2] P. Visciano, M. Schirone, R. Tofalo, and G. Suzzi, “Histamine poisoning and control measures in ﬁsh and ﬁshery products,” Frontiers in Microbiology, vol. 5, no. 1–3, 2014. [3] F. M. Colombo, P. Cattaneo, E. Confalonieri, and C. Bernardi, “Histamine food poisonings: a systematic review and metaanalysis,” Critical Reviews in Food Science and Nutrition, vol. 58, no. 7, pp. 1131–1151, 2018. [4] V. Tortorella, P. Masciari, M. Pezzi et al., “Histamine poisoning from ingestion of ﬁsh or scombroid syndrome,” Case Reports in Emergency Medicine, vol. 2014, Article ID 482531, 4 pages, 2014. [5] C. Stan, Standard for Fish Sauce, Food and Agriculture Organization, Rome, Italy, 2012. [6] K. B. Biji, C. N. Ravishankar, R. Venkateswarlu, C. O. Mohan, and T. K. S. Gopal, “Biogenic amines in seafood: a review,” Journal of Food Science and Technology, vol. 53, no. 5, pp. 2210–2218, 2016. [7] EC: Commission Regulation (EU), No 1019/2013 of 23 October 2013 amending Annex I to Regulation (EC) No 2073/ 2005 as Regards Histamine in Fishery Products Text with EEA Relevance. https://eur-lex.europa.eu/eli/reg/2013/1019/oj. [8] FDA, Fish and Fishery Products Hazards and Controls Guidance, Scombrotoxin (Histamine) Formation, FDA, Silver Spring, MD, USA, 2020, https://www.fda.gov/media/80288/ download Chapter 7, 4th edition. [9] EC, Commission Regulation (EC) No 1441/2007, Oﬃcial Journal of the European Union. 322, 2007. [10] A. Gildberg, J. Wichaphon, S. Lertsiri, A. Assavanig, N. K. Sørensen, and C. Thongthai, “Chemical and organoleptic comparison of ﬁsh sauce made from cold water species and typical Thai ﬁsh sauce,” Journal of Aquatic Food Product Technology, vol. 16, no. 3, pp. 31–42, 2007. [11] Y. Wang, C. Li, L. Li et al., “Application of UHPLC-Q/TOFMS-based metabolomics in the evaluation of metabolites and taste quality of Chinese ﬁsh sauce (Yu-lu) during fermentation,” Food Chemistry, vol. 296, pp. 132–141, 2019. [12] FAO, Estimating the Risk of Developing Histamine Poisoning from the Consumption of Thai Fish Sauces, FAO, Rome, Italy, 2011. [13] J. Yongsawatdigul, Y. J. Choi, and S. Udomporn, “Biogenic amines formation in ﬁsh sauce prepared from fresh and temperature-abused Indian anchovy (Stolephorus indicus),” Journal of Food Science, vol. 69, pp. 312–319, 2004. [14] S. Brillantes and W. Samosorn, “Determination of histamine in ﬁsh sauce from Thailand using a solid phase extraction and high-performance liquid chromatography,” Fisheries Science, vol. 67, no. 6, pp. 1163–1168, 2001. [15] B. Padilah, S. Bahruddin, A. Fazilah, and R. R. A. Gulam, “Biogenic amines analysis in shrimp pastes belacan obtained from the Northern States of Peninsular Malaysia,” International Food Research Journal, vol. 25, pp. 1893–1899, 2018. [16] Y. L. T. Dang, T. H. T. Tran, L. D. T. Nguyen, A. H. Nguyen, and T. T. T. Nguyen, “Isolation and screening of histamineproducing bacteria from the ﬁrst six months of the cat hai ﬁsh sauce fermentation process,” Vietnam Journal of Agricultural Sciences, vol. 1, no. 3, pp. 220–229, 2019. International Journal of Analytical Chemistry [17] F. Kvasnička, S. Kavková, and A. Honzlová, “Electrophoretic determination of histamine,” Journal of Chromatography A, vol. 1588, pp. 180–184, 2019. [18] Z. Bajc and K. Š. Gačnik, “Densitometric TLC analysis of histamine in ﬁsh and ﬁshery products,” Journal of Planar Chromatography—Modern TLC, vol. 22, no. 1, pp. 15–17, 2009. [19] E. López-Rituerto, A. Avenoza, J. H. Busto, and J. M. Peregrina, “NMR study of histidine metabolism during alcoholic and malolactic fermentations of wine and their inﬂuence on histamine production,” Journal of Agricultural and Food Chemistry, vol. 61, no. 39, pp. 9464–9469, 2013. [20] S. Khan, L. S. A. Carneiro, M. S. Vianna, E. C. Romani, and R. Q. Aucelio, “Determination of histamine in tuna ﬁsh by photoluminescence sensing using thioglycolic acid modiﬁed CdTe quantum dots and cationic solid phase extraction,” Journal of Luminescence, vol. 182, pp. 71–78, 2018. [21] S. B. Patange, M. K. Mukundan, and K. Ashok Kumar, “A simple and rapid method for colorimetric determination of histamine in ﬁsh ﬂesh,” Food Control, vol. 16, no. 5, pp. 465–472, 2005. [22] TCVN 8352 : 2010, Fish and Fishery Products—Determination of Histamine Content—Method Using High-Performance Liquid Chromatography, 2010. [23] T. Janči, D. Valinger, J. Gajdoš Kljusurić, L. Mikac, S. Vidaček, and M. Ivanda, “Determination of histamine in ﬁsh by surface enhanced Raman spectroscopy using silver colloid SERS substrates,” Food Chemistry, vol. 224, pp. 48–54, 2017. [24] A. Veseli, M. Vasjari, T. Arbneshi et al., “Electrochemical determination of histamine in ﬁsh sauce using heterogeneous carbon electrodes modiﬁed with rhenium (IV) oxide,” Sensors and Actuators B: Chemical, vol. 228, pp. 774–781, 2016. [25] C. Huang, S. Wang, W. Zhao et al., “Visual and photometric determination of histamine using unmodiﬁed gold nanoparticles,” Microchimica Acta, vol. 184, no. 7, pp. 2249–2254, 2017. [26] A. Ali, K. N. Waheed, A. Hadaiyt, and I. Begum, “Determination of histamine levels by LC-MS/MS in various ﬁsh species available in the local markets of Punjab, Pakistan,” International Journal of Fisheries and Aquatic Studies, vol. 4, pp. 128–132, 2016. [27] M. E. Lame, E. E. Chambers, and K. J. Fountain, “Development of a quantitative UPLC/MS/MS Assay for the simultaneous quantiﬁcation of acetylcholine, histamine, and their metabolites in human cerebrospinal ﬂuid (CSF) using a CORTECS UPLC HILIC column to improve peak resolution, sensitivity, and speed O,” 2013, https://www.waters.com/ webassets/cms/library/docs/720004722en.pdf. [28] AOAC, AOAC Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals, AOAC, Rockville, MD, USA, 2002. [29] A. Kruve, R. Rebane, K. Kipper et al., “Tutorial review on validation of liquid chromatography-mass spectrometry methods: part I,” Analytica Chimica Acta, vol. 870, pp. 29–44, 2015. [30] B. K. Matuszewski, M. L. Constanzer, and C. M. Chavez-Eng, “Strategies for the assessment of matrix eﬀect in quantitative bioanalytical methods based on HPLC−MS/MS,” Analytical Chemistry, vol. 75, no. 13, pp. 3019–3030, 2003. [31] AOAC, “AOAC international guidelines for validation of botanical identiﬁcation methods,” Journal of AOAC International, vol. 95, pp. 268–272, 2012. [32] N. Byrd, “Quick, easy and reliable detection of histamine in food using the agilent 6490 triple quadrupole LC/MS with jet 9 [33] [34] [35] [36] [37] [38] stream technology,” Agilent Technologies, Santa Clara, CA, USA, 2013. Y. Ohtsubo, H. Kurooka, H. Tada, and N. Manabe, “Method for determination of histamine in food by LC-MS/MS,” Food Hygiene and Safety Science (Shokuhin Eiseigaku Zasshi), vol. 55, no. 2, pp. 103–109, 2014. S. Sentellas, Ó. Núñez, and J. Saurina, “Recent advances in the determination of biogenic amines in food samples by (U) HPLC,” Journal of Agricultural and Food Chemistry, vol. 64, no. 41, pp. 7667–7678, 2016. M. Nadeem, T. Naveed, F. Rehman, and Z. Xu, “Determination of histamine in ﬁsh without derivatization by indirect reverse phase-HPLC method,” Microchemical Journal, vol. 144, pp. 209–214, 2019. AOAC Oﬃcial Method 977.13, “Histamine in seafood— ﬂuorometric method,” Oﬃcials Methods of Analysis of AOAC International, vol. 35, pp. 17–19, 2002. E. Peralta and A. Serrano Jr, “Histamine formation and microbiological quality of deboned milkﬁsh (Chanos chanos) during ambient storage,” ELBA Bioﬂux, vol. 7, pp. 1–8, 2015. N. A. Dung, Regional Survey of Histamine in Fish and Fish Products, SEAFDEC, HCM City, Vietnam, 2008, http:// repository.seafdec.org/handle/20.500.12066/4410http://repository. seafdec.org/handle/20.500.12066/4410.
W2551019733.txt	https://download.atlantis-press.com/article/25868754.pdf	en		An efficient computational approach for solving type-2 intuitionistic fuzzy numbers based Transportation Problems	The International journal of computational intelligence systems/International journal of computational intelligence systems	2,016	cc-by	15,062	International Journal of Computational Intelligence Systems, Vol. 9, No. 6 (2016) 1154-1173 An efficient computational approach for solving type-2 intuitionistic fuzzy numbers based Transportation Problems Ali Ebrahimnejad 1, ∗, Jose Luis Verdegay 2 1 2 Department of Mathematics, Qaemshahr Branch Islamic Azad University, Qaemshahr, P.O. Box 163, Iran E-mails: a.ebrahimnejad@qaemiau.ac.ir and aemarzoun@gmail.com Department of Computer Science and Artificial Intelligence, Universidad de Granada 18014 Granada, Spain E-mail: verdegay@decsai.ugr.es Received 5 August 2015 Accepted 20 June 2016 Abstract This paper is concerned with the solution procedure of a Transportation Problem in which costs are triangular intuitionistic fuzzy numbers (TIFN) and availabilities and demands are taken as exact numerical values. According to the existing solution approach, TIFN are first ordered by using an accuracy function defined on score functions for membership and non-membership functions of TIFN. Then this ordering is used to develop methods for finding an initial basic feasible solution and the optimal solution of intuitionistic fuzzy Transportation Problems in terms of triangular intuitionistic fuzzy numbers. This solution approach, in spite of its merits, requires a lot of fuzzy arithmetic operations, such as additions and subtractions of TIFN, as well as a lot of comparisons on TIFN. In this paper an efficient computational solution approach is proposed for solving intuitionistic fuzzy Transportation Problems based on classical transportation algorithms to overcome the shortcomings of the aforementioned solution approach. In the approach here presented, the comparison of triangular intuitionistic fuzzy costs is done once and all arithmetic operations are done on real numbers. Finally, for the sake of illustration, two intuitionistic fuzzy Transportation Problems are solved herein to demonstrate the usages and advantages of the proposed solution approach. Keywords: Intuitionistic fuzzy transportation problem; Triangular intuitionistic fuzzy number; Accuracy function. 1. Introduction A useful, effective and operative tool to handle imprecise data is that of fuzzy sets as defined by Zadeh 1 . In such a case, the main drawback to be pointed out is the fact that the accomplishment of the property defining the postulated fuzzy set is to be measured by means of an “exact” and unique real number. Therefore using fuzzy sets is not suitable in cases where there may be a hesitation or uncertainty about the accomplishment degree of the element in a set. To tackle this drawback, Atanassov 2 introduced the concept of Intuitionistic Fuzzy Set (IFS) that seems to suitably describe an imprecise concept and incorporates the mentioned hesitation in the membership degrees. In IFS, not only the degree of acceptance is defined by a membership function, but ∗ Corresponding author. Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1154 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach also the degree of rejection is considered by a nonmembership function so that the sum of both degrees should be smaller than one. Because of these facts, it is expected that IFS can cope with the presence of vagueness and hesitancy originating from imprecise knowledge or information. IFS Theory has been applied in many important and essential areas, including multi attribute decision-making models 3,4 , multi attribute group decision-making problems 5,6 , image restoration 7 , medical diagnosis 8 , game theory 9,10 and pattern recognitions 11 among others. As is well known, nowadays Transportation Problems (TP) because of their recent relevant applications (optimization problems associated to different models of smart cities, multimodal transports, etc.) are of upmost importance and in almost all the cases imprecise data are omnipresent. Hence, Fuzzy Sets Theory and IFS Theory could be used to capture linguistic uncertainty in optimization problems (see Refs. 12–18), particularly in TP. Basically, the central question in TP is to find the least total transportation cost of a commodity in order to satisfy demands at destinations using available supplies at origins. In classical TP it is assumed that the transportation costs and values of supplies and demands are exactly known. However, in many situations the decision maker has no precise information about the coefficients defining the TP. In these cases, the corresponding coefficients or elements defining the problem can be specified by means of fuzzy sets, and a Fuzzy Transportation Problem (FTP) appears in a natural way. From this point of view, numerous researchers have devoted their efforts to using fuzzy numbers in real life TP. Thus, for instance, Jimenez and Verdegay 19,20 studied Fuzzy Solid TP in which supplies, demands and conveyance capacities are represented by trapezoidal fuzzy numbers and presented a parametric approach for finding a fuzzy solution. Also, an FTP in which supplies and demands are trapezoidal fuzzy numbers was formulated by Gani and Razak 21 and a parametric approach for finding a fuzzy solution with the aim of minimizing the sum of the transportation costs in the two stages was analysed by them. Dinagar and Palanivel 22 considered an FTP with trapezoidal fuzzy numbers and proposed a fuzzy modified distribution method to obtain the optimal solution in terms of fuzzy numbers. A new algorithm, namely the fuzzy zero point method for finding the fuzzy optimal solution for FTP, in which the transportation cost, supplies and demands are represented by trapezoidal fuzzy numbers was introduced by Pandian and Natarajan 23 . A systematic procedure for solving all types of FTP whether to maximize or to minimize the objective function was proposed by Basirzadeh 24 , and a new method to find the solution of a linear multiobjective TP, by representing all the parameters as interval-valued fuzzy numbers, was considered by Gupta and Kumar 25 . Shanmugasundari and Ganesan 26 developed the fuzzy version of Vogel’s and MODI methods for obtaining the fuzzy initial basic feasible solution and the fuzzy optimal solution respectively, and also Kaur and Kumar 27 proposed a new method based on ranking functions for solving FTP by assuming that the parameters of the TP are represented by generalized trapezoidal fuzzy numbers. Moreover, Kaur and Kumar 28 approached a special type of FTP where transportation costs are represented by generalized trapezoidal fuzzy numbers. More recently, Ebrahimnjead 29 presented a simplified approach to find the optimal solution of the FTP studied by Kaur and Kumar 28 . Sudhagar and Ganesan 30 proposed an algorithm to find an optimal solution of a FTP with all parameters represented by fuzzy numbers. It was shown by Ebrahimnjead 31 that the algorithm considered by Sudhagar and Ganesan 30 does not always lead to a fuzzy optimal solution. In addition, Ebrahimnejad 32 proposed a two-step method for solving FTP where all of the parameters are represented by non-negative triangular fuzzy numbers. In spite of this, although fuzzy numbers are commonly used for modeling imprecise data when one has to cope with real TP, this may not be suitable for situations where one has to deal with uncertainty as well as with hesitation. In such situations, Intuitionistic Fuzzy Numbers are used to represent the imprecise parameters of the TP under consideration. The resulting problem is therefore referred to as an Intuitionistic Fuzzy Transportation Programming Problem (IFTP). Despite the importance of the problem Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1155 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach there are few studies in the current literature facing the practical solving of IFTP. Hussain and Kumar 33 focused on TP in which supplies and demands are intuitionistic fuzzy numbers. Then, they analysed an intuitionistic fuzzy zero point method to find the optimal solution in terms of triangular intuitionistic fuzzy numbers. Nagoorgani and Abbas 34 proposed another method based on ranking functions for finding an optimal solution of the same IFTP. Singh and Yadav 35 presented intuitionistic fuzzy methods to find the starting basic feasible solution in terms of triangular intuitionistic fuzzy numbers, and also proposed an intuitionistic fuzzy modified distribution method to find optimal solution of the same IFTP. Antony et al. 36 considered solving TP with triangular intuitionistic fuzzy numbers using Vogel’s approximation method. Finally, a new method for solving TP has been approached by Singh and Yadav 37 in which transportation costs are triangular intuitionistic fuzzy numbers (TIFNs) and availabilities and demands are taken as exact numerical values. The method, first ranks TIFNs using an accuracy function defined on score functions for membership and non-membership functions of TIFNs. Then, one uses this ordering to develop methods for finding an initial basic feasible solution and an optimal solution of IFTP in terms of triangular intuitionistic fuzzy numbers. However the method proposed by Singh and Yadav 37 , in spite of its merits, requires a lot of fuzzy arithmetic operations such as additions and subtractions of TIFNs and a lot of comparisons on TIFNs. For that reason in this paper one proposes an efficient computational solution approach for solving the same problem based on classical transportation algorithms. In the proposed approach, comparison of triangular intuitionistic fuzzy costs is done once, and all arithmetic operations are done on real numbers. Consequently the rest of the paper is organized as follows: In Section 2, some basic concepts of fuzzy sets theory and intuitionistic fuzzy sets theory are reviewed. In Section 3, the TP with intuitionistic fuzzy transportation costs is formulated and the aforementioned approach to find the optimal solution is summarized. In Section 4, an efficient computational solution approach based on classical transportation algorithms is proposed for solving IFTP and to diminish the amount of computations of the existing approach. Finally, in Section 5, the application of the proposed method is illustrated by using two numerical examples and the obtained results are discussed. Section 6, including the main conclusions as well as some interesting future research lines, ends the paper. 2. Preliminaries This section briefly introduces some basic concepts including fuzzy sets theory and intuitionistic fuzzy sets which are applied throughout this paper (see Refs. 2 and 37). Definition 1: Let X denote the universe set. A e in X is defined by a set of ordered pairs fuzzy{(set A ) } e A = x, µAe(x) ; x ∈ X where µAe(x) ∈ [0, 1] repree and is called sents the membership degree of x in A, e the membership function of A. Definition 2: Let Xdenote the universe set. An intueI in X is defined by a set itionistic fuzzy set (IFS){⟨A ⟩ } I e = x, µ eI (x), υ eI (x) ; x ∈ X of ordered triple A A A where the functions µAeI (x) : X → [0, 1]andυAeI (x) : X → [0, 1], respectively, represent the membership e such degree and non-membership degree of x in A that for each element x ∈ X, 0 6 µAeI (x)+ υAeI (x) 6 1. Definition fuzzy set {⟨ 3: For each ⟩ intuitionistic } eI = x, µ eI (x), υ eI (x) ; x ∈ X in X, the value A A A hAeI (x) = 1 − µAeI (x) − υAeI (x) is called degree of heseI . itancy of x to A eI = Definition 4: An⟩ intuitionistic fuzzy set A {⟨ } x, µAeI (x), υAeI (x) ; x ∈ X is called intuitionistic fuzzy normal if there is any x◦ ∈ X such that µAeI (x◦ ) = 1 (so υAeI (x◦ ) = 0). eI = Definition 5: An⟩ intuitionistic fuzzy set A {⟨ } x, µAeI (x), υAeI (x) ; x ∈ X is called intuitionistic fuzzy convex if its membership function is fuzzy convex and its non-membership function is concave, i.e. ∀x1 , x2 ∈ X, ∀λ ∈ [0, 1], µAeI (λ x1 + (1 − λ )x2 ) > Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1156 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach { } min {µAeI (x1 ), µAeI (x2 ) }and υAeI (λ x1 + (1 − λ )x2 ) 6 max υAeI (x1 ), υAeI (x2 ) . eI = Definition 6: An⟩ intuitionistic fuzzy set A {⟨ } x, µAeI (x), υAeI (x) ; x ∈ R of the real number R is called an intuitionistic fuzzy number if eI is intuitionistic fuzzy normal and intuition(i) A istic fuzzy convex. (ii) µAeI is upper semi continuous and υAeI is semi lower continuous. { } eI = x ∈ R; υ eI (x) < 1 is bounded. (iii) Supp A A Definition 7: A triangular intuitionistic fuzzy numeI is a special IFN with the memberber (TIFN) A ship function non-membership function defined as follows:  x−a  a2 −a11 , a1 < x 6 a2 a3 −x , a2 6 x < a3 µAeI (x) =  a3 −a2 0, Otherwise. and  a −x ′ 2   a2 −a′1 , a1 < x 6 a2 x−a2 ′ υAeI (x) = a′3 −a2 , a2 6 x < a3   1, Otherwise. ′ where a1 6 a1 < a2 < a3 6 a′3 . This TIFN is denoted eI = (a1 , a2 , a3 ; a′ , a2 , a′ ). by A 1 3 eI = Definition 8: Given two TIFNs A ′ ′ I ′ (a1 , a2 , a3 ; a1 , a2 , a3 ) and Be = (b1 , b2 , b3 ; b1 , b2 , b′3 ), eI and BeI can some arithmetic operations between A be defined as follows: eI ⊕ BeI = (a1 + b1 , a2 + b2 , a3 + b3 ; a′ + (i) A 1 b′1 , a2 + b2 , a′3 + b′3 ), eI ⊖BeI = (a1 − b3 , a2 − b2 , a3 − b1 ; a′ − (ii) A 1 b′3 , a2 − b2 , a′3 − b′1 ), eI (ka1 , ka2 , ka3 ; ka′ , ka2 , ka′ ), k > 0, (iii) kA 1 3 eI = (ka3 , ka2 , ka1 ; ka′ , ka2 , ka′ ), k < 0. (iv) kA 3 1 eI = (a1 , a2 , a3 ; a′ , a2 , a′ ), Definition 9: For TIFN A 1 3 its accuracy function is defined as follows: ′ ′ eI ) = (a1 + 2a2 + a3 ) + ( a1 + 2 a2 + a3 ) f (A 8 (1) Singh and Yadav 37 proved that the accuracy function f : IF(R) → R, where IF(R) is a set of TIFNs defined on a set of real numbers, is a linear function. They used this linear function for comeI = (a1 , a2 , a3 ; a′ , a2 , a′ ) and paring two TIFNs A 1 3 I ′ Be = (b1 , b2 , b3 ; b1 , b2 , b′3 ). eI = Definition 10: Assume two TIFNs A ′ ′ I ′ e (a1 , a2 , a3 ; a1 , a2 , a3 ) and B = (b1 , b2 , b3 ; b1 , b2 , b′3 ), eI ) and f (BeI ) as their accuracy functions, rewith f (A spectively. Then eI > BeI if f (A eI ) > f (BeI ), (i) A eI 6 BeI if f (A eI ) 6 f (BeI ), (ii) A eI = BeI if f (A eI ) = f (BeI ). (iii) A 3. Intuitionistic Fuzzy Balanced Transportation Problems Singh and Yadav 37 applied an interesting methodology in solving a TP having uncertainty as well as hesitation in prediction of the transportation costs. One defines a TP having intuitionistic fuzzy transportation costs but crisp availabilities and demands as an intuitionistic Fuzzy Balanced Transportation Problem of type-2 (IFBTP-2). The IFBTP-2, in which a decision maker considers the cost as TIFN to deal efficiently with the uncertainty as well as hesitation arising in prediction of transportation cost, but (s)he is sure about the availability and demand of the product, can be formulated as follows (see Ref. 37): m n min ZeI = ∑ ∑ ceIi j xi j i=1 j=1 n s.t. ∑ xi j = ai , i = 1, 2, ..., m, ∑ xi j = b j , j = 1, 2, ..., n, j=1 m (2) i=1 xi j > 0, i = 1, 2, ..., m, j = 1, 2, ..., n. where ai is: the total availability of the product at ith source; b j : the total demand of the product at ′ ′ jth destination; ceIi j = (ci1j , ci2j , ci3j ; ci1j , ci2j , ci3j ): the intuitionistic cost for transporting one unit quantity Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1157 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach of the product from the ith source to the jth destination; xi j : the quantity transported from the ith source to the jth destination or decision variables; n eIi j xi j : total intuitionistic fuzzy transporta∑m i=1 ∑ j=1 c tion cost. Singh and Yadav 37 considered the linear ordering given in (1) to develop methods for obtaining the initial basic feasible solution (IBFS) of the IFBTP-2 given in (2). Then one generalizes an intuitionistic fuzzy method to test the optimality of the obtained IBFS using the same ordering. To do so, they rewrote the IFBTP-2 given in (2) as an Intuitionistic Fuzzy Linear Programming problem and proved some theorems concerning optimality conditions and duality properties. Let ueIi = ′ ′ ′ ′ (ui1 , ui2 , ui3 ; ui1 , ui2 , ui3 ) and veIj = (v1j , v2j , v3j ; v1j , v2j , v3j ) be the intuitionistic fuzzy dual variables associated with ith row and jth column constraints, respectively, then the intuitionistic fuzzy dual of the IFBTP-2 given in Eq. (2) is defined as follows (see Ref. 37): m n i=1 j=1 max ZeDI = ∑ ai ueIi ⊕ ∑ b j veIj ueIi ⊕ veIj 6 ceIi j i = 1, 2, ..., m, j = 1, 2, ..., n. (3) Based on the linear function given in (1), Singh and Yadav 37 introduced three methods, namely intuitionistic fuzzy North West corner method (IFNWCM), intuitionistic fuzzy least-cost method (IFLCM) and intuitionistic fuzzy Vogel’s approximation method (IFVAM) to find the initial IBFS for the IFBTP-2 given in Eq. (2). Then, they utilized the intuitionistic fuzzy modified distribution method (IFMODIM) to find the fuzzy optimal solution for the IFBTP-2 (2) with the help of IBFS. They also proved some theorems to provide optimality criteria for the obtained IBFS. The resulting theorem is summarized as follows (see Ref. 37): s.t. Theorem 1. Let IFBTP-2 (2) has a basic feasible solution (BFS) with B as a basis matrix. If deiIj = ueIi ⊕ veIj 6 ceIi j for all non-basic variables, the current BFS is optimal. In the proposed approach by Singh and Yadav 37 all arithmetic operations are performed on the trian- gular intuitionistic fuzzy numbers, i.e., ueIi , veIj and ceIi j . In the following section, we show that it is possible to find the same solution as the IFBTP-2 (2) without solving any intuitionistic fuzzy problems and so all arithmetic operations are done on real numbers instead of triangular intuitionistic fuzzy numbers. 4. An efficient computational approach In this section an efficient computational solution approach is proposed for solving intuitionistic fuzzy TP, based on classical transportation algorithms to diminish the amount of computations of the Sing and Yadav’s solution approach. In the algorithm proposed by Singh and Yadav 37 , the linear accuracy function (1) has been used to compare between triangular intuitionistic fuzzy numbers. In such a case by using this linear ranking function it is possible to define a rank for each triangular intuitionistic fuzzy number. In eI = (a1 , a2 , a3 ; a′ , a2 , a′ ) is a fact, assuming that A 1 3 eI ) = triangular intuitionistic fuzzy number, then f (A (a1 +2a2 +a3 )+( a′1 +2 a2 +a′3 ) . This equation enables us to 8 convert the IFBTP-2 (2) into a crisp TP. To do this, we substitute the rank of each triangular intuitionistic fuzzy number instead of the corresponding triangular intuitionistic fuzzy number in the problem under consideration. This leads to an equivalent crisp TP that can be solved by traditional transportation algorithms. In summary, once the ranking function is chosen, the intuitionistic fuzzy TP under consideration is converted into a crisp one, which is easily solved by the existing transportation simplex methods. Therefore, it is possible to obtain the optimal solution of the IFBTP-2 (2) problem, without the need for a fuzzy approach. As a result, the computational amount is decreased significantly in our proposed approach. Our main contribution in this study is the reduction of the amount of computations for solving the IFBTP-2 (2) compared to the intuitionistic fuzzy transportation algorithm proposed by Singh and Yadav 37 . In particular, in what follows it is shown in detail that our method needs fewer elementary operations such as additions, subtractions and compar- Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1158 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach isons than the aforementioned method. Two main steps of the intuitionistic fuzzy transportation algorithm proposed by Singh and Yadav 37 for solving IFBTP-2 (2) are summarized as follows: Step 1: Find an initial BFS using IFNWCM, IFLCM or IFVAM. Step 2: Find the optimal solution of IFBTP-2 using the intuitionistic fuzzy modified distribution method (IFMODIM). First, the computation effort required for finding an initial BFS using the proposed method and the method of Singh and Yadav 37 are compared. It should be noted that in the IFBTP-2 (2) only the transportation cost is represented by triangular intuitionistic fuzzy numbers. It can be seen that in the IFNWCM proposed by Singh and Yadav 37 , the intuitionistic fuzzy costs have no role on finding initial BFS. Thus this method needs the same amount of computations compared to the standard North West corner method for finding an initial BFS. In addition, in the IFLCM proposed by them it is required to determine the smallest intuitionistic fuzzy cost in the IFBTP table using the accuracy function given in (1) for each iteration. Thus in this approach no addition and subtraction operations are done on intuitionistic fuzzy costs to obtain an initial BFS. In this approach, the comparison between intuitionistic fuzzy costs is only done once using the accuracy function given in (1) to determine the smallest one. However, to find an initial BFS according to IFVAM, it is required to (1) Compute the intuitionistic penalty for each row and each column of the IFBTP table of order m × n, (2) Select the highest intuitionistic penalty using the accuracy function, (3) Determine the cell with the smallest intuitionistic cost in the selected row or column using the accuracy function, (4) Allocate as much as possible to the variable corresponding to the determined cell, adjust the supply and demand and delete the satisfied row or column, (5) Repeat the process for the reduced table until the table is reduced to1 × 1. Remark 1: The penalty measure for each row (column) is determined by subtracting the smallest unit intuitionistic fuzzy cost element in the row (column) from the next smallest unit intuitionistic fuzzy cost element in the same row (column). Regarding Remark 1 and the process of IFVAM, the main drawback of this method is that it requires a lot of fuzzy subtractions of TIFNs and a lot of comparisons based on accuracy function on TIFNs to compute the intuitionistic penalty measures of each row and each column and to identify the row or column with the largest intuitionistic penalty. In sum, our proposed method and the existing method of Singh and Yadav 37 have a same computation effort if IFNWCM or IFLCM is used to obtain the initial BFS of IFBTP-2 (2). However, in our approach all the triangular intuitionistic fuzzy costs are changed to crisp numbers according to the accuracy function. Thus, comparison of triangular intuitionistic fuzzy costs is done once and all arithmetic operations are performed on real numbers. Due to these facts, our method here needs less computation effort in comparison to the method proposed by Singh and Yadav 37 if IFVAM is used to obtain the initial BFS of IFBTP-2 (2). Now, the computation effort required for finding the optimal solution of IFBTP-2 (2) using the presented method and the method proposed by Singh and Yadav 37 is compared. Let us suppose that an initial BFS obtained using IFNWCM, IFLCM or IFVAM with basis B is at hand. The main steps of the intuitionistic fuzzy modified distribution method (IFMODIM) proposed by Singh and Yadav 37 to find the intuitionistic fuzzy optimal solution for the IFBTP-2 (2) are summarized as follows: Step 1: For each cell (i, j), define intuitionistic ′ ′ fuzzy dual variables ueIi = (ui1 , ui2 , ui3 ; ui1 , ui2 , ui3 ) and ′ ′ veIj = (v1j , v2j , v3j ; v1j , v2j , v3j ) associated with ith row and jth column, respectively. Step 2: Solve the intuitionistic fuzzy system ueIi ⊕ veIj = ceIi j for each basic cell (i, j). cIi j for each nonStep 3: Compute dei j = ueIi ⊕ veIj ⊖e basic cell (i, j). If f (dei j ) 6 0 for each non-basic cell Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1159 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach (i, j), then stop, the current BFS is optimal. Otherwise select an entering cell (a non-basic cell with the most positive f (deiIj )). Step 4: Determine an existing cell, obtain the new BFS using the standard transportation methods and repeat Step 1. Our main contribution here is the reduction of the computational complexity of the method proposed by Singh and Yadav 37 . In particular, it is shown that our proposed method needs a lower number of elementary operations such as additions, multiplications, and comparisons as compared to the method proposed by Singh and Yadav 37 for testing the optimality conditions. According to the method proposed by Singh and Yadav 37 , to carry out Step 2 of the IFMODIM it is required to solve the intuitionistic fuzzy system ueIi + veIj = ceIi j with m +n −1 intuitionistic fuzzy equations corresponding to basic cells. After solving this intuitionistic fuzzy system, the intuitionistic fuzzy value deiIj for each non-basic cell is obtained based on dei j = ueIi ⊕ veIj ⊖e cIi j . Finally the entering cell is determined according to the most positive rank of deiIj . These facts ensure that this step requires a lot of intuitionistic fuzzy additions and subtractions on TIFNs. While based on our proposed methods, the optimality criteria are checked without solving any intuitionistic fuzzy system, without any intuitionistic fuzzy arithmetic operations and without any comparison of TIFNs. These results confirm that the proposed method is simpler and computationally more efficient than the method proposed by Singh and Yadav 37 . As a final point, in the next theorem we mathematically prove that the results of the method proposed by Singh and Yadav 37 and the proposed method for solving IFBTP-2 (2) are the same. Theorem 2. The optimal solution of the IFBTP-2 (2) according to the existing method and the proposed method is the same. Proof. According to the proposed approach, using the linear accuracy function (1) we substitute the rank of each triangular intuitionistic fuzzy transportation cost instead of the corresponding triangular intuitionistic fuzzy transportation cost in IFBTP- 2 (2). This leads to the following crisp TP: m min n f (ZeI ) = ∑ ∑ f (e cIi j )xi j i=1 j=1 n s.t. ∑ xi j = ai , i = 1, 2, ..., m, ∑ xi j = b j , j = 1, 2, ..., n, j=1 m (4) i=1 xi j > 0, i = 1, 2, ..., m, j = 1, 2, ..., n. Let f (e uIi ) and f (e vIj ) be the dual variables associated with ith row and jth column constraints, respectively. In this case, the dual of the TP (4) is given as follows: m n i=1 j=1 max f (ZeDI ) = ∑ ai f (e uIi ) + ∑ b j f (e vIj ) s.t. f (e uIi ) + f (e vIj ) 6 f (e cIi j ), i = 1, 2, ..., m, j = 1, 2, ..., n. (5) Therefore, if the basis B is the optimal basis of the crisp TP (4), then we have f (e uIi ) ⊕ f (e vIj ) 6 f (e cIi j ) as the optimality conditions of TP (4). It should be noted that in order to obtain the optimal solution according to the method proposed by Singh and Yadav 37 and our proposed method, the IFBTP-2 (2) and the crisp TP (4) are solved, respectively. If we show that these problems have the same optimal solution, we conclude that the results of our proposed approach are matched with those obtained based on the method proposed by Singh and Yadav 37 . Note that both problems have a same feasible space. Thus, it is sufficient to show that both problems have the same optimality conditions. In fact, if the basis B is the optimal basis of the IFBTP-2 (2), then it will be the optimal basis of the equivalent crisp TP (4). To do this, suppose that x∗ = (xi∗j )1×mn is an optimal solution of the IFBTP-2 (2) with B as the optimal basis. Thus, according to Theorem 1 we have ueIi ⊕ veIj 6 ceIi j for all non-basic variables. With regard to Definition 10, these conditions are equivcIi j ). Since the accuracy alent to f (e uIi ⊕ veIj ) 6 f (e uIi ) + function is linear, we have f (e uIi ⊕ veIj ) = f (e Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1160 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach Table 1. Summary of the intuitionistic FTP. D1 D2 D3 D4 ai S1 (2, 4, 5; 1, 4, 6) (2, 5, 7; 1, 5, 8) (4, 6, 8; 3, 6, 9) (4, 7, 8; 3, 7, 9) 11 S2 S3 S4 (4, 6, 8; 3, 6, 9) (3, 4, 6; 1, 4, 8) (2, 4, 6; 1, 4, 7) (3, 7, 12; 2, 7, 13) (8, 10, 13; 5, 10, 16) (3, 9, 10; 2, 9, 12) (10, 15, 20; 8, 15, 22) (2, 3, 5; 1, 3, 6) (3, 6, 10; 2, 6, 12) (11, 12, 13; 10, 12, 14) (6, 10, 14; 5, 10, 15) (3, 4, 5; 2, 4, 8) 11 11 12 bj 16 10 8 11 45 f (e vIj ). This means that ueIi ⊕ veIj 6 ceIi j if and only if f (e uIi ) + f (e vIj ) 6 f (e cIi j ). Thus, we conclude that ∗ ∗ x = (xi j )1×mn is the optimal solution of the TP (4). This completes the proof. 5. Numerical examples In this section, in order to demonstrate the effectiveness of the proposed method, two intuitionistic fuzzy TP taken from Singh and Yadav 37 are considered. Example 5.1: Table 1 gives the crisp availability (ai ) of the product available at four origins Si , i = 1, 2, 3, 4 and the crisp demand (b j ) at four destinations D j , j = 1, 2, 3, 4. The transportation costs from origins to destinations are represented by intuitionistic fuzzy triangular fuzzy numbers. The aim is to find the least total intuitionistic fuzzy transportation cost of the commodity in order to satisfy demands at destinations using available availabilities at origins. According to the accuracy function given in (1), we substitute the rank order of each intuitionistic fuzzy transportation cost (given in Table 1) with its corresponding intuitionistic fuzzy number to obtain the classical transportation problem. The results are given in Table 2. Table 2. Summary of the classical transportation problem. S1 S2 S3 S4 bj D1 D2 D3 D4 ai 30 8 48 8 34 8 32 8 38 8 58 8 82 8 63 8 48 8 120 8 26 8 51 8 52 8 96 8 80 8 34 8 11 11 11 16 10 8 11 12 45 In what follows, we derive the optimal solution of the given intuitionistic fuzzy transportation prob- lem based on the method proposed by Singh and Yadav 37 according to Table 1 and our proposed method according to Table 2. The initial BFS of Table 1 can be found by any one of the IFNWCM, IFLCM or IFVAM methods proposed by Singh and Yadav 37 . Also, the initial BFS of Table 2 can be found by any one of the classical North West corner method (NWCM), least-cost method (LCM) or Vogel’s approximation method (VAM) that correspond to our proposed method. 5.1. Results based on IFNWCM and NWCM Both the IFNWCM proposed by Singh and Yadav 37 and the classical NWCM give the initial BFS given in Table 3. Table 3. Initial BFS by IFNWCM. D1 D2 D3 D4 ai S1 S2 S3 11 5 - 6 4 7 - 11 11 11 S4 bj 16 10 1 8 11 11 12 45 5.1.1. Iteration 1(IFNWCM) According to Step 1 and Step 2 of the IFMODIM proposed by Singh and Yadav 37 the following intuitionistic fuzzy system should be solved to test the optimality of the initial BFS given in Table 3: Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1161 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach ′ ′ ′ ′ (u11 , u12 , u13 ; u11 , u12 , u13 ) + (v11 , v12 , v13 ; v11 , v12 , v13 ) ceI11 = = (2, 4, 5; 1, 4, 6) ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v11 , v12 , v13 ; v11 , v12 , v13 ) = ceI21 = (4, 6, 8; 3, 6, 9) ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI22 = (3, 7, 12; 2, 7, 13) ′ ′ ′ ′ (u31 , u32 , u33 ; u31 , u32 , u33 ) + (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI32 = (8, 10, 13; 5, 10, 16) ′ ′ ′ ′ (u31 , u32 , u33 ; u31 , u32 , u33 ) + (v31 , v32 , v33 ; v31 , v32 , v33 ) = ceI33 = (2, 3, 5; 1, 3, 6) ′ ′ ′ ′ (u41 , u42 , u43 ; u41 , u42 , u43 ) + (v31 , v32 , v33 ; v31 , v32 , v33 ) = ceI43 = (3, 6, 10; 2, 6, 12) ′ ′ ′ ′ (u41 , u42 , u43 ; u41 , u42 , u43 ) + (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI44 = (3, 4, 5; 2, 4, 8) (6) The intuitionistic fuzzy solution of this system is given as follows (see Ref. 37): ′ ′ ueI1 = (u11 , u12 , u13 ; u11 , u12 , u13 ) = (−24, −8, 7; −33, −8, 15), ′ ′ I ve1 = (v11 , v12 , v13 ; v11 , v12 , v13 ) = (−2, 12, 26; −9, 12, 34) ′ ′ I ue2 = (u21 , u22 , u23 ; u21 , u22 , u23 ) = (−18, −6, 6; −25, −6, 12), ′ ′ I ve2 = (v21 , v22 , v23 ; v21 , v22 , v23 ) = (6, 13, 21; 1, 13, 27) ′ ′ I ue3 = (u31 , u32 , u33 ; u31 , u32 , u33 ) = (−8, −3, 2; −11, −3, 4), ′ ′ veI3 = (v31 , v32 , v33 ; v31 , v32 , v33 ) = (3, 6, 10; 2, 6, 12), ′ ′ ueI4 = (u41 , u42 , u43 ; u41 , u42 , u43 ) = (0, 0, 0; 0, 0, 0), ′ ′ veI4 = (v41 , v42 , v43 ; v41 , v42 , v43 ) = (3, 4, 5; 2, 4, 8) (7) According to Step 3 of the IFMODIM proposed by Singh and Yadav 37 , it is required to compute cIi j for each non-basic cell(i, j). Thus, deiIj = ueIi ⊕ veIj ⊖e we have: I =u eI1 ⊕ veI2 ⊖e de12 cI12 = (−24, −8, 7; −33, −8, 15)⊕ (6, 13, 21; 1, 13, 27)⊖(2, 5, 7; 1, 5, 8) = (−25, 0, 26; −40, 0, 41), I =u eI1 ⊕ veI3 ⊖e de13 cI13 = (−24, −8, 7; −33, −8, 15)⊕ (3, 6, 10; 2, 6, 12)⊖(4, 6, 8; 3, 6, 9) = (−29, −8, 13; −40, −8, 24), I =u eI1 ⊕ veI4 ⊖e de14 cI14 = (−24, −8, 7; −33, −8, 15)⊕ (3, 4, 5; 2, 4, 8)⊖(4, 7, 8; 3, 7, 9) = (−29, −11, 8; −40, −11, 20), I =u eI2 ⊕ veI3 ⊖e de23 cI23 = (−18, −6, 6; −25, −6, 12)⊕ (3, 6, 10; 2, 6, 12)⊖(10, 15, 20; 8, 15, 22) = (−35, −15, 6; −45, −15, 16), I =u eI2 ⊕ veI4 ⊖e de24 cI24 = (−18, −6, 6; −25, −6, 12)⊕ (3, 4, 5; 2, 4, 8)⊖(11, 12, 13; 10, 12, 14) = (−28, −14, 0; −37, −14, 10), I =u eI3 ⊕ veI1 ⊖e de31 cI31 = (−8, −3, 2; −11, −3, 4)⊕ (−2, 12, 26; −9, 12, 34)⊖(3, 4, 6; 1, 4, 8) = (−16, 5, 25; −28, 5, 37), I =u eI3 ⊕ veI4 ⊖e de34 cI34 = (−8, −3, 2; −11, −3, 4)⊕ (3, 4, 5; 2, 4, 8)⊖(6, 10, 14; 5, 10, 15) = (−19, −9, 1; −24, −9, 7), I =u eI4 ⊕ veI1 ⊖e de41 cI41 = (0, 0, 0; 0, 0, 0)⊕ (−2, 12, 26; −9, 12, 34)⊖(2, 4, 6; 1, 4, 7) = (−8, 8, 24; −16, 8, 33), I =u eI4 ⊕ veI2 ⊖e de42 cI42 = (0, 0, 0; 0, 0, 0)⊕ (6, 13, 21; 1, 13, 27)⊖(3, 9, 10; 2, 9, 12) = (−4, 4, 18; −11, 4, 25). (8) Also, I ) = 2 , f (deI ) = −8, f (deI ) = −85 , f (de12 13 14 8 8 I ) = −118 , f (deI ) = −111 , f (deI ) = 38 , f (de23 24 31 8 8 8 I ) = −71 , f (deI ) = 65 , f (deI ) = 44 , f (de34 41 42 8 8 8 (9) Since f (deiIj ) ̸6 0 for all non-basic cells (i, j), then the current BFS is not optimal. Thus, a non-basic cell with the most positive f (deiIj ), i.e., x41 is selected as the entering variable. According to the classical transportation algorithm, x43 is selected as the leaving variable and the new BFS is found as given in Table 4. Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1162 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach Table 4. Improved solution-1. D1 D2 D3 D4 ai S1 11 - - - 11 S2 S3 S4 4 1 7 3 - 8 - 11 11 11 12 bj 16 10 8 11 45 5.1.2. Iteration 1(NWCM) According to the classical modified distribution method (MODIM) applied by our proposed approach the following crisp system should be solved to test the optimality of the initial BFS given in Table 3: u1 + v1 = c11 = u2 + v2 = c22 = u3 + v3 = c33 = u4 + v4 = c44 = 30 8 , u2 + v1 58 8 , u3 + v2 26 8 , u4 + v3 34 8 = c21 = 6, = c32 = 82 8 , = c43 = 51 8 , (10) The solution of this crisp system is given as follows: −49 −25 u1 = −67 8 , u2 = 8 , u3 = 8 , u4 = 0 97 107 51 v1 = 8 , v2 = 8 , v3 = 8 , v4 = 34 8 (11) To test the optimality of the solution given in Table 4 it is required to compute di j = ui + v j − ci j for each non-basic cell (i, j). Thus, we have: 107 38 2 d12 = u1 + v2 − c12 = −67 8 + 8 − 8 = 8, 51 48 d13 = u1 + v3 − c13 = −67 8 + 8 − 8 = 8, −67 34 −85 d14 = u1 + v4 − c14 = 8 + 8 − 52 8 = 8 , −49 51 120 d23 = u2 + v3 − c23 = 8 + 8 − 8 = −118 8 , 34 96 −111 d24 = u2 + v4 − c24 = −49 + − = 8 8 8 8 , 97 34 38 + − = d31 = u3 + v1 − c31 = −25 8 8 8 8 , −25 34 80 −71 d34 = u3 + v4 − c34 = 8 + 8 − 8 = 8 , 32 65 d41 = u4 + v1 − c41 = 0 + 97 8 − 8 = 8 , 63 −44 d42 = u4 + v2 − c42 = 0 + 107 8 − 8 = 8 . (12) Since di j ̸6 0 for all non-basic cells (i, j), then the current BFS is not optimal. Thus, according to the classical transportation algorithm in crisp environment, x41 and x43 are selected as the entering variable and the leaving variable, respectively, and thus the new BFS is found as given in Table 4 matching with the result of the improved solution-1 of the method proposed by Singh and Yadav 37 . However, to find the improved solution-1 given in Table 4 it is necessary to solve the intuitionistic fuzzy system (6). After solving this system, the intuitionistic fuzzy value deiIj = ueIi ⊕ veIj ⊖e cIi j for each non-basic cell is calculated according to (8). As we see, these two steps require a large number of fuzzy additions and subtractions on TIFNs. While based on our proposed approach, these steps are done with solving the crisp system (10) and with calculating the crisp values given in (12) using the elementary operation on real numbers. Moreover, to choose the entering variable according to the method proposed by Singh and Yadav 37 it is necessary to compare the intuitionistic fuzzy value deiIj for each non-basic cell as done in (9). While according to our proposed method, the entering variable is selected without any comparison of TIFNs. Due to these facts, our proposed method is preferred to the method proposed by Singh and Yadav 37 from the computational attempt point of view. 5.1.3. Iteration 2(IFNWCM) According to Step 1 and Step 2 of the IFMODIM proposed by Singh and Yadav 37 the following intuitionistic fuzzy system should be solved to test the optimality of the improved solution-1 given in Table 4: ′ ′ ′ ′ (u11 , u12 , u13 ; u11 , u12 , u13 ) + (v11 , v12 , v13 ; v11 , v12 , v13 ) = ceI11 = (2, 4, 5; 1, 4, 6), ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v11 , v12 , v13 ; v11 , v12 , v13 ) = ceI21 = (4, 6, 8; 3, 6, 9), ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI22 = (3, 7, 12; 2, 7, 13), ′ ′ ′ ′ (u31 , u32 , u33 ; u31 , u32 , u33 ) + (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI32 = (8, 10, 13; 5, 10, 16), ′ ′ ′ ′ (u31 , u32 , u33 ; u31 , u32 , u33 ) + (v31 , v32 , v33 ; v31 , v32 , v33 ) = ceI33 = (2, 3, 5; 1, 3, 6), ′ ′ ′ ′ (u41 , u42 , u43 ; u41 , u42 , u43 ) + (v11 , v12 , v13 ; v11 , v12 , v13 ) = ceI41 = (2, 4, 6; 1, 4, 7), ′ ′ ′ ′ (u41 , u42 , u43 ; u41 , u42 , u43 ) + (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI44 = (3, 4, 5; 2, 4, 8). (13) Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1163 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach The intuitionistic fuzzy solution of intuitionistic fuzzy system (13) is given as follows (see Ref. 37): ueI1 = (−6, −2, 1; −8, −2, 3), veI1 = (4, 6, 8; 3, 6, 9) ueI2 = (0, 0, 0; 0, 0, 0), veI2 = (3, 7, 12; 2, 7, 13) ueI3 = (−4, −3, 10; −8, 3, 14), veI3 = (−8, 0, 9; −13, 0, 14) ueI4 = (−6, −2, 2; −8, −2, 4), veI4 = (1, 6, 11; −2, 6, 16) (14) Now, according to Step 3 of the IFMODIM proposed by Singh and Yadav 37 , the intuitionistic fuzzy value deiIj = ueIi ⊕ veIj ⊖e cIi j for each non-basic cell is calculated as follows I =u eI1 ⊕ veI2 ⊖e de12 cI12 = (−6, −2, 1; −8, −2, 3) ⊕ (3, 7, 12; 2, 7, 13) ⊖(2, 5, 7; 1, 5, 8) = (−10, 0, 11; −14, 0, 15), I =u eI1 ⊕ veI3 ⊖e de13 cI13 = (−6, −2, 1; −8, −2, 3) ⊕ (−8, 0, 9; −13, 0, 14) ⊖(4, 6, 8; 3, 6, 9) = (−22, −8, 6; −30, −8, 14), I =u eI1 ⊕ veI4 ⊖e de14 cI14 = (−6, −2, 1; −8, −2, 3) ⊕ (1, 6, 11; −2, 6, 16) ⊖(4, 7, 8; 3, 7, 9) = (−13, −3, 8; −19, −3, 16), I =u eI2 ⊕ veI3 ⊖e de23 cI23 = (0, 0, 0; 0, 0, 0) ⊕ (−8, 0, 9; −13, 0, 14) ⊖(10, 15, 20; 8, 15, 22) = (−28, −15, −1; −35, −15, 6), I =u eI2 ⊕ veI4 ⊖e de24 cI24 = (0, 0, 0; 0, 0, 0) ⊕ (1, 6, 11; −2, 6, 16) ⊖(11, 12, 13; 10, 12, 14) = (−12, −6, 0; −16, −6, 6), I =u eI3 ⊕ veI1 ⊖e de31 cI31 = (−4, 3, 10; −8, 3, 14) ⊕ (4, 6, 8; 3, 6, 9) ⊖(3, 4, 6; 1, 4, 8) = (−6, 5, 15; −13, 5, 22), I =u eI3 ⊕ veI4 ⊖e de34 cI34 = (−4, 3, 10; −8, 3, 14) ⊕ (1, 6, 11; −2, 6, 16) ⊖(6, 10, 14; 5, 10, 15) = (−17, −1, 15; −25, −1, 25), I =u eI4 ⊕ veI2 ⊖e de42 cI42 = (−6, −2, 2; −8, −2, 4) ⊕ (3, 7, 12; 2, 7, 13) ⊖(3, 9, 10; 2, 9, 12) = (−13, −4, 11; −18, −4, 15), I =u eI4 ⊕ veI3 ⊖e de43 cI43 = (−6, −2, 2; −8, −2, 4) ⊕ (−8, 0, 9; −13, 0, 14) ⊖(3, 6, 10; 2, 6, 12) = (−24, −8, 8; −33, −8, 16). (15) To test the optimality of the improved solution-1, the rank order of the intuitionistic fuzzy value deiIj given in (13) should be obtained using the accuracy function as follows: I ) = 2 , f (deI ) = −8, f (deI ) = −20 , f (de12 13 14 8 8 I ) = −118 , f (deI ) = −46 , f (deI ) = 38 , f (de23 24 31 8 8 8 I ) = −6 , f (deI ) = −21 , f (deI ) = −65 . f (de34 42 43 8 8 8 (16) Since f (deiIj ) ̸6 0 for all non-basic cells (i, j), then the improved solution-1 is not optimal. According to the IFMODIM proposed by Singh and Yadav 37 the improved solution-2 given in Table 5 is obtained. Table 5. Improved solution-2. D1 D2 D3 D4 ai S1 S2 S3 11 1 3 10 - 8 - 11 11 11 S4 bj 1 16 10 8 11 11 12 45 5.1.4. Iteration 2(NWCM) Again it is demonstrated that the same improved solution can be obtained by our proposed approach and without solving any intuitionistic fuzzy system, without doing an arithmetic operations on the intuitionistic triangular fuzzy numbers and without any comparison of ITFNs. According to the classical modified distribution method (MODIM) applied by our proposed approach the following crisp system should be solved to test the optimality of the improved solution-1 given in Table 4: u1 + v1 = c11 = u2 + v2 = c22 = u3 + v3 = c33 = u4 + v4 = c44 = 30 8 , u2 + v1 58 8 , u3 + v2 26 8 , u4 + v1 34 8. = c21 = 6, = c32 = 82 8, = c41 = 32 8, (17) The solution of this crisp system is given as follows: u1 = −18 8 , u2 = 0, u3 = 3, u4 = −2 2 50 v1 = 6, v2 = 58 8 , v3 = 8 , v4 = 8 (18) The crisp value of di j = ui + v j − ci j for each nonbasic cell (i, j) is calculated as follows: Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1164 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach Table 7. Initial BFS by IFLCM and LCM. 58 38 2 d12 = u1 + v2 − c12 = −18 8 + 8 − 8 = 8, −18 2 48 d13 = u1 + v3 − c13 = 8 + 8 − 8 = 8, 50 52 −20 d14 = u1 + v4 − c14 = −18 8 + 8 − 8 = 8 , −118 d23 = u2 + v3 − c23 = 0 + 28 − 120 8 = 8 , 50 96 d24 = u2 + v4 − c24 = 0 + 8 − 8 = −46 8 , 34 38 d31 = u3 + v1 − c31 = 3 + 6 − 8 = 8 , 80 −6 d34 = u3 + v4 − c34 = 3 + 50 8 − 8 = 8 , 63 −21 d42 = u4 + v2 − c42 = −2 + 58 8 − 8 = 8 , 2 51 −65 d43 = u4 + v3 − c43 = −2 + 8 − 8 = 8 . (19) I e Since f (di j ) ̸6 0 for all non-basic cells (i, j), then the improved solution-1 is not optimal. According to the classical MODIM applied by our proposed approach, the improved solution-2 given in Table 5 is obtained. Thus, the results of our proposed approach are matched with those obtained based on the method proposed by Singh and Yadav 37 . However, regarding the process of finding the improved solutions, the method proposed in this study is far simpler and computationally much more efficient than the corresponding one proposed by Singh and Yadav 37 . It should be noted that in the next iteration, both our proposed method and the method proposed by Singh and Yadav 37 give the same optimal solution shown in Table 6 and the same total intuitionistic fuzzy transportation cost ZeI = (126, 204, 282; 78, 204, 352). Table 6. Optimal solution. D1 D2 D3 D4 ai S1 1 10 - - 11 S2 S3 11 3 - 8 - 11 11 S4 bj 1 16 10 8 11 11 12 45 5.2. Results based on IFLCM and LCM According to the IFLCM proposed by Singh and Yadav 37 and the classical LCM applied by our proposed approach the initial BFS given in Table 7 is obtained. D1 D2 D3 D4 ai S1 11 - - - 11 S2 S3 S4 5 10 - 8 - 1 3 7 11 11 12 bj 16 10 8 11 45 5.2.1. Iteration 1(IFLCM) According to Step 1 and Step 2 of the IFMODIM proposed by Singh and Yadav 37 the following intuitionistic fuzzy system is solved to test the optimality of the initial BFS given in Table 7: ′ ′ ′ ′ (u11 , u12 , u13 ; u11 , u12 , u13 ) + (v11 , v12 , v13 ; v11 , v12 , v13 ) = ceI11 = (2, 4, 5; 1, 4, 6), ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI22 = (3, 7, 12; 2, 7, 13), ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI24 = (11, 12, 13; 10, 12, 14), ′ ′ ′ ′ (u31 , u32 , u33 ; u31 , u32 , u33 ) + (v31 , v32 , v33 ; v31 , v32 , v33 ) = ceI33 = (2, 3, 5; 1, 3, 6), ′ ′ ′ ′ (u31 , u32 , u33 ; u31 , u32 , u33 ) + (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI34 = (6, 10, 14; 5, 10, 15), ′ ′ ′ ′ (u41 , u42 , u43 ; u41 , u42 , u43 ) + (v11 , v12 , v13 ; v11 , v12 , v13 ) = ceI41 = (2, 4, 6; 1, 4, 7), ′ ′ ′ ′ (u41 , u42 , u43 ; u41 , u42 , u43 ) + (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI44 = (3, 4, 5; 2, 4, 8). (20) The intuitionistic fuzzy solution of this system is given as follows: ′ ′ ueI1 = (u11 , u12 , u13 ; u11 , u12 , u13 ) = (−1, 4, 8; −4, 4, 13), ′ ′ veI1 = (v11 , v12 , v13 ; v11 , v12 , v13 ) = (−3, 0, 3; −7, 0, 5), ′ ′ ueI2 = (u21 , u22 , u23 ; u21 , u22 , u23 ) = (11, 12, 13; 10, 12, 14), ′ ′ I ve2 = (v21 , v22 , v23 ; v21 , v22 , v23 ) = (−10, −5, 1; −12, −5, 3), ′ ′ I ue3 = (u31 , u32 , u33 ; u31 , u32 , u33 ) = (6, 10, 14; 5, 10, 15), ′ ′ veI3 = (v31 , v32 , v33 ; v31 , v32 , v33 ) = (−12, −7, −1; −14, −7, 1), ′ ′ ueI4 = (u41 , u42 , u43 ; u41 , u42 , u43 ) = (3, 4, 5; 2, 4, 8), ′ ′ veI4 = (v41 , v42 , v43 ; v41 , v42 , v43 ) = (0, 0, 0; 0, 0, 0). (21) Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1165 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach According to Step 3 of the IFMODIM proposed by Singh and Yadav 37 , the intuitionistic fuzzy value of deiIj = ueIi ⊕ veIj ⊖e cIi j for each non-basic cell (i, j) is computed as follows: I =u eI1 ⊕ veI2 ⊖e de12 cI12 = (−1, 4, 8; −4, 4, 13)⊕ (−10, −5, 1; −12, −5, 3)⊖(2, 5, 7; 1, 5, 8) = (−18, −6, 7; −24, −6, 15), I =u eI1 ⊕ veI3 ⊖e de13 cI13 = (−1, 4, 8; −4, 4, 13)⊕ (−12, −7, −1; −14, −7, 1)⊖(4, 6, 8; 3, 6, 9) = (−21, −9, 3; −27, −9, 11), I =u eI1 ⊕ veI4 ⊖e de14 cI14 = (−1, 4, 8; −4, 4, 13)⊕ (0, 0, 0; 0, 0, 0)⊖(4, 7, 8; 3, 7, 9) = (−9, −3, 4; −13, −3, 10), I =u eI2 ⊕ veI1 ⊖e de21 cI21 = (11, 12, 13; 10, 12, 14)⊕ (−3, 0, 3; −7, 0, 5)⊖(4, 6, 8; 3, 6, 9) = (0, 6, 12; −6, 6, 16), I =u eI2 ⊕ veI3 ⊖e de23 cI23 = (11, 12, 13; 10, 12, 14)⊕ (−12, −7, −1; −14, −7, 1)⊖(10, 15, 20; 8, 15, 22) = (−21, −10, 2; −26, −10, 7), I =u eI3 ⊕ veI1 ⊖e de31 cI31 = (6, 10, 14; 5, 10, 15)⊕ (−3, 0, 3; −7, 0, 5)⊖(3, 4, 6; 1, 4, 8) = (−3, 6, 14; −10, 6, 19), I =u eI3 ⊕ veI2 ⊖e de32 cI32 = (6, 10, 14; 5, 10, 15)⊕ (−10, −5, 1; −12, −5, 3)⊖(8, 10, 13; 5, 10, 16) = (−17, −5, 7; −23, −5, 13), I =u eI4 ⊕ veI2 ⊖e de42 cI42 = (3, 4, 5; 2, 4, 8)⊕ (−10, −5, 1; −12, −5, 3)⊖(3, 9, 10; 2, 9, 12) = (−17, −10, 3; −22, −10, 9), I =u eI4 ⊕ veI3 ⊖e de43 cI43 = (3, 4, 5; 2, 4, 8)⊕ (−12, −7, −1; −14, −7, 1)⊖(3, 6, 10; 2, 6, 12) = (−19, −9, 1; −24, −9, 7). (22) Now, the rank order of the intuitionistic fuzzy value of deiIj given in (22) should be computed to test the optimality of the initial BFS shown in Table 7. So, we have I )= f (de12 I )= f (de21 I )= f (de32 −70 −20 −44 eI eI 8 , f (d13 ) = 8 , f (d14 ) = 8 , 46 −78 44 eI eI 8 , f (d24 ) = 8 ,, f (d31 ) = 8 , −40 −67 −71 I I e e 8 , f (d42 ) = 8 , f (d43 ) = 8 . (23) Since f (deiIj ) ̸6 0 for all non-basic cells (i, j), then the current BFS is not optimal. Thus, a non-basic cell with the most positive f (deiIj ), i.e., x21 is selected as the entering variable. According to the classical transportation algorithm, x24 is selected as the leaving variable and the new BFS is found as given in Table 8. Table 8. The improved BFS-1. D1 D2 D3 D4 ai S1 11 - - - 11 S2 S3 S4 1 4 10 - 8 - 3 8 11 11 12 bj 16 10 8 11 45 5.2.2. Iteration 1(LCM) According to the classical modified distribution method (MODIM) applied by our proposed approach the following crisp system is solved to test the optimality of the initial BFS given in Table 7: u1 + v1 = c11 = u2 + v4 = c24 = u3 + v4 = c34 = u4 + v4 = c44 = 30 8 , u2 + v2 96 8 , u3 + v3 80 8 , u4 + v1 34 8. = c22 = = c33 = = c41 = 58 8, 26 8, 32 8, (24) The solution of this crisp system is given as follows: u1 = 32 8 , u2 = −2 v1 = 8 , v2 = 96 80 34 8 , u3 = 8 , u4 = 8 , −38 −54 8 , v3 = 8 , v4 = 0. (25) To test the optimality of the solution given in Table 7 it is required to compute di j = ui + v j − ci j for each non-basic cell(i, j). Thus, we have: d12 = u1 + v2 − c12 = d13 = u1 + v3 − c13 = d14 = u1 + v4 − c14 = d21 = u2 + v1 − c21 = d23 = u2 + v3 − c23 = d31 = u3 + v1 − c31 = d32 = u3 + v2 − c32 = d42 = u4 + v2 − c42 = d43 = u4 + v3 − c43 = Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1166 32 8 32 8 32 8 96 8 96 8 80 8 80 8 34 8 34 8 38 −44 + −38 8 − 8 = 8 , 48 −70 + −54 8 − 8 = 8 , 52 −20 +0 − 8 = 8 , 48 46 + −2 8 − 8 = 8 , −54 120 + 8 − 8 = −78 8 , −2 34 44 + 8 − 8 = 8, 82 −40 + −38 8 − 8 = 8 , −38 63 −67 + 8 − 8 = 8 , 51 −71 + −54 8 − 8 = 8 . (26) A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach Table 9. A real life intuitionistic FTP. D1 D2 D3 D4 ai S1 (210,250,270;200,250,280) (600,700,750;600,700,800) (950,1000,1050;900,1000,1100) (3500, 3700, 3900;3400, 3700,4100) 4500 S2 (650,750, 800;600,750,850) (350,400,450;340,400,480) (1000,1050,1100;950,1050,1150) (3600, 3900, 4600;3500, 3900,4600) 3500 S3 (2600,2800,3000; 2500,2800,3100) (2100,2200,2300;2100,2200,2350) (2900,3100,3300;2800,3100,3400) (5400, 5600, 5800;5300, 5600,6000) 2000 bj 3500 3000 2000 1500 10000 Since di j ̸6 0 for all non-basic cells (i, j), then the current BFS is not optimal. Thus, according to the classical transportation algorithm in crisp environment, x21 and x24 are selected as the entering variable and the leaving variable, respectively and thus the new BFS is found as given in Table 8 matching with the result of the improved solution-1 of themethod proposed by Singh and Yadav 37 . However, to find the improved solution-1 given in Table 8 it is necessary to solve the intuitionistic fuzzy system (20), to calculate the intuitionistic fuzzy value deiIj = ueIi ⊕ veIj ⊖e cIi j for each non-basic cell according to (22) and to compare the intuitionistic fuzzy value deiIj for each non-basic cell as done in (23). It can be seen that these steps require a large number of intuitionistic fuzzy additions, intuitionistic fuzzy subtractions and comparison on TIFNs. However, based on our proposed approach, these steps are done with solving crisp system (24) and using the elementary operations and comparison on real numbers. These results confirm that our proposed approach is more effective than the method proposed by Singh and Yadav 37 from the computational point of view. It is worthwhile to note that on using IFMODIM after three more iterations, the optimal solution given in Table 5 is obtained. This means that to get the optimal solution of the IFBTP given in Table 1 by the help of initial BFS obtained based on IFLCM it is required to solve three more intuitionistic fuzzy systems similar to (20), to calculate the intuitioniscIi j for each non-basic tic fuzzy value deiIj = ueIi ⊕ veIj ⊖e cell similar to (22) three more times and to compare the intuitionistic fuzzy value deiIj for each non-basic cell three more times. The same optimal solution is found easily using our proposed approach and according to classical transportation algorithms. A similar discussion can be done by comparing the results obtained from using IFMODIM proposed by Singh and Yadav 37 on initial BFS found by IFVAM and MODIM applied by our proposed approach on initial BFS found by VAM. In the next example a real life intuitionistic fuzzy TP given in Ref. 37 is solved and the results obtained are discussed and compared in details. Example 5.2: The data shown in Table 9 are collected from a trader of Chandigarh, India, which supplies the commodity TMT (Thermo mechanically treated) steel from three plants S1 , S2 and S3 to four different companies D1 , D2 , D3 and D4 . The trader is certain about the availabilities and demands of the materials, but (s)he is uncertain about the transportation cost from different sources to different destinations due to some uncontrollable factors such as weather in Hilly areas. In this kind of situation, the usual way is to obtain the triangular intuitionistic fuzzy numbers after a thorough discussion based upon past experience or expert advice. The aim is to determine the optimal transportation of products so that the total intuitionistic fuzzy transportation cost is minimized. According to the accuracy function given in (1), the rank order of each intuitionistic fuzzy transportation cost (given in Table 9) is substituted by the corresponding intuitionistic fuzzy numbers to obtain the classical TP. The results are given in Table 10. Table 10. A real life crisp TP. D1 D2 D3 D4 ai S1 S2 245 737.5 693.75 402.5 1000 1050 3712.5 3987.5 4500 3500 S3 bj 2800 3500 2206.25 3000 3100 2000 5612.5 1500 2000 10000 Now, we obtain the initial BFS for the problems given in Table 9 and Table 10 according to IFVAM and VAM, respectively and explore the obtained results. Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1167 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach Table 11. First assignment in IFVAM. D1 D2 D3 D4 RI Pei S1 (210,250,270;200,250,280) (600,700,750;600,700,800) (950,1000,1050;900,1000,1100) (3500, 3700, 3900;3400, 3700,4100) (330,450,540;320,450,600) S2 (650,750, 800;600,750,850) (350,400,450;340,400,480) (1000,1050,1100;950,1050,1150) (3600, 3900, 4600;3500, 3900,4600) (200,350,450;120,350,510) CI Pej (380,500,590;320,500,650) (150,300,400;120,300,460) (-50,50,150;-150,50,250) (-300,200,1100;-600,200,1200) Table 12. Second assignment in IFVAM. D2 D3 D4 RI Pei S1 (600,700,750;600,700,800) (950,1000,1050;900,1000,1100) (3500, 3700, 3900;3400, 3700,4100) (330,450,540;320,450,600) S2 (350,400,450;340,400,480) (1000,1050,1100;950,1050,1150) (3600, 3900, 4600;3500, 3900,4600) (200,350,450;120,350,510) CI Pej (150,300,400;120,300,460) (-50,50,150;-150,50,250) (-300,200,1100;-600,200,1200) 5.3. Initial BFS based on IFVAM and VAM According to Step (1) of the IFVAM proposed by Singh and Yadav 37 , it is required to compute the intuitionistic penalty regarding to Remark 1 for each row and each column of Table 9. The row intuitionistic penalty (RIP) and the column intuitionistic penalty (CIP) of Table 9 are obtained as follows: cI11 =(600, 700, 750; 600, 700, 800)⊖ RI Pe1I = ceI12 ⊖e (210, 250, 270;200, 250, 280) = (330, 450, 540; 320, 450, 600) cI22 =(650, 750, 800;600, 750, 850)⊖ RI Pe2I = ceI21 ⊖e (350, 400, 450; 340, 400, 480) = (200, 350, 450; 120, 350, 510) cI32 = (2600, 2800, 3000; 2500, 2800, 3100)⊖ RI Pe3I = ceI31 ⊖e (2100, 2200, 2300; 2100, 2200, 2350) = (300, 600, 900; 150, 600, 1000), cI11 = (650, 750, 800;600, 750, 850)⊖ CI Pe1I = ceI21 ⊖e (210, 250, 270; 200, 250, 280) = (380, 500, 590; 320, 500, 650), cI22 = (600, 700, 750; 600, 700, 800)⊖ CI Pe2I = ceI12 ⊖e (350, 400, 450; 340, 400, 480) = (150, 300, 400; 120, 300, 460), cI13 = (1000, 1050, 1100; 950, 1050, 1150)⊖ CI Pe3I = ceI23 ⊖e (950, 1000, 1050; 900, 1000, 1100) = (−50, 50, 150; −150, 50, 250), CI Pe4I = ceI24 ⊖e cI14 = (3600, 3900, 4600; 3500, 3900, 4600)⊖ (3500, 3700, 3900; 3400, 3700, 4100) = (−300, 200, 1100; −600, 200, 1200). Because row 3 has the highest intuitionistic penalty based on the accuracy function, and the cell (3, 2) has the smallest intuitionistic cost in this row, the amount 2000 is assigned to x32 = 2000. Now, row 3 is satisfied and should be deleted. The reduced table is given as Table 11. Now, in a similar way the new intuitionistic penalties are recomputed as in Table 11. Now according to the accuracy function, column 1 in Table 11 has the highest intuitionistic penalty and cell (1, 1) has the smallest intuitionistic cost in this column. Thus, the amount 3500 is assigned to x11 = 3500. In this case, column 1 is satisfied and should be deleted. The reduced table and new computed intuitionistic penalties are given as Table 12. This process is continued and after four more iterations the initial BFS given in Table 13 is found. Table 13. Initial BFS by IFVAM. D1 D2 D3 D4 ai S1 S2 3500 − − 1000 − 2000 1000 500 4500 3500 S3 bj − 3500 2000 3000 − 2000 − 1500 2000 10000 It should be noted that the same initial BFS (given in Table 13) can be obtained for Table 10 by Vogel’s approximation method (VAM) corresponding to our proposed method without doing any arithmetic operations on the intuitionistic triangular fuzzy numbers and without any comparison of ITFNs. This confirms that our proposed method needs less computational effort for finding initial BFS compared to the method proposed by Singh and Yadav 37 . In what follows, it is shown that the same result can be concluded for finding the optimal solution. 5.4. Optimal solution based on IFMODIM According to Step 1 and Step 2 of the IFMODIM given in Ref. 37 the following intuitionistic fuzzy Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1168 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach system is solved to test the optimality of the initial BFS given in Table 13: ′ ′ ′ ′ (u11 , u12 , u13 ; u11 , u12 , u13 ) + (v11 , v12 , v13 ; v11 , v12 , v13 ) = ceI11 = (210, 250, 270; 200, 250, 280), ′ ′ ′ ′ (u11 , u12 , u13 ; u11 , u12 , u13 ) + (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI14 = (3500, 3700, 3900; 3400, 3700, 4100), ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI22 = (350, 400, 450; 340, 400, 480), ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v31 , v32 , v33 ; v31 , v32 , v33 ) = ceI23 = (1000, 1050, 1100; 950, 1050, 1150), ′ ′ ′ ′ (u21 , u22 , u23 ; u21 , u22 , u23 ) + (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI24 = (3600, 3900, 4600; 3500, 3900, 4600), ′ ′ ′ ′ (u31 , u32 , u33 ; u31 , u32 , u33 ) + (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI32 = (2100, 2200, 2300; 2100, 2200, 2350) (27) The intuitionistic fuzzy solution of this system is given as follows: 1′ Now, the rank order of the intuitionistic fuzzy value of deiIj given in (29) should be computed to test the optimality of the initial BFS shown in Table 13. So, we have I ) = −566.25, f (deI ) = −225, f (de12 13 I ) = −217.5, f (deI ) = −476.25, f (de21 31 I ) = −246.25, f (deI ) = 178.75. f (de33 34 I ) = 178.75 ̸6 0, then the current BFS is Since f (de34 not optimal and x34 is selected as the entering variable. According to Step 5 of IFMODIM proposed by Singh and Yadav 37 , x24 is selected as the leaving variable and the new BFS is found as given in Table 14. Table 14. Improved BFS-1 (optimal solution). 1′ ueI1 = (u11 , u12 , u13 ; u1 , u12 , u3 ) = (−1100, −200, 300; −1200, −200, 600), ′ ′ veI1 = (v11 , v12 , v13 ; v11 , v12 , v13 ) = (−90, 450, 1370; −400, 450, 1480), ′ ′ ueI2 = (u21 , u22 , u23 ; u12 , u22 , u23 ) = (0, 0, 0; 0, 0, 0), ′ ′ veI2 = (v21 , v22 , v23 ; v21 , v22 , v23 ) = (350, 400, 450; 340, 400, 480) ′ ′ I 3 ue3 = (u1 , u32 , u33 ; u31 , u32 , u33 ) = (1650, 1800, 1950; 1620, 1800, 2010), ′ ′ I 3 ve3 = (v1 , v32 , v33 ; v31 , v32 , v33 ) = (1000, 1050, 1100; 950, 1050, 1150), ′ ′ I 4 ve4 = (v1 , v42 , v43 ; v41 , v42 , v43 ) = (3600, 3900, 4600; 3500, 3900, 4600). (28) According to Step 3 of the IFMODIM given in Ref. 37, the intuitionistic fuzzy value of deiIj = ueIi ⊕ cIi j for each non-basic cell (i, j) is computed as veIj ⊖e follows: I = (−1500, −500, 150; −1660, −500, 480), de12 I de13 = (−1150, −150, 450; −1350, −150, 850), I = (−890, −300, 720; −1250, −300, 880), de21 I = (−1440, −550, 720; −1880, −550, 990), de31 I = (−650, −250, 150; −830, −250, 360) de33 I = (−550, 100, 1150; −880, 100, 1310). de34 (29) (30) D1 D2 D3 D4 ai S1 S2 3500 − − 1500 − 2000 1000 − 4500 3500 S3 bj − 3500 1500 3000 − 2000 500 1500 2000 10000 According to Step 1 and Step 2 of the IFMODIM given in Ref. 37, the following intuitionistic fuzzy system is solved to test the optimality of the improved solution-1 given in Table 14: ′ ′ ′ ′ (u11 , u12 , u13 ; u11 , u12 , u13 ) ⊕ (v11 , v12 , v13 ; v11 , v12 , v13 ) = ceI11 = (210, 250, 270; 200, 250, 280), ′ ′ ′ ′ 1 1 1 (u1 , u2 , u3 ; u11 , u12 , u13 ) ⊕ (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI14 = (3500, 3700, 3900; 3400, 3700, 4100), ′ ′ ′ ′ 2 2 2 (u1 , u2 , u3 ; u21 , u22 , u23 ) ⊕ (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI22 = (350, 400, 450; 340, 400, 480), ′ ′ ′ ′ 2 2 2 (u1 , u2 , u3 ; u21 , u22 , u23 ) ⊕ (v31 , v32 , v33 ; v31 , v32 , v33 ) = ceI23 = (1000, 1050, 1100; 950, 1050, 1150), ′ ′ ′ ′ 3 3 3 (u1 , u2 , u3 ; u31 , u32 , u33 ) ⊕ (v21 , v22 , v23 ; v21 , v22 , v23 ) = ceI32 = (2100, 2200, 2300; 2100, 2200, 2350), ′ ′ ′ ′ (u31 , u32 , u33 ; u31 , u32 , u33 ) ⊕ (v41 , v42 , v43 ; v41 , v42 , v43 ) = ceI34 = (5400, 5600, 5800; 5300, 5600, 6000). (31) The intuitionistic fuzzy solution of this system is Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1169 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach This means that the optimal solution is given by given as follows: ′ ′ ueI1 = (u11 , u12 , u13 ; u11 , u12 , u13 ) = (−650, −100, 450; −980, −100, 810), ′ ′ veI1 = (v11 , v12 , v13 ; v11 , v12 , v13 ) = (−240, 350, 920; −610, 350, 1260), ′ ′ ueI2 = (u21 , u22 , u23 ; u21 , u22 , u23 ) = (0, 0, 0; 0, 0, 0), ′ ′ veI2 = (v21 , v22 , v23 ; v21 , v22 , v23 ) = (350, 400, 450; 340, 400, 480) ′ ′ ueI3 = (u31 , u32 , u33 ; u31 , u32 , u33 ) = (1650, 1800, 1950; 1620, 1800, 2010), ′ ′ veI3 = (v31 , v32 , v33 ; v31 , v32 , v33 ) = (1000, 1050, 1100; 950, 1050, 1150), ′ ′ veI4 = (v41 , v42 , v43 ; v41 , v42 , v43 ) = (3450, 3800, 4150; 3290, 3800, 4380). (32) According to Step 3 of the IFMODIM proposed by Singh and Yadav 37 , the intuitionistic fuzzy value of deiIj = ueIi ⊕ veIj ⊖e cIi j for each non-basic cell (i, j) is computed as follows: I =u eI1 ⊕ veI2 ⊖e de12 cI12 = (−1050, −400, 300; −1440, −400, 690), I =u eI1 ⊕ veI3 ⊖e de13 cI13 = (−700, −50, 600; −1130, −50, 1060), I =u eI2 ⊕ veI1 ⊖e de21 cI21 = (−1040, −400, 270; −1460, −400, 660), I =u eI2 ⊕ veI4 ⊖e de24 cI24 = (−1150, −100, 550; −1310, −100, 880), I =u eI3 ⊕ veI1 ⊖e de31 cI31 = (−1590, −650, 270; −2090, −650, 770), I =u eI3 ⊕ veI3 ⊖e de33 cI33 = (−650, −250, 150; −830, −250, 360). (33) Now, the rank order of the intuitionistic fuzzy value of deiIj given in (33) is obtained to test the optimality of the improved solution-1 shown in Table 14 as follows: I ) = −387.5, f (deI ) = −46.25, f (de12 13 I ) = −396.25, f (deI ) = −178.75, f (de21 24 I ) = −655, f (deI ) = −246.25. f (de31 33 (34) Since f (deiIj ) 6 0 for all non-basic cells (i, j), the improved solution-1, shown in Table 14 is optimal. x11 = 3500, x14 = 1000,x22 = 1500, x23 = 2000,x32 = 1500, x34 = 500. (35) Putting the values of the optimal solution (35) in the objective function of the intuitionistic fuzzy TP in Table 9, the total intuitionistic fuzzy transportation cost achieved is: 3 4 I ZeOPI = ∑ ∑ ceIi j xi j = i=1 j=1 (12610000, 13375000, 14070000; 12310000, 13375000, 14625000) (36) It is worthwhile to note that the optimal solution and total intuitionistic fuzzy transportation cost of the real life intuitionistic fuzzy TP (Table 9) obtained by the method proposed by Singh and Yadav 37 , are as follows: Optimal Solution of the method given in Ref. 37 : x11 = 3500, x13 = 1000, x22 = 2500, x23 = 1000, x32 = 500, x34 = 1500. (37) I ZeOPI−existing = method proposed by Singh and Yadav 37 : (12710000, 13425000, 14070000; 12400000, 13425000, 14605000) (38) By comparing the intuitionistic fuzzy objective value (36) with the intuitionistic fuzzy objective value given in (38), we conclude that the solution obtained by Singh and Yadav 37 is not the optimal solution. If fact, we have I ZeOPI = (12610000, 13375000, 14070000; 12310000, 13375000, 14625000) ≺ I e ZOPI−method proposed by Singh and Yadav37 = (12710000, 13425000, 14070000; 12400000, 13425000, 14605000) 5.5. Optimal solution based on MODIM Here, it is shown that the same optimal solution can be found for the real life intuitionistic fuzzy TP (Table 9) according to our proposed approach and using traditional MODIM on initial BFS given in Table 13. Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1170 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach According to classical MODIM applied by our proposed approach, the following crisp system is solved to test the optimality of the initial BFS given in Table 13: u1 + v1 = c11 = 245, u1 + v4 = c24 = 3712.5, u2 + v2 = c22 = 402.5, u2 + v3 = c23 = 1050, u2 + v4 = c24 = 3987.5, u3 + v2 = c32 = 2206.25. (39) The solution of this crisp system is given as follows: u1 = −275, u2 = 0, u3 = 1803.75, v1 = 520, v2 = 402.5, v3 = 1050, v4 = 3987.5. (40) To test the optimality of the solution given in Table 13, it is required to compute di j = ui + v j − ci j for each non-basic cell (i, j). Thus, we have: d12 = u1 + v2 − c12 = −275 + 402.5 − 693.75 = −566.25, d13 = u1 + v3 − c13 = −275 + 1050 − 1000 = −225, d21 = u2 + v1 − c21 = 0 + 520 − 737.5 = −217.5, d31 = u3 + v1 − c31 = 1803.75 + 520 − 2800 = −476.25, d33 = u3 + v3 − c33 = 1803.75 + 1050 − 3100 = −246.25, d34 = u3 + v4 − c34 = 1803.75 + 3987.5 − 5612.5 = 178.75. (41) Since d34 = 178.75 ̸6 0, then the current BFS is not optimal. Thus, according to the classical transportation algorithm in crisp environment, x34 and x24 are selected as the entering variable and the leaving variable, respectively, and thus the new solution is found as given in Table 14 matching with the result of the improved solution-1 of the method proposed by Singh and Yadav 37 . Now, the following crisp system is solved to test the optimality of the improved solution-1 given in Table 14: u1 + v1 = c11 = 245, u1 + v4 = c24 = 3712.5, u2 + v2 = c22 = 402.5, u2 + v3 = c23 = 1050, u3 + v2 = c32 = 2206.25, u3 + v4 = c34 = 5612.5. (42) The intuitionistic fuzzy solution of this system is given as follows: u1 = −96.25, u2 = 0, u3 = 1803.75, v1 = 341.25, v2 = 402.5, v3 = 1050, v4 = 3808.75. (43) The value of di j = ui + v j − ci j for each non-basic cell (i, j) is obtained as follows: d12 = u1 + v2 − c12 = −387.5, d13 = u1 + v3 − c13 = −46.25, d21 = u2 + v1 − c21 = −396.25, d24 = u2 + v4 − c24 = −178.75, d31 = u3 + v1 − c31 = −655, d33 = u3 + v3 − c33 = −246.25. (44) Thus, the improved-solution 1 (Table 14) is optimal. This means that our proposed method and the method proposed by Singh and Yadav 37 have the same results. However, the method proposed by Singh and Yadav 37 requires a large number of intuitionistic fuzzy additions, intuitionistic fuzzy subtractions and comparison on TIFNs. While based on our proposed approach, all elementary operations and comparison are done on real numbers. These results confirm that our proposed approach should be preferred to the method proposed by Singh and Yadav 37 in terms of the computational point of view. 6. Conclusions and future work In this paper, a TP having uncertainty as well as hesitation in prediction of the transportation cost has been investigated. In the TP considered in this study, the values of transportation costs are represented by triangular intuitionistic fuzzy numbers and the values of supply and demand of the products are represented by real numbers. Here, we proposed an efficient computational solution approach for solving intuitionistic fuzzy TP based on classical transportation algorithms. In contrast to the method proposed by Singh and Yadav 37 where all elementary operations and comparisons are done on triangular intuitionistic fuzzy numbers, in the proposed algorithm in this paper all arithmetic calculations and comparisons are performed on crisp numbers. Therefore, Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1171 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach the complexity of computation is reduced very much compared to the method proposed by Singh and Yadav 37 . Here, we shall point out that the IFTP studied in this paper is not in the form of a problem whose demands and supplies are as triangular intuitionistic fuzzy numbers too. Therefore, further research on extending the proposed method to overcome these shortcomings is an interesting stream of future research. We shall report the significant results of these ongoing projects in the near future by extending the proposed methods by Ebrahimnjead 38,39 utilized for solving fully FTP and interval-valued FTP. 8. 9. 10. 11. Acknowledgments Research supported in part under grant TIN201455024-P from the Spanish Govern and TIC8001 from the Andalusian Govern, both including FEDER funds. The authors would like to thank the anonymous reviewers and the editor for their insightful comments and suggestions. The first author would also like to thank the office of Vice Chancellor for Research and Technology at Islamic Azad University, Qaemshahr Branch, for the financial support. 12. 13. 14. 15. References 16. 1. L. A. Zadeh, Fuzzy sets, Information and Computation. 8 (3) (1965) 338–353. 2. K. T. Atanassov, Intuitionistic fuzzy sets, Fuzzy Sets and Systems. 20 (1) (1986) 87–96. 3. D-F. Li, Multiattribute decision making models and methods using intuitionistic fuzzy sets, Journal of Computer and System Sciences. 70 (1) (2005) 73–85. 4. D-F. Li, Linear programming method for MADM with interval-valued intuitionistic fuzzy sets, Expert Systems with Applications. 37 (8) (2010) 5939–5945. 5. D-F. Li, Y-C. Wang, S. Liu and F. Shen, Fractional programming methodology for multi-attribute group decision-making using IFS, Applied Soft Computing. 9 (8) (2009) 219–225. 6. D-F. Li, G-H. Chen and Z-H. Huang, Linear programming method for multiattribute group decision making using IF sets, Information Sciences. 180 (9) (2010) 1591–1609. 7. H. Bustince, E. Barrenechea, M. Pagola, and R. Orduna, Image thresholding computation using Atanassov’s intuitionistic fuzzy sets, Journal of Ad- 17. 18. 19. 20. 21. vanced Computational Intelligence and Intelligent Informatics. 11 2) (2007) 187–194. S. K. De, R. Biswas, and A. R. Roy, An application of intuitionistic fuzzy sets in medical diagnoses, Fuzzy Sets and Systems. 117 (2) (2001) 209–213. J-X, Nan, D-F. Li, and M-J. Zhang, A lexicographic method for matrix games with payoffs of triangular intuitionistic fuzzy numbers, International Journal of Computational Intelligence Systems. 3 (2) (2010) 280–289. J-X, Nan and D-F. Li,. Linear programming approach to matrix games with intuitionistic fuzzy goals, International Journal of Computational Intelligence Systems. 6 (1) (2013) 186–197. D-F. Li and C. Cheng, New similarity measures of intuitionistic fuzzy sets and application to pattern recognitions, Pattern Recognition Letters. 23 (1–3) (2002) 221–225. J. L. Verdegay, A dual approach to solve the fuzzy linear programming problem, Fuzzy Sets and Systems. 14 (2) (1984) 131–141. E. Cascetta, M. Gallo and B. Montella, Models and algorithms for the optimization of signal settings on urban networks with stochastic assignment models, Annals of Operations Research. 144 (1) (2006) 301–328. K. Ganesan and P. Veeramani, Fuzzy linear programs with trapezoidal fuzzy numbers, Annals of Operations Research. 143 (1) (2006) 305–315. M. Kumar, S. P. Yadav and S. Kumar, A new approach for analyzing the fuzzy system reliability using intuitionistic fuzzy number, International Journal of Industrial and System Engineering. 8 (2) (2011) 135– 156. S. K. Sing and S. P. Yadav, Modeling and optimization of multi objective non-linear programming problem in intuitionistic fuzzy environment, Applied Mathematical Modelling. 29 (16) (2015) 4617–4629. S. K. Sing and S. P. Yadav, Intuitionistic fuzzy non linear programming problems: modeling and optimization in manufacturing systems, Journal of Intelligent and Fuzzy Systems. 28 (3) (2015) 1421–1433. A. Ebrahimnejad, A duality approach for solving bounded linear programming with fuzzy variables based on ranking functions and its application in transportation problems, International Journal of Systems Science. 46 (11) (2015) 2048–2060. F. Jimenez and J. L. Verdegay, Uncertain solid transportation problem, Fuzzy Sets and Systems. 100 (1–3) (1998) 45–57. F. Jimenez and J. L. Verdegay, Solving fuzzy solid transportation problems by an evolutionary algorithm based parametric approach, European Journal of Operational Research. 117 (3) (1999) 485–510. A.Gani and K. A. Razak, Two stage fuzzy transportation problem, Journal of Physical Sciences, 10 (1) Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1172 A. Ebrahimnejad and J.L. Verdegay / An efficient computational approach (2006) 63–69. 22. D. S. Dinagar and K. Palanivel, The transportation problem in fuzzy environment, International Journal of Algorithms, Computing and Mathematics. 2 (1) (2006) 65–71. 23. P. Pandian and G. Natarajan, A new algorithm for finding a fuzzy optimal solution for fuzzy transportation problems, Applied Mathematical Sciences. 4 (2) (2010) 79–90. 24. H. Basirzadeh, An approach for solving fuzzy transportation problem, Applied Mathematical Sciences. 5 (32) (2011) 1549–1566. 25. A. Gupta and A. Kumar, A new method for solving linear multi-objective transportation problems with fuzzy parameters, Applied Mathematical Modelling. 36 (4) (2012) 1421–1430. 26. M. Shanmugasundari and K. Ganesan, A novel approach for the fuzzy optimal solution of fuzzy transportation problem, International Journal of Engineering Research and Applications. 3 (1) (2013) 1416– 1421. 27. A. Kaur and A. Kumar, A new method for solving fuzzy transportation problems using ranking function, Applied Mathematical Modelling. 35 (12) (2011) 5652–5661. 28. A. Kaur and A. Kumar, A new approach for solving fuzzy transportation problems using generalized trapezoidal fuzzy numbers, Applied Soft Computing. 12 (3) (2012) 1201–1213. 29. A. Ebrahimnejad, A simplified new approach for solving fuzzy transportation problems with generalized trapezoidal fuzzy numbers, Applied Soft Computing. 19 (2014) 171–176. 30. S. Sudhagar and K. Ganesan, A fuzzy approach to transport optimization problem, Optimization and Engineering. (2012) DOI: 10.1007/s11081–012–9202– 6. 31. A. Ebrahimnejad, Note on A fuzzy approach to transport optimization problem, Optimization and Engineering. (2015) DOI: 10.1007/s11081–015–9277–y. 32. A. Ebrahimnejad, An improved approach for solving fuzzy transportation problem with triangular fuzzy numbers, Journal of Intelligent and Fuzzy Systems. 29 (2) (2015) 963–974. 33. R. J. Hussain and P. S. Kumar, Algorithmic approach for solving intuitionistic fuzzy transportation problem, Applied Mathematical Sciences. 6 (80) (2012) 3981– 3989. 34. A. Nagoorgani and S. Abbas, A new method for solving intuitionistic fuzzy transportation problem,Applied Mathematical Science. 7 (28) (2013) 1357–1365. 35. S. K. Singh and S. P. Yadav, Efficient approach for solving type-1 intuitionistic fuzzy transportation problem, International Journal of System Assurance Engineering and Management. 6 (3) (2015) 259–267. 36. R. J. P. Antony, S. J. Savarimuthu and T. Pathinathan, Method for solving the transportation problem using triangular intuitionistic fuzzy number, International Journal of Computing Algorithm. 3 (1) (2014) 590– 605. 37. S. K. Singh and S. P. Yadav, A new approach for solving intuitionistic fuzzy transportation problem of type2, Annals of Operations Research. 43 (1) (2016) 349– 363. 38. A. Ebrahimnejad, New method for solving fuzzy transportation problem with LR flat fuzzy numbers. Information Sciences. 37 (2016) 108–124. 39. A. Ebrahimnejad, Fuzzy linear programming approach for solving transportation problems with interval-valued trapezoidal fuzzy numbers, Sadhana. 41 (3) (2016) 299–316. Co-published by Atlantis Press and Taylor & Francis Copyright: the authors 1173
https://openalex.org/W4295926449	https://air.unimi.it/bitstream/2434/938170/2/vetsci-09-00505.pdf	English	null	Critically Appraised Topic on Low-Level Laser Therapy (LLLT) in Dogs: An Advisable Treatment for Skin Diseases?	Veterinary sciences	2,022	cc-by	10,790	Citation: Perego, R.; Mazzeo, M.; Spada, E.; Proverbio, D. Critically Appraised Topic on Low-Level Laser Therapy (LLLT) in Dogs: An Advisable Treatment for Skin Diseases? Vet. Sci. 2022, 9, 505. https://doi.org/10.3390/ vetsci9090505 Academic Editor: Justina Prada Received: 10 August 2022 Accepted: 12 September 2022 Published: 14 September 2022 Citation: Perego, R.; Mazzeo, M.; Spada, E.; Proverbio, D. Critically Appraised Topic on Low-Level Laser Therapy (LLLT) in Dogs: An Advisable Treatment for Skin Diseases? Vet. Sci. 2022, 9, 505. https://doi.org/10.3390/ vetsci9090505 Keywords: photobiomodulation; low-level laser therapy; ﬂuorescence biomodulation; photodynamic therapy; therapeutic laser; dermatology; dogs Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Roberta Perego * , Martina Mazzeo, Eva Spada * and Daniela Proverbio Department of Veterinary Medicine and Animal Sciences (DIVAS), University of Milan, 26900 Lodi, Italy * Correspondence: roberta.perego@unimi.it (R.P.); eva.spada@unimi.it (E.S.) Simple Summary: Low-level laser therapy (LLLT) is a therapeutic technique with reported regenera- tive, anti-inﬂammatory, antibacterial, and analgesic effects. In the last few years, LLLT has been used in dogs for the management of different skin lesions and diseases. This study reports a literature review using the critically appraised topic (CAT) method to determine the canine skin diseases for which LLLT is an advisable treatment. Only primary clinical prospective studies were considered. A meticulous literature search revealed 19 signiﬁcant clinical trials, and these were critically analyzed. The evaluation of the best accessible evidence in July 2022 suggests that LLLT can be a promising and effective adjunctive treatment in combination with systemic antibiotic therapy for canine interdigital pyoderma and canine deep pyoderma. Furthermore, the use of LLLT is not recommended as a therapy for pedal pruritus secondary to canine atopic dermatitis. In other canine skin diseases, there is a possible helpful effect of LLLT; however, the evidence for its use is not currently convincing. Abstract: Low-level laser therapy (LLLT) is a therapeutic option that stimulates cellular function through intracellular photobiological and photochemical reactions, promoting better tissue repair and an anti-inﬂammatory, antibacterial, and analgesic effect. Previous studies in human and veterinary medicine have shown the clinical efﬁcacy of LLLT in many ﬁelds. In this study, the literature was reviewed using the critically appraised topic (CAT) method to determine the canine skin diseases for which LLLT is an advisable treatment. A meticulous literature search revealed 19 signiﬁcant clinical trials, which were critically analyzed. The evaluation of the best accessible evidence in July 2022 suggests that ﬂuorescence biomodulation (FBM), a type of LLLT, can, in combination with systemic antibiotic therapy, be a promising and effective adjunctive treatment for canine interdigital pyoderma and canine deep pyoderma. Furthermore, the evidence suggests that the use of LLLT is not recommended as a therapy for pedal pruritus secondary to canine atopic dermatitis. For other canine skin diseases included in the CAT, although LLLT appears to be a promising treatment, there is not yet good scientiﬁc evidence to recommend its use. veterinary sciences veterinary sciences Article Critically Appraised Topic on Low-Level Laser Therapy (LLLT) in Dogs: An Advisable Treatment for Skin Diseases? Roberta Perego * , Martina Mazzeo, Eva Spada * and Daniela Proverbio veterinary sciences veterinary sciences veterinary sciences 1. Introduction Low-level laser therapy (LLLT) is a noninvasive, easy-to-apply therapeutic option, with minimal side-effects. It uses photons at diverse wavelengths via a nonthermal mecha- nism to affect biological activity [1]. The use of LLLT is increasing in human and veterinary medicine; LLLT has been studied in a number of species, and a variety of clinical uses in veterinary medicine have recently been reviewed [2–4]. However, to date the precise biochemical mechanism of LLLT is not totally understood [1]. LLLT does not exploit thermal or ablative mechanisms but instead stimulates cellular function. The photons emitted by the laser or LED are absorbed by the mitochondrial chromophores (particularly cytochrome c-oxidase) or by the chromophores contained in the photoconverting substrate (applied prior to exposure to the light source), stimulating oxidative phosphorylation to Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/vetsci Vet. Sci. 2022, 9, 505. https://doi.org/10.3390/vetsci9090505 Vet. Sci. 2022, 9, 505 2 of 16 increase ATP production and reduce oxidative stress [5,6]. These effects improve tissue repair, while having anti-inﬂammatory, anti-edema, antibacterial, and analgesic activity [7]. Different techniques are included under the term LLLT, such as photobiomodulation (PBM), ﬂuorescence biomodulation (FBM), and photodynamic therapy (PDT). PBM involves the application of a coherent (laser) or noncoherent (ﬁltered lamps or light-emitting diode (LED)) light source with a wavelength between 600 and 900/1200 nm [1]. A variety of substrates have been used to create the lasers used for PBM [8]. FBM involves direct application to the lesions to be treated of a photoconverting gel holding chromophores, which is subsequently illuminated by a blue LED lamp and, thus, generates ﬂuorescence, which acts on the target tissues [2]. PDT consists of the application of a photosensitizing agent (such as aminolaevulinic acid, the precursor of porphyrins) to the pathological tissue, followed by illumination with light with a wavelength in the visible spectrum. The ensuing oxygen-dependent reaction generates reactive oxygen species (ROS) which have cytotoxic or immunomodulatory results [9]. Considering the widespread use of LLLT in veterinary medicine in the last two decades, particularly in the dermatological ﬁeld, it would be useful to know the skin diseases in dog for which LLLT is really an advisable therapy. 2. Materials and Methods Clinical scenario: The owner of a 4 year old Labrador Retriever dog affected by canine atopic dermatitis, suffering from recurrent otitis, pododermatitis, and pedal pruritus, asks the veterinarian for information about LLLT for dermatological diseases. This is a therapy which he has read about on the internet. Structured question: In which canine skin diseases is LLLT an advisable therapy with good scientiﬁc evidence? Search strategy: The PUBMED, Google Scholar, Web of Science (Science Citation Index Expanded), Agricola and CAB Abstract databases were examined during July 2022 using the following string: (photobiomodulation OR ﬂuorescence biomodulation OR therapy therapeutic laser OR low level laser therapy OR low level light therapy OR low light therapy OR photodynamic therapy OR ﬂuorescent light) and (dog OR dogs OR canine) and (veterinary) and (skin OR dermatology OR dermatological). Filters were set relating to the language used (English) and the publication period (from 1999 onward). Congress proceedings, book chapters, and reviews were excluded, and only prospective clinical studies in dogs were considered. Two case reports of two aged mixed-breed dogs No difference between treated and control wounds for all parameters, as well as in terms of histology. Gender may impact wound healing in intact dogs. 3. Results Wound planimetry carried out on the caudal wounds, from which percentage contraction and percent epithelialization were estimated. Histologic features were assessed at 7 timepoints from cranial wound biopsies. Obtained data were also correlated to wounds from a female control cohort of a previous study. No differe control wo well as in may impa intact dog Table 1. Articles included in the critically appraised topic. Ref. Disease Treated Type of Therapy Authors and Year Study Population and Study Design I [10] Chronic wounds PBM Lucroy et al., 1999 Single case report of an 8 year old castrated Whippet dog Ir w d c im [11] Chronic wounds PBM Hoisang et al., 2021 RCT of 21 owned dogs of different breeds, genders, and ages D g a ir r w S h a d a [12] Acute traumatic wounds FBM Marchegiani et al., 2020 Two case reports of two aged mixed-breed dogs F p th c d w [13] Bilateral trunk wounds, surgically created PBM Kurach et al., 2015 RCT of 10 adult (13–18 months of age), purpose-bred, male Beagle dogs E s w c w e fe c w fe Table 1. Articles included in the critically appraised topic. 3. Results The literature search in PUBMED identiﬁed 19 articles consistent with the desired characteristics, relevant to the clinical question and compliant with the inclusion criteria in the study. Other databases, such as Google Scholar, Web of Science, CAB Abstract, and Agricola were also searched, but no other relevant articles were found in addition to those previously identiﬁed on PUBMED. The selected articles, all written in English, consisted of prospective clinical studies, published from 1999 to 2022 (Table 1). Vet. Sci. 2022, 9, 505 3 of 16 Table 1. Articles included in the critically appraised topic. Ref. Disease Treated Type of Therapy Authors and Year Study Population and Study Design Interventions and Outcomes Results [10] Chronic wounds PBM Lucroy et al., 1999 Single case report of an 8 year old castrated Whippet dog Irradiation on the awake dog with 630 nM wavelength once daily for 4 successive days. Changes in wound surface zone calculated by computer analysis of digital images of the wound. The woun the trial an by day 21. complicati [11] Chronic wounds PBM Hoisang et al., 2021 RCT of 21 owned dogs of different breeds, genders, and ages Dogs assigned into three groups: (1) control group (C) managed with irrigated saline and without PBM (n = 7); (2) L1 group with irrigated saline together with PBM radiation at 830 nm (n = 7); (3) L2 group with irrigated saline together with SPMW-PBM radiation (n = 7). Wound healing estimated on wound size decrease as a percentage of wound zone every 2nd day for 15 days employing image analysis software. A signiﬁca percentage was record groups at (15 days). wound siz PBM and n percentage was signiﬁ the PBM a 7 (p < 0.05 [12] Acute traumatic wounds FBM Marchegiani et al., 2020 Two case reports of two aged mixed-breed dogs FBM therapy began 5 days after the initial presentation in both dogs. The wound was then covered with a bandage to avoid contamination. The whole process was duplicated once a week until wound healing. Wound clo were fulﬁl treatments re-epitheli small degr did not res and appar dogs (no s tendency t [13] Bilateral trunk wounds, surgically created PBM Kurach et al., 2015 RCT of 10 adult (13–18 months of age), purpose-bred, male Beagle dogs Each side randomized to get LLLT or standard-of-care management 3 times weekly for 32 days. FBM therapy began 5 days after the initial presentation in both dogs. The wound was then covered with a bandage to avoid contamination. The whole process was duplicated once a week until wound healing. RCT of 21 owned dogs of different breeds, genders, and ages Results Results Irradiation on the awake dog with 630 nM wavelength once daily for 4 successive days. Changes in wound surface zone calculated by computer analysis of digital images of the wound. The wound diminished in size during the trial and was completely healed by day 21. No post-treatment complications occurred. A signiﬁcant difference in the percentage of wound area reduction was recorded between the C and PBM groups at the end of the study (15 days). A consistent decrease in wound size was observed in both PBM and non-PBM groups. The percentage of wound area reduction was signiﬁcantly different between the PBM and non-PBM groups on day 7 (p < 0.05). Dogs assigned into three groups: (1) control group (C) managed with irrigated saline and without PBM (n = 7); (2) L1 group with irrigated saline together with PBM radiation at 830 nm (n = 7); (3) L2 group with irrigated saline together with SPMW-PBM radiation (n = 7). Wound healing estimated on wound size decrease as a percentage of wound zone every 2nd day for 15 days employing image analysis software. RCT of 21 owned dogs of different breeds, genders, and ages Wound closure and wound healing were fulﬁlled after 9 and 16 weekly treatments, respectively, with a total re-epithelization of the skin. The small degree of wound contraction did not restrict the free movements and apparently did not disturb the dogs (no signs of suffering or tendency to self-trauma). FBM therapy began 5 days after the initial presentation in both dogs. The wound was then covered with a bandage to avoid contamination. The whole process was duplicated once a week until wound healing. Each side randomized to get LLLT or standard-of-care management 3 times weekly for 32 days. Wound planimetry carried out on the caudal wounds, from which percentage contraction and percent epithelialization were estimated. Histologic features were assessed at 7 timepoints from cranial wound biopsies. Obtained data were also correlated to wounds from a female control cohort of a previous study. No difference between treated and control wounds for all parameters, as well as in terms of histology. Gender may impact wound healing in intact dogs. Vet. Sci. 2022, 9, 505 4 of 16 Table 1. Cont. Ref. Results Almost all treated areas had considerable and visible clinical improvement compared to control areas. Nevertheless, statistical analysis revealed that there was no signiﬁcant difference between the two groups at D4. No adverse reactions were reported. One-half of the wound randomly selected and managed with LLLT and the other left untreated. The protocol was twice daily, 6 min, laser treatments for 5 days. The treated and control areas assessed with a clinical score on the ﬁrst day (D0) and at the end of laser treatment (D4). RCT of seven client-owned dogs of different age and breed that underwent ovariectomy for elective sterilization Dogs were subjected to bilateral ﬂank ovariectomy procedures and open wounds generated bilaterally with a punch biopsy. Each side of the dog (open wound and incision) was randomly allocated to the treatment group or the control group. The treatment group received LLLT once daily for 5 days with a 980 nm laser. The control group received a fake treatment (laser turned off) for an equal amount of time each day. Wounds evaluated visually and biopsied on postoperative days 7 and 14. First three dogs utilized to develop a standardized scar scale to score the other dogs’ incision healing. The other 9 dogs randomly allocated to either receive laser therapy once a day for 7 days or the control group (untreated). Incision healing scored using a scar scale from 0 to 5, with 0 denoting a fresh incision and 5 denoting completely healed with scar contraction and hair growth. Photographs were collected within 24 h of surgery and 1, 3, 5, 7, and 21 days postoperatively. There was no difference between groups for subjective evaluation of healing time and wound measurements. There was no difference in histopathologic evaluation except that the control group at day 7 had more necrosis and perivascular lymphocytes and macrophages. First three dogs utilized to develop a standardized scar scale to score the other dogs’ incision healing. The other 9 dogs randomly allocated to either receive laser therapy once a day for 7 days or the control group (untreated). Incision healing scored using a scar scale from 0 to 5, with 0 denoting a fresh incision and 5 denoting completely healed with scar contraction and hair growth. Photographs were collected within 24 h of surgery and 1, 3, 5, 7, and 21 days postoperatively. All scar scores signiﬁcantly improved with extending time from surgery. Results Disease Treated Type of Therapy Authors and Year Study Population and Study Design [14] Post-neutering surgical skin wounds of at least 3 cm in length PBM Perego et al., 2016 RCT of seven client-owned dogs of different age and breed that underwent ovariectomy for elective sterilization [15] Surgically closed incisions and surgically created open wounds PBM Gammel et al., 2018 RCT of 10 dogs of different breeds that underwent bilateral ﬂank ovariectomy procedures, aged 6 months to 5 years [16] Surgical wounds PBM Wardlaw et al., 2019 RCT of 12 Dachshund dogs that underwent thoraco-lumbar hemilaminectomies for intervertebral disc disease Table 1. Cont. Interventions and Outcomes RCT of 10 dogs of different breeds that underwent bilateral ﬂank ovariectomy procedures, aged 6 months to 5 years There was no difference between groups for subjective evaluation of healing time and wound measurements. There was no difference in histopathologic evaluation except that the control group at day 7 had more necrosis and perivascular lymphocytes and macrophages. There were no signiﬁcant dissimilarities in LCADSS or LPVAS between LLLT and placebo treatments between weeks 0 and 5. However, LCADSS and LPVAS signiﬁcantly decreased from week 0 at weeks 2, 4, and 5 in both LLLT and placebo groups. RCT of 30 client-owned dogs which satisﬁed at least six of the seven diagnostic criteria for atopic dermatitis RCT of 10 healthy client-owned dogs of different genders, ages, and breeds that underwent orthopedic surgery Results Good inter-rater reliability. Laser therapy increased the scar scale score and revealed improved cosmetic healing, by day 7 to day 21, compared to control dogs. [16] Surgical wounds PBM Wardlaw et al., 2019 RCT of 12 Dachshund dogs that underwent thoraco-lumbar hemilaminectomies for intervertebral disc disease 5 of 16 Vet. Sci. 2022, 9, 505 Table 1. Cont. Ref. Disease Treated Type of Therapy Authors and Year Study Population and Study Design [17] Surgical wounds FBM Salvaggio et al., 2019 RCT of 10 healthy client-owned dogs of different genders, ages, and breeds that underwent orthopedic surgery [18] Symmetrical pedal pruritus PBM Stich et al., 2014 RCT of 30 client-owned dogs which satisﬁed at least six of the seven diagnostic criteria for atopic dermatitis Table 1. Cont. Interventions and Outcomes There were no signiﬁcant dissimilarities in median LVAS, lesion/ulcer size, or thickness of the lesion between TG and CG. There was a signiﬁcantly bigger increase (24%) in hair growth in TG compared to CG. Results Disease Treated Type of Therapy Authors and Year Study Population and Study Design Interventions and Outcomes Results [19] Acral lick dermatitis PBM Schnedeker et al., 2021 RCT of 13 owned dogs of different breeds, genders, and ages Dogs were treated with systemic antibiotics and trazodone and randomly allocated to two groups. The treatment group (TG) received LLLT by laser (130 mW, 2 min) with blue and red light-emitting diodes (LEDs), while the control group (CG) had fake therapy (laser/LEDs off). Treatments managed three times weekly for 2 weeks, then twice weekly for 2 weeks for a total of 10 times. The licking visual analog scale (LVAS) was developed. The LVAS questionnaire was completed by the owner at each visit until study end, and the scores were registered by the unblinded examiner. There were dissimilarit lesion/ulce lesion betw a signiﬁcan in hair grow [20] Multiple lesions of sterile pyogranulomatous pododermatitis PBM Perego et al., 2016 RCT of ﬁve client-owned dogs of different genders, ages, and breeds One lesion randomly allocated as control (treated with a 0.0584% hydrocortisone aceponate spray), and one or more other lesions managed with LLLT daily for 5 days. Lesions clinically scored before treatment (D0), at the end (D4), 16 days after the last laser treatment (D20), and after 2 months (D65). There was a difference a treated and treated grou statistically between D0 recurrence than 50% of No adverse [21] Interdigital pyoderma FBM Marchegiani et al., 2019 RCT of 36 privately owned dogs of different genders, ages, and breeds Dogs randomly and blindly assigned to treatment groups of either antibiotic alone (control group) or antibiotic and twice-weekly FBM treatment (FBM group). Dogs scored over a 12 weeks period on the basis of two evaluated parameters: a global lesion score comprised of four different lesions types and neutrophil engulﬁng bacterial scores. A statistica recorded by parameters to control g time-to-reso 4.3 weeks in 10.4 weeks Results RCT of ﬁve client-owned dogs of different genders, ages, and breeds Results Results The FBM areas treated reached lower histology scores, with complete re-epithelialization, less inﬂammation of the dermal layer, and bigger and more regular collagen deposition. As revealed by immunohistochemistry, expression of factor VIII, decorin, collagen III, epidural growth factor, and Ki67 raised in treated compared with untreated tissues. Half of the length of each surgical wound randomly assigned to treatment with FBM, and the remaining 50% was handled with saline solution on the ﬁrst day after surgery and every 3 days until day 13. Wound healing of treated and control areas evaluated with macroscopic assessment and histological and immunohistochemical analysis. The surgeon and the pathologists were blind to the treatment designations. Dogs randomly assigned to one of two study groups. Group A: LLLT on the right paw and placebo on the right paw; Group B: placebo on the left paw and LLLT on the left paw. The principal investigator and owners were unaware of the group designations. Each dog experienced three laser sessions per week over the course of weeks 1 and 2, two laser sessions per week in weeks 3 and 4, and no laser treatments in week 5. At weeks 0, 2, 4, and 5, dogs assigned a score (localized canine atopic dermatitis severity score—LCADSS) by the principal investigator and a score (localized pruritic visual analog score—LPVAS) by the owner, and cytology assessed. The primary outcome assessment was a >50% reduction from baseline of the LCADSS and LPVAS. [18] Symmetrical pedal pruritus PBM Stich et al., 2014 RCT of 30 client-owned dogs which satisﬁed at least six of the seven diagnostic criteria for atopic dermatitis [18] Symmetrical pedal pruritus PBM Stich et al., 2014 [18] Vet. Sci. 2022, 9, 505 6 of 16 Table 1. Cont. Ref. Disease Treated Type of Therapy Authors and Year Study Population and Study Design [19] Acral lick dermatitis PBM Schnedeker et al., 2021 RCT of 13 owned dogs of different breeds, genders, and ages [20] Multiple lesions of sterile pyogranulomatous pododermatitis PBM Perego et al., 2016 RCT of ﬁve client-owned dogs of different genders, ages, and breeds [21] Interdigital pyoderma FBM Marchegiani et al., 2019 RCT of 36 privately owned dogs of different genders, ages, and breeds Table 1. Cont. Table 1. Cont. Ref. Interventions and Outcomes Interventions and Outcomes RCT of 35 privately owned dogs of different genders, ages, and breeds All dogs got better with FBM, achieving a signiﬁcant reduction in vocalization, straining, and licking after 2 weeks. After 5 weeks of therapy, lesional areas had signiﬁcantly decreased. Only one dog required more than seven treatments. No adverse events were noted. Results Dog scores compared with the results obtained from twice-weekly FBM application in a previous study with same inclusion/exclusion criteria, blinding scheme, scoring method, and clinical evaluation [21] Once-weekly FBM applicati the same beneﬁcial effect of interdigital foruncolosis hea twice weekly. [23] Deep pyoderma FBM Marchegiani et al., 2021 RCT of 35 privately owned dogs of different genders, ages, and breeds Dogs randomly and blindly assigned to treatment groups of either antibiotic alone (control group) or antibiotic and twice-weekly FBM application (FBM group). Assessments began weekly for 8 weeks and every 2 weeks thereafter until 12 weeks after admission. After 8 weeks of treatment, percentage of dogs that reac clinical resolution was 35.0% 88.0% for control group and group, respectively. Lesions showed highly statistically s difference in favor of FBM gr week 3 to 8, and neutrophil bacteria scores showed stati difference from week 2 forw favor of FBM group. [24] Perianal ﬁstulas FBM Marchegiani et al., 2020 Case series of four owned dogs of different genders and ages FBM used as only therapy once a week with two consecutive applications in the same session for each dog until clinical signs had signiﬁcantly improved, with weekly evaluations for a 6 week period. Dogs were evaluated by measuring the size of lesions at the start of the study and then weekly for 6 weeks, using software. Owners recorded vocalization and distress frequency scores during their pet’s defecation, as well as perianal licking frequency on a 0–5 point scale to evaluate the response to therapy. All dogs got better with FBM achieving a signiﬁcant redu vocalization, straining, and after 2 weeks. After 5 weeks therapy, lesional areas had signiﬁcantly decreased. Onl required more than seven tr No adverse events were not Results Results Dogs were treated with systemic antibiotics and trazodone and randomly allocated to two groups. The treatment group (TG) received LLLT by laser (130 mW, 2 min) with blue and red light-emitting diodes (LEDs), while the control group (CG) had fake therapy (laser/LEDs off). Treatments managed three times weekly for 2 weeks, then twice weekly for 2 weeks for a total of 10 times. The licking visual analog scale (LVAS) was developed. The LVAS questionnaire was completed by the owner at each visit until study end, and the scores were registered by the unblinded examiner. There were no signiﬁcant dissimilarities in median LVAS, lesion/ulcer size, or thickness of the lesion between TG and CG. There was a signiﬁcantly bigger increase (24%) in hair growth in TG compared to CG. RCT of 13 owned dogs of different breeds, genders, and ages There was a statistically signiﬁcant difference at D4 and D20 between treated and control groups; in the treated group over time, there was a statistically signiﬁcant advancement between D0, D4, and D20. Lesion recurrence was not present in more than 50% of the treated lesions at D65. No adverse reactions were recorded. One lesion randomly allocated as control (treated with a 0.0584% hydrocortisone aceponate spray), and one or more other lesions managed with LLLT daily for 5 days. Lesions clinically scored before treatment (D0), at the end (D4), 16 days after the last laser treatment (D20), and after 2 months (D65). Dogs randomly and blindly assigned to treatment groups of either antibiotic alone (control group) or antibiotic and twice-weekly FBM treatment (FBM group). Dogs scored over a 12 weeks period on the basis of two evaluated parameters: a global lesion score comprised of four different lesions types and neutrophil engulﬁng bacterial scores. A statistically signiﬁcant decrease recorded by week 3 in both measured parameters for FBM group compared to control group. The mean time-to-resolution of lesions was 4.3 weeks in FBM group and 10.4 weeks in control group. 7 of 16 7 of 16 Vet. Sci. 2022, 9, 505 Table 1. Cont. Ref. Disease Treated Type of Therapy Authors and Year Study Population and Study Design Interventions and Outcomes Results [22] Interdigital pyoderma FBM Marchegiani et al., 2022 Cases series of 12 privately owned dogs of different genders, ages, and breeds Dogs received antibiotic and once weekly FBM. Case series of four owned dogs of different genders and ages All groups achieved improvement during the study. The greatest clinical score reduction appaired in Group BW. BW obtained a clinically relevant effect level at T3, QW reached it at T4, and C did not reach it. No differences between groups were noted in the reduction in CFU/mL (T0–T5). Interventions and Outcomes Results Results Dogs received antibiotic and once weekly FBM. Dog scores compared with the results obtained from twice-weekly FBM application in a previous study with same inclusion/exclusion criteria, blinding scheme, scoring method, and clinical evaluation [21] Once-weekly FBM application exerts the same beneﬁcial effect of interdigital foruncolosis healing as twice weekly. Cases series of 12 privately owned dogs of different genders, ages, and breeds After 8 weeks of treatment, the percentage of dogs that reached clinical resolution was 35.0% and 88.0% for control group and FBM group, respectively. Lesions cores showed highly statistically signiﬁcant difference in favor of FBM group from week 3 to 8, and neutrophil engulﬁng bacteria scores showed statistical difference from week 2 forward in favor of FBM group. Dogs randomly and blindly assigned to treatment groups of either antibiotic alone (control group) or antibiotic and twice-weekly FBM application (FBM group). Assessments began weekly for 8 weeks and every 2 weeks thereafter until 12 weeks after admission. RCT of 35 privately owned dogs of different genders, ages, and breeds FBM used as only therapy once a week with two consecutive applications in the same session for each dog until clinical signs had signiﬁcantly improved, with weekly evaluations for a 6 week period. Dogs were evaluated by measuring the size of lesions at the start of the study and then weekly for 6 weeks, using software. Owners recorded vocalization and distress frequency scores during their pet’s defecation, as well as perianal licking frequency on a 0–5 point scale to evaluate the response to therapy. All dogs got better with FBM, achieving a signiﬁcant reduction in vocalization, straining, and licking after 2 weeks. After 5 weeks of therapy, lesional areas had signiﬁcantly decreased. Only one dog required more than seven treatments. No adverse events were noted. Vet. Sci. 2022, 9, 505 8 of 16 8 of 16 Table 1. Cont. Ref. Disease Treated Type of Therapy Authors and Year Study Population and Study Design Interventions and Outcomes Results [25] Spontaneous otitis externa PDT Sellera et al., 2019 Single case report of a 5 year old Lhasa apso dog Unilateral otitis externa caused by carbapenem-resistant P. aeruginosa treated with antimicrobial photodynamic therapy (aPDT) using methylene blue as photosensitizer. The isolated bacterial strain also checked for susceptibility to in vitro aPDT. For decolonization, probiotic supplements were orally used (once daily) for 14 days. Results Effectiveness of probiotics and photodynamic therapy evaluated by clinical and microbiological culture assays. Total resolutio reached by da obtained imm 14 days follow VIM-2-produc and rectal swa 14, and 21 afte validated effe decolonizatio [26] Spontaneous otitis externa FBM Tambella et al., 2020 RCT of 37 owned dogs of different genders, ages, and breeds Dogs randomly assigned to three groups: group QW with a topical LED-illuminated gel (LIG) once weekly; group BW with LIG twice weekly; group C with enroﬂoxacin and silver sulfadiazine twice daily. The estimation protocol (T0 to T5) considered clinical assessment (OTIS-3 index scoring system; pruritus severity scale; pain severity score; aural temperature), cytological scoring system, and quali-quantitative bacteriologic evaluation. All groups ach during the stu score reductio BW. BW obtai effect level at and C did not between grou reduction in C Table 1. Cont. Results Unilateral otitis externa caused by carbapenem-resistant P. aeruginosa treated with antimicrobial photodynamic therapy (aPDT) using methylene blue as photosensitizer. The isolated bacterial strain also checked for susceptibility to in vitro aPDT. For decolonization, probiotic supplements were orally used (once daily) for 14 days. Effectiveness of probiotics and photodynamic therapy evaluated by clinical and microbiological culture assays. Total resolution of clinical signs reached by day 7 after aPDT. Samples obtained immediately and after 7 and 14 days following aPDT negative for VIM-2-producing P. aeruginosa. Oral and rectal swabs obtained on days 7, 14, and 21 after probiotic therapy validated effective gastrointestinal decolonization. Dogs randomly assigned to three groups: group QW with a topical LED-illuminated gel (LIG) once weekly; group BW with LIG twice weekly; group C with enroﬂoxacin and silver sulfadiazine twice daily. The estimation protocol (T0 to T5) considered clinical assessment (OTIS-3 index scoring system; pruritus severity scale; pain severity score; aural temperature), cytological scoring system, and quali-quantitative bacteriologic evaluation. All groups achieved improvement during the study. The greatest clinical score reduction appaired in Group BW. BW obtained a clinically relevant effect level at T3, QW reached it at T4, and C did not reach it. No differences between groups were noted in the reduction in CFU/mL (T0–T5). [26] Vet. Sci. 2022, 9, 505 9 of 16 9 of 16 Table 1. Cont. Table 1. Cont. Ref. Disease Treated Type of Therapy Authors and Year Study Population and Study Design Interventions and Outcomes R [27] Noninﬂammatory alopecia PBM Olivieri et al., 2014 Case series of seven privately owned dogs of different ages, genders, and breeds Unsedated dog treated twice weekly for a maximum of 2 months with therapeutic laser with three different wavelengths. A ﬁxed alopecic area left untreated and used as a control area. The efﬁcacy assessed clinically by visual examination. Areas documented and with photographs and graded at the start of the study, after eight applications (4 weeks) and at the end of the study. From one dog, post-treatment biopsies of treated and untreated sites achieved for histological determination of hair density and the percentage of haired and non-haired follicles. A g i t 1 t f t c [28] Calcinosis cutis with secondary pyoderma FBM Apostolopoulos et al., 2020 Single case report of a 15 year old male Golden Retriever dog FBM used as an auxiliary to systemic antimicrobial and topical therapies. Results Part of the lesions covered with a towel and not exposed to FBM, to determine clinical and cytological efﬁcacy. C i l u Interventions and Outcomes Results Results Unsedated dog treated twice weekly for a maximum of 2 months with therapeutic laser with three different wavelengths. A ﬁxed alopecic area left untreated and used as a control area. The efﬁcacy assessed clinically by visual examination. Areas documented and with photographs and graded at the start of the study, after eight applications (4 weeks) and at the end of the study. From one dog, post-treatment biopsies of treated and untreated sites achieved for histological determination of hair density and the percentage of haired and non-haired follicles. At the end of the study, coat regrowth greatly improved in 6/7 animals and improved in 1/7. Via morphometry, the area occupied by hair follicles was 18% in the treated sample and 11% in the untreated one (11%); haired follicles were (per area) 93% in the treated sample and only 9% in the control sample. Cytology supported increased improvement of the illuminated lesions compared with unexposed lesions. Single case report of a 15 year old male Golden Retriever dog Vet. Sci. 2022, 9, 505 10 of 16 10 of 16 RCT: randomized controlled trial; PBM: photobiomodulation, FBM: ﬂuorescence biomodulation; PDT: photodynamic therapy; SPMW: simultaneous superpulsed and multi- ple wavelengths.These studies met the inclusion criteria and addressed the clinical question but had very dissimilar study designs. The scientiﬁc quality of each study was analyzed employing the following parameters to establish the risk-of-bias assessment of treatment efﬁcacy [29], as summarized in Table 2: - Levels of evidence: assigned according to the previously identiﬁed criteria for thera- peutic studies [30,31]. Brieﬂy, level IA = systematic review (with homogeneity) of randomized control trials (RCTs); level IB = individual RCT (with narrow conﬁdence intervals); level IC = all or none study; level IIA = systematic review (with homo- geneity) of cohort studies; level IIB = individual cohort study; level 2C = “outcomes” ecological studies; level IIIA = systematic review (with homogeneity) of case–control studies; level IIIB = individual case–control study; level IV = case series (or poor- quality cohort and case–control study); level V = expert opinion without explicit critical appraisal or based on physiology bench research or “ﬁrst principles” [31]. - Randomization: presence of a method of randomization and hiding of the allocation of subjects to the intervention groups to the people recruiting the participants. Score: 0 (no)–1 (yes). y - Blinding: trial investigator(s) blinded to the treatment allocation. Score: 0 (no)–1 (yes). Results 2022, 9, 505 11 of 16 11 of 16 Table 2. Assessment of scientiﬁc evidence of the studies. The studies are categorized as conclusive evidence (total score 8–10), highly suggestive evidence (total score 6–7), suggestive evidence (total score 4–5), or inconclusive evidence (total score ≤3). evidence (total score 8 10), highly suggestive evidence (total score 6 7), suggestive evidence (total score 4–5), or inconclusive evidence (total score ≤3). Year Reference Level of Evidence Randomization Blinding Group Size Similarity between Groups Equal Treatment of Groups Follow-Up (>12 Months) Total Score Grading of Evidence 1999 Lucroy et al. [10] IV 0 0 0 0 0 0 0 Inconclusive 2014 Stich et al. [18] IB 1 1 2 2 2 0 8 Conclusive 2014 Olivieri et al. [27] IV 0 0 0 1 1 0 2 Inconclusive 2015 Kurach et al. 2015 [13] IB 1 1 1 2 2 0 7 Highly suggestive 2016 Perego et al. [20] IB 1 1 0 2 2 0 6 Highly suggestive 2016 Perego et al. [14] IB 1 0 0 1 2 0 4 Suggestive 2018 Gammel et al. [15] IB 1 1 1 0 2 0 5 Suggestive 2019 Wardlaw et al. [16] IB 1 1 1 2 1 0 6 Highly suggestive 2019 Sellera et al. [25] IV 0 0 0 0 0 0 0 Inconclusive 2019 Salvaggio et al. [17] IB 1 1 1 1 2 0 6 Highly suggestive 2019 Marchegiani et al. [21] IB 1 1 2 2 2 0 8 Conclusive 2020 Tambella et al. [26] IB 1 0 3 0 1 0 5 Suggestive 2020 Marchegiani et al. [12] IV 0 0 0 0 0 0 0 Inconclusive 2020 Apostopoulos et al. [28] IV 0 0 0 0 0 0 0 Inconclusive 2020 Marchegiani et al. [24] IV 0 0 0 0 0 0 0 Inconclusive 2021 Schnedeker et al. [19] IB 1 1 1 0 2 0 5 Suggestive 2021 Hoisang et al. [11] IB 1 0 2 0 2 0 5 Suggestive 2021 Marchegiani et al. [23] IB 1 1 2 2 2 0 8 Conclusive 2022 Marchegiani et al. [22] IV 0 1 1 2 2 0 6 Highly suggestive IB: randomized controlled trial, IV: poor-quality case–control study or cases series. Results g g ( ) ( ) (y ) - Similarity between groups: populations allocated to different groups in the trial share the same characteristics at the start and throughout the study. Score: 0 (no), 1 (deduced from the text), 2 (yes). y - Equal treatment of groups: populations allocated to different groups in the trial treated similarly except for the therapy. Score: 0 (no), 1 (deduced from the text), 2 (yes). y p py - Presence of at least 12 months follow-up: score: 0 (no)–1 (yes). f f p ( ) (y ) Group size: score: 0 (<10 subjects), 1 (10–20 subjects), 2 (21–40 subjects), 3 (>40 subjects). f f p y - Group size: score: 0 (<10 subjects), 1 (10–20 subjects), 2 (21–40 subjects), 3 (>40 subjects) As described in Table 2, at the end of this step of quality assessment, each study was graded [32] according to total score: conclusive evidence (total score 8–10) [18,21,23], highly suggestive evidence (total score 6–7) [13,16,17,20,22], suggestive evidence (total score 4–5) [11,14,15,19,26], or inconclusive evidence (total score ≤3) [10,12,24,25,27,28]. Studies showing inconclusive evidence were excluded from further assessment. To emphasize the overall strength of chosen studies, we evaluated conclusive, highly suggestive, and suggestive studies for each dermatological pathology treated (Table 3), considering the following variables: - Type of laser used: diode laser, dual diode laser, solid medium diode laser, or LED lamp with photoconverter gel, with indication of wavelength, energy density, and power, when reported. p - Treatment frequency (posology): twice a day, daily, every other day, twice a week, or weekly administration. y - Study limits reported in the article: presence/absence. - Ease of administration: yes: good acceptance of therapy in most subjects; no: difﬁcult to administer, annoying, or painful. y g p - Adverse effects: score 0 = none; score 1 = yes, mild or rare (<10%); score 2 = yes moderate or common (≥10%); score 3 = yes, common and moderate or severe. y - Number of administrations carried out: score 1 = ≤5 administrations; score 2 = 6–10 administrations; score 3 = >10 administrations. - Efﬁcacy of the treatment: WCG = only treatment group without control group; NS = no statistical difference between treatment group and control group; SD = statistical difference between treatment and control group. All studies showed a score of 0 (none) for adverse effects; hence, this category is omitted from Table 3. Vet. Sci. Results Randomization score: 0 (no)–1 (yes); blinding score: 0 (no)–1 (yes); group size score: 0 (<10 subjects), 1 (10–20 subjects), 2 (21–40 subjects), 3 (>40 subjects); similarity between groups score: 0 (no), 1 (deduced from the text), 2 (yes); equal treatment of groups score: 0 (no), 1 (deduced from the text), 2 (yes); follow-up (>12 months) score: 0 (no)–1 (yes). Articles with conclusive evidence grading are shown in bold. Table 3. Overall strength of LLLT in different dermatological conditions. Only conclusive, highly suggestive, and suggestive studies are analyzed. Ref. Disease Treated Type of Laser Used Treatment Frequency Easy to Use Number of Administrations Score Reported Study Limits Efﬁcacy Score [18] Pedal pruritus in AD Solid medium diode laser, 12 W (maximum power) laser with dual-wavelength output 980 nm (80%) and 810 nm (20%), power 4.5 mW 30 s EOD for the 2 weeks, then twice a week for 2 weeks Yes 2 Application protocol derived from human studies and independent of the hair length/type; absence of untreated group; possible placebo or systemic effect on the untreated paw NS [20] Pododermatitis Solid medium diode laser, wavelength 808 nm, power 250 mW, energy density 0.9 J/min/cm2 1.5 + 6 min on the ﬁrst day, then 6 min daily for 5 days Yes 1 Small sample, one treatment per day SD [21] Interdigital pyoderma LED lamp with photoconverter gel, peak wavelength between 440 and 460 nm, energy density between 55 and 129 mW/cm2 2 min, twice weekly until clinical resolution Yes 3 Pruritus not evaluated as response to therapy SD IB: randomized controlled trial, IV: poor-quality case–control study or cases series. Randomization score: 0 (no)–1 (yes); blinding score: 0 (no)–1 (yes); group size score: 0 (<10 subjects), 1 (10–20 subjects), 2 (21–40 subjects), 3 (>40 subjects); similarity between groups score: 0 (no), 1 (deduced from the text), 2 (yes); equal treatment of groups score: 0 (no), 1 (deduced from the text), 2 (yes); follow-up (>12 months) score: 0 (no)–1 (yes). Articles with conclusive evidence grading are shown in bold. Table 3. Overall strength of LLLT in different dermatological conditions. Only conclusive, highly suggestive, and suggestive studies are analyzed. Ref. Results Disease Treated Type of Laser Used Treatment Frequency Easy to Use Number of Administrations Score Reported Study Limits Efﬁcacy Score [18] Pedal pruritus in AD Solid medium diode laser, 12 W (maximum power) laser with dual-wavelength output 980 nm (80%) and 810 nm (20%), power 4.5 mW 30 s EOD for the 2 weeks, then twice a week for 2 weeks Yes 2 Application protocol derived from human studies and independent of the hair length/type; absence of untreated group; possible placebo or systemic effect on the untreated paw NS [20] Pododermatitis Solid medium diode laser, wavelength 808 nm, power 250 mW, energy density 0.9 J/min/cm2 1.5 + 6 min on the ﬁrst day, then 6 min daily for 5 days Yes 1 Small sample, one treatment per day SD [21] Interdigital pyoderma LED lamp with photoconverter gel, peak wavelength between 440 and 460 nm, energy density between 55 and 129 mW/cm2 2 min, twice weekly until clinical resolution Yes 3 Pruritus not evaluated as response to therapy SD Table 3. Overall strength of LLLT in different dermatological conditions. Only conclusive, highly suggestive, and suggestive studies are analyzed. Vet. Sci. 2022, 9, 505 12 of 16 Table 3. Cont. Table 3. Cont. Ref. Results Disease Treated Type of Laser Used Treatment Frequency Easy to Use Number of Administrations Score Reported Study Limits Efﬁcacy Score [22] Interdigital furuncolosis LED lamp with photoconverter gel, peak wavelength between 440 and 460 nm, energy density between 55 and 129 mW/cm2 2 + 2 min with 1 min rest between one illumination and the other, once weekly until clinical resolution Yes 3 Not reported SD [23] Deep pyoderma LED lamp with photoconverter gel, peak wavelength between 440 and 460 nm, energy density between 55 and 129 mW/cm2 2 min, twice weekly until clinical resolution Yes 3 Pruritus not evaluated as response to therapy SD [13] Wound Dual diode laser (7.5 mW/diode), wavelength 635 nm, total energy density 1.125 J/cm2 5 min EOD until complete reepithelization Yes 3 Use of historical control group NS [14] Wound Solid medium diode laser, wavelength 808 nm, power 250 mW, energy density 0.9 J/min/cm2 6 min twice daily for 5 days Yes 2 Possible spillover effect; no clinical healing follow-up NS [15] Wound Type of laser not reported, wavelength 980 nm, energy density 5 J/cm2, power 2–3.5 W 1.33–2.00 min daily for 5 days Yes 1 Small sample; no medical history; no conclusion for traumatic, chronic, infected, or delayed healing wound; failure to rule out systemic effect of LLLT; open wound assessed only subjectively NS [16] Wound Diode laser, wavelength 850 nm, energy density 8 J/cm2 Min not reported; daily for 7 days Yes 2 Small sample; no recorded time of treatment for each patient; inability to draw conclusion about many risk factors due to abnormal wound healing SD [17] Wound LED lamp with photoconverter gel, wavelength between 440 and 460 nm, energy density between 55 and 129 mW/cm2 2 min for 5 times until 13th day Yes 1 Small sample; no conclusion for traumatic, chronic, infected wound SD [11] Wound Group L1: diode laser 830 nm, 4 J/cm2, 200 mW Group L2: diode laser 830 nm, 4 J/cm2, 200 mW + super pulsed diode laser 100 mW of 660 nm, 250 mW of 850 nm, and 50 mW of 905 nm Group L1: 3.45–41.40 min EOD for 2 weeks Group L2: 3.45–41.40 min EOD for 2 weeks + 1 min/4 cm2 wound area one time Yes Group L1: 2 Group L2: 2 + 1 Small sample size; different wound size; variability of subjects; absence of histopathological analysis; small aperture of probe SD [26] Otitis externa LED lamp with photoconverter gel, wavelength between 440 and 460 nm 30 s soft + 1 min high power Group QW: weekly for 6 weeks Group BW: twice weekly for 6 weeks Yes Group QW: 2 Group BW: 3 Different OTIS-3 score between group at D0; clinical assessment not blinded; intact tympanic membrane for inclusion SD [19] Acral dermatitis Diode laser, wavelength between 470 and 640, average power 130 mW with a dose of 15.6 J and ﬂuence of 3.93 J/cm2 2 min EOD for 2 weeks, then twice weekly for 2 weeks Yes 2 Small sample size; empirical selection of dose and frequency of laser administration NS Ref: reference; AD: atopic dermatitis; EOD: every other day. Results Number of administrations score 1 = ≤5 adminis trations, score 2 = 6–10 administrations, score 3 = >10 administrations; efﬁcacy of the treatment score WCG = Ref: reference; AD: atopic dermatitis; EOD: every other day. Number of administrations score 1 = ≤5 adminis- trations, score 2 = 6–10 administrations, score 3 = >10 administrations; efﬁcacy of the treatment score WCG = only treatment group without control group, NS = no statistical difference between treatment group and control group, SD = statistical difference between treatment and control group. Articles with conclusive results are shown in bold. Ref: reference; AD: atopic dermatitis; EOD: every other day. Number of administrations score 1 = ≤5 adminis- trations, score 2 = 6–10 administrations, score 3 = >10 administrations; efﬁcacy of the treatment score WCG = only treatment group without control group, NS = no statistical difference between treatment group and control group, SD = statistical difference between treatment and control group. Articles with conclusive results are shown in bold. 4. Discussion Despite the variety of published articles, in the international literature, there are few prospective clinical studies conducted in vivo that provide good scientiﬁc evidence to evaluate the effectiveness of LLLT as a treatment for canine skin diseases. Vet. Sci. 2022, 9, 505 13 of 16 13 of 16 On the basis of the results obtained from this CAT, only three of the 19 studies were considered conclusive. The study of Marchegiani et al. [21] on canine interdigital pyoderma is one of the three, with a total score of 8, characterized by good methodological quality and scientiﬁc evidence. For this reason, FBM can be recommended for the therapy of interdigital pyoderma in dogs. This prospective randomized blinded clinical study on 36 dogs evaluated the effect of a LED lamp with a photoconverter gel system, used for 2 min twice weekly until clinical resolution, in combination with systemic antibiotics on clinical manifestations of canine interdigital pyoderma. This was compared to dogs treated with antibiotics alone as control group. A statistically signiﬁcant decrease was noted in measured parameters for the treatment group compared to the control group. The mean time to lesion resolution was 4.3 weeks in treatment group and 10.4 weeks in control group. The second study [23] was performed by the same authors and with the same exper- imental design, but on 35 dogs affected by deep pyoderma, regardless of body location. The total score was 8, and the study was characterized by good methodological quality and scientiﬁc evidence. It was a prospective randomized blinded clinical trial evaluating the effect of a LED lamp with a photoconverter gel system, used for 2 min twice weekly until clinical resolution in combination with systemic antibiotics on clinical manifestations of canine deep pyoderma. This was compared to control dogs treated with antibiotics alone. A statistically signiﬁcant decrease was recorded in the measured parameters for the treatment group compared to the control group. The mean time to resolution of lesions was 5.7 weeks in the treatment group and 11.7 weeks in the control group. For this reason, FBM can be recommended for the therapy of deep pyoderma in dogs. It is interesting to note that, in both these trials, the duration of the course of systemic an- tibiotic therapy was significantly reduced if FBM was administered as an additional treatment. py g y The study by Stich at al. 4. Discussion [18] is the third study with good methodological quality and scientiﬁc evidence that was rated with “conclusive evidence”, with a total score of 8. This study demonstrated that the use of PBM is not beneﬁcial as a treatment for pedal pruritus secondary to canine atopic dermatitis. This was a prospective, randomized, double- blinded, intraindividual study (with each dog serving as their own placebo control) on 30 client- owned dogs with symmetrical pedal pruritus secondary to canine atopic dermatitis. PBM was not effective as a localized treatment; only 38% of patients treated with PBM had a reduction of more than 50% in pruritus or lesion scores for the treated paw compared to baseline values, and there was no signiﬁcant difference in scores between the paws of individual dogs treated with PBM and placebo laser. Scores decreased signiﬁcantly for untreated paws, as well as in the treatment group, and the authors postulated that this improvement probably represented a placebo effect (the principal investigator and the owners were informed that one paw was being treated with PBM), although it is also possible that PBM caused a systemic effect for both treated and untreated paws in the same subject [18]. j With regard to the other canine skin diseases investigated in this critically appraised topic, all LLLT methods were promising in many of the included studies. In fact, in most of the articles analyzed, LLLT led to an improvement in symptoms of treated subjects and often to their complete resolution [10–12,16,17,20,22,24–28]. In four studies (three in surgical or surgically created wounds [13–15] and one [19] in acral lick dermatitis), the LLLT did not show any signiﬁcant clinical efﬁcacy in the treated subjects. However, all clinical studies, unlike the studies by Stich et al. [18] and Marchegiani et al. [21,23], were characterized by insufﬁcient scientiﬁc evidence for an incomplete and inappropriate methodology, as listed below. Indeed, it is important to distinguish the clinical outcome of treated subjects with the scientiﬁc-based evidence of a study, which is the purpose of a CAT. In the ﬁeld of LLLT used on dogs with skin diseases, many of the studies published were unfortunately clinical cases series, thus strongly limiting their scientiﬁc evidence. 4. Discussion y g y g It is not possible to conclusively recommend or not recommended the use of LLLT for management of surgical wounds and incisions, because the relevant studies [13–17], although all randomized and blinded studies, were rated only with “highly suggestive or Vet. Sci. 2022, 9, 505 14 of 16 14 of 16 suggestive evidence” mainly due to the small number of subjects treated and the lack of adequate follow-up. For acute traumatic and chronic wounds, two studies [10,12] were not randomized or blinded and, therefore, categorized with “inconclusive evidence”, while another study [11], randomized but not blinded, was rated as “suggestive evidence”. In the case of otitis externa, one study, not blinded or randomized, was categorized with “inconclusive evidence” [25], while the other, randomized but not blinded, had “suggestive evidence” [26]. For acral lick dermatitis [19] and sterile pyogranulomatous pododermatitis [20], the studies were categorized with “highly suggestive or suggestive evidence”, while, for perianal ﬁstulas, the study was graded as “inconclusive evidence”, due to the lack of a control group [24]. In noninﬂammatory alopecia, the study was graded as “inconclusive evidence”, due to a lack of randomization and blinding [27]; for bacterial skin infection associated with calcinosis cutis [28], the study was rated as “inconclusive evidence” due to the lack of a control group, randomization, and blindness. g p A separate case is the very recent study by Marchegiani et al. [22], an update on FBM for the treatment of interdigital furunculosis. The trial tested the once-weekly adminis- tration on 12 dogs affected by interdigital pyoderma by comparing the results obtained with those of a previous study of 2019 [21] with identical structure (inclusion/exclusion criteria, blinding scheme, scoring system, etc.) but twice-weekly administration. Unlike the 2019 study, which was “conclusive”, this new study was categorized only as “highly suggestive”, due to the lack of randomization and the fewer subjects treated. This, therefore, suggests that once-weekly administration of FBM for interdigital furunculosis cannot be recommended at the moment. In all evaluated studies, PBM, FBM, and PDT proved to be noninvasive, easy to deliver in unsedated dogs, and without side-effects during the investigation period. Further research in this ﬁeld is indicated to increase our understanding of this new ther- apeutic option in the veterinary dermatological ﬁeld. Generalizable, in vivo, randomized, double-blind, and controlled studies are required on a sufﬁciently large scale. 4. Discussion The subject inclusion criteria, the group randomization process, and the blinding procedure should always be described in detail in future studies. Moreover, to allow the comparison of results, all new studies evaluating the efﬁcacy of LLLT as a treatment for canine dermatological diseases should follow the same clinical criteria for the inclusion of subjects, use the same clinical score when monitoring healing, and follow up subjects after treatment for at least 12 months, so as to be able to identify any relapses. Through further studies, it would also be interesting to investigate the potential of LLLT to reduce the use of antibiotics, a potential advantage that emerged from the studies by Marchegiani et al. [21,23], in which duration of systemic antibiotics was reduced following concurrent FBM. The antimicrobial effect of LLLT has not yet been totally proven, although some human in vitro dentistry studies have highlighted this aspect [33,34]. The use of antibiotics is a very hotly debated topic with regard to the development of antimicrobial resistance, which is considered a global public health crisis that threatens our ability to successfully treat bacterial infections [35]. Further studies are also required to draft standardized protocols, which are currently inadequate, relating to the optimal parameters of the therapeutic lasers to be used and the posology for the treatment of each pathology. Factors such as spot size, wavelength, energy density, power density, pulse structure, total energy, total power, delivery mode (contact, point, and wide beam), the duration of the treatment, and the treatment intervals might inﬂuence the success of the LLLT. It is evident that, to obtain positive results with LLLT, each of these dosimetric parameters must be controlled within a limited range of values [36]. 5. Conclusions In this critically appraised topic on the use of LLLT as a treatment for canine skin dis- eases, good scientiﬁc evidence was identiﬁed only for the recommendation of ﬂuorescence Vet. Sci. 2022, 9, 505 15 of 16 15 of 16 biomodulation (FBM) for management of canine interdigital pyoderma and canine deep pyoderma, in combination with systemic antibiotic therapy. biomodulation (FBM) for management of canine interdigital pyoderma and canine deep pyoderma, in combination with systemic antibiotic therapy. py y py LLLT has the potential to be a promising treatment for many canine skin diseases. However, additional valid and generalizable clinical studies, with good scientiﬁc evidence, are required to investigate its actual efﬁcacy and potential antimicrobic effect, as well as to produce scientiﬁcally validated standardized therapeutic protocols. Author Contributions: Conceptualization, D.P., M.M. and R.P.; methodology, D.P., E.S. and R.P.; data curation, R.P., M.M. and D.P.; writing—original draft preparation, M.M. and R.P.; writing—review and editing, R.P., M.M., D.P. and E.S.; project administration, R.P. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Data Availability Statement: All data discussed are contained in the manuscript. Data Availability Statement: All data discussed are contained in the manuscript. Acknowledgments: The authors acknowledge the support of the APC central fund of the University of Milan. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Avci, P.; Gupta, A.; Sadasivam, M.; Vecchio, D.; Pam, Z.; Pam, N.; Hamblin, M.R. Low-level laser (light) therapy (LLLT) in skin: Stimulating, healing, restoring. Semin. Cutan. Med. Surg. 2013, 32, 41–52. [PubMed] 2. Marchegiani, A.; Spaterna, A.; Cerquetella, M. Current Applications and Future Pe Biomodulation in Veterinary Medicine. Vet. Sci. 2021, 8, 20. [CrossRef] [PubMed] 2. Marchegiani, A.; Spaterna, A.; Cerquetella, M. Current Applications and Future Perspectives of Fluorescence Light Energy Biomodulation in Veterinary Medicine. Vet. Sci. 2021, 8, 20. [CrossRef] [PubMed] 3. Fesseha, H. Laser Therapy and its Potential Application in Veterinary Practice—A Review. J. Light Laser Curr. Trends 2020, 3, 1–9. 4. Hochman, L. Photobiomodulation Therapy in Veterinary Medicine: A Review. Top. Companion Anim. Med. 2018, 33, 83–88. [CrossRef] 4. Hochman, L. Photobiomodulation Therapy in Veterinary Medicine: A Review. Top. Companion Anim. Med. 2018, 33, 83–88. [CrossRef] [ ] 5. Prindeze, N.J.; Moffatt, L.T.; Shupp, J.W. Mechanisms of action for light therapy: A review of molecular interactions. Exp. Biol. Med. 2012, 237, 1241–1248. [CrossRef] 6. Houreld, N.N.; Masha, R.T.; Abrahamse, H. Low-intensity laser irradiation at 660 nm stimulates cytochrome c oxidase in stressed ﬁbroblast cells. Lasers Surg. Med. 2012, 44, 429–434. [CrossRef] 7. Enwemeka, C.S.; Parker, J.C.; Dowdy, D.S.; Harkness, E.E.; Sanford, L.E.; Woodruff, L.D. The efﬁcacy of low-power lasers in tissue repair and pain control: A meta-analysis study. Photomed. Laser Surg. 2004, 22, 323–329. [CrossRef] D.A.; Dover, J.S.; Arndt, K.A.; Silapunt, S.; Alam, M. Low-level laser therapy for wound healing: Mechanism ol. Surg. 2005, 31, 334–340. [CrossRef] 8. Posten, W.; Wrone, D.A.; Dover, J.S.; Arndt, K.A.; Silapunt, S.; Alam, M. Low-level laser therapy for wo and efﬁcacy. Dermatol. Surg. 2005, 31, 334–340. [CrossRef] 9. Babilas, P.; Schreml, S.; Landthaler, M.; Szeimies, R.M. Photodynamic therapy in dermatology: State-of-the-art. Photodermatol. Photoimmunol. Photomed. 2010, 26, 118–132. [CrossRef] 10. Lucroy, M.D.; Edwards, B.F.; Madewell, B.R. Low-intensity laser light-induced closure of a chronic wound in a dog. Vet. Surg. 1999, 28, 292–295. [CrossRef] 11. Hoisang, S.; Kampa, N.; Seesupa, S.; Jitpean, S. Assessment of wound area reduction on chronic wounds in dogs with photo- biomodulation therapy: A randomized controlled clinical trial. Vet. World 2021, 14, 2251–2259. [CrossRef] [PubMed] 12. Marchegiani, A.; Spaterna, A.; Piccionello, A.P.; Meligrana, M.; Fruganti, A.; Tambella, A.M. Fluorescence biomodulation in the management of acute traumatic wounds in two aged dogs. Vet. Med. 2020, 65, 215–220. [CrossRef] 13. References Kurach, L.M.; Stanley, B.J.; Gazzola, K.M.; Fritz, M.C.; Steﬁcek, B.A.; Hauptman, J.G.; Seymour, K.J. T Therapy on the Healing of Open Wounds in Dogs. Vet. Surg. 2015, 44, 988–996. [CrossRef] [PubMe py g p g g 14. Perego, R.; Proverbio, D.; Spada, E.; Zuccaro, A. First Experience with Photobiomodulation (Pbm) in Post-Surgical Wound Healing in Dogs. J. Vet. Clin. Pract. Pet Care 2016, 1, 1–6. [CrossRef] g g J , , [ ] 15. Gammel, J.E.; Biskup, J.J.; Drum, M.G.; Newkirk, K.; Lux, C.N. Effects of low-level laser therapy on the healing of surgically closed incisions and surgically created open wounds in dogs. Vet. Surg. 2018, 47, 499–506. [CrossRef] g y p g g 16. Wardlaw, J.L.; Gazzola, K.M.; Wagoner, A.; Brinkman, E.; Burt, J.; Butler, R.; Gunter, J.M.; Senter, L.H. Laser therapy for incision healing in 9 dogs. Front. Vet. Sci. 2019, 5, 349. [CrossRef] g g 17. Salvaggio, A.; Magi, G.E.; Rossi, G.; Tambella, A.M.; Vullo, C.; Marchegiani, A.; Botto, R.; Palumbo Piccionello, A. Effect of the topical Klox ﬂuorescence biomodulation system on the healing of canine surgical wounds. Vet. Surg. 2020, 49, 719–727. [CrossRef] Vet. Sci. 2022, 9, 505 16 of 16 16 of 16 18. Stich, A.N.; Rosenkrantz, W.S.; Grifﬁn, C.E. Clinical efﬁcacy of low-level laser therapy on localized canine atopic dermatitis severity score and localized pruritic visual analog score in pedal pruritus due to canine atopic dermatitis. Vet. Dermatol. 2014, 25, 464-e74. [CrossRef] [ ] 19. Schnedeker, A.H.; Cole, L.K.; Diaz, S.F.; Lorch, G.; Rajala-Shultz, P.J.; Jennings, R.N.; Hostnik, E.T.; Daniels, J.B. Is low-level laser therapy useful as an adjunctive treatment for canine acral lick dermatitis? A randomized, double-blinded, sham-controlled study. Vet. Dermatol. 2021, 32, 148-e35. [CrossRef] 20. Perego, R.; Proverbio, D.; Zuccaro, A.; Spada, E. Low-level laser therapy: Case-control study in dogs with sterile pyogranuloma- tous pododermatitis. Vet. World 2016, 9, 882. [CrossRef] 21. Marchegiani, A.; Spaterna, A.; Cerquetella, M.; Tambella, A.M.; Fruganti, A.; Paterson, S. Fluorescence biomodulation in the management of canine interdigital pyoderma cases: A prospective, single-blinded, randomized and controlled clinical study. Vet. Dermatol. 2019, 30, 371-e109. [CrossRef] [PubMed] 22. Marchegiani, A.; Fruganti, A.; Gavazza, A.; Spaterna, A.; Cerquetella, M. Fluorescence Biomodulation for Canine Interdigital Furunculosis: Updates for Once-Weekly Schedule. Front. Vet. Sci. 2022, 9, 880349. [CrossRef] [PubMed] 23. Marchegiani, A.; Fruganti, A.; Spaterna, A.; Cerquetella, M.; Tambella, A.M.; Paterson, S. References The Effectiveness of Fluorescent Light Energy as Adjunct Therapy in Canine Deep Pyoderma: A Randomized Clinical Trial. Vet. Med. Int. 2021, 2021, 6643416. [CrossRef] 24. Marchegiani, A.; Tambella, A.M.; Fruganti, A.; Spaterna, A.; Cerquetella, M.; Paterson, S. Management of canine perianal ﬁstula with ﬂuorescence light energy: Preliminary ﬁndings. Vet. Dermatol. 2020, 31, 460-e122. [CrossRef] 25. Sellera, F.P.; Fernandes, M.R.; Sabino, C.P.; de Freitas, L.M.; da Silva, L.C.B.A.; Pogliani, F.C.; Ribeiro, M.S.; Hamblin, M.R.; Lincopan, N. Effective treatment and decolonization of a dog infected with carbapenemase (VIM-2)-producing Pseudomonas aeruginosa using probiotic and photodynamic therapies. Vet. Dermatol. 2019, 30, 170-e52. [CrossRef] [PubMed] 26. Tambella, A.M.; Attili, A.R.; Beribè, F.; Galosi, M.; Marchegiani, A.; Cerquetella, M.; Palumbo Piccionello, A.; Vullo, C.; Spaterna, A.; Fruganti, A. Management of otitis externa with an led-illuminated gel: A randomized controlled clinical trial in dogs. BMC Vet. Res. 2020, 16, 91. [CrossRef] [PubMed] g 27. Olivieri, L.; Cavina, D.; Radicchi, G.; Miragliotta, V.; Abramo, F. Efﬁcacy of low-level laser therapy on hair regrowth in dogs with noninﬂammatory alopecia: A pilot study. Vet. Dermatol. 2015, 26, 35–39.e11. [CrossRef] 28. Apostolopoulos, N.; Mayer, U. Use of ﬂuorescent light energy for the management of bacterial skin infection associated with canine calcinosis cutis lesions. Vet. Rec. Case Rep. 2020, 8, e001285. [CrossRef] 29. Straus, S.E.; Richardson, W.S.; Glasziou, P.; Haynes, R.B. Evidence-Based Medicine, 3rd Edition—How to Practice and Teach EBM, 3rd ed.; Elsevier Limited: Philadelphia, PA, USA, 2005. p R.; Kelly, A.M.; Cronin, P. How to Write a Critically Appraised Topic (CAT). Acad. Radiol. 2012, 19, 872–888 30. Sadigh, G.; Parker, R.; Kelly, A.M.; Cronin, P. How to Write a Critically Appraised Topic (CAT). Acad. [CrossRef] 31. Burns, P.B.; Rohrich, R.J.; Chung, K.C. The Levels of Evidence and their role in Evidence-Based Medicine. Plast. Reconstr. Surg. 2012, 128, 305–310. [CrossRef] 32. Wendt, O.; Lloyd, L.L. Grading Evidence and Critically Appraised Topics (CATs). In Proceedings of the Ninth Biennial Research Symposium of the International Society for Augmentative and Alternative Communication (ISAAC); Lage, D., Ed.; International Society for Augmentative and Alternative Communication (ISAAC): Toronto, ON, Canada, 2008; Volume 10, pp. 88–95. 33. Tonin, M.H.; Brites, F.C.; Mariano, J.R.; Freitas, K.M.S.; Ortiz, M.A.L.; Salmeron, S. Low-Level Laser and Antimicrobial Photo- dynamic Therapy Reduce Peri-implantitis–related Microorganisms Grown In Vitro. Eur. J. Dent. 2022, 16, 161–166. [CrossRef] [PubMed] 34. Cai, Z.; Li, Y.; Wang, Y.; Chen, S.; Jiang, S.; Ge, H.; Lei, L.; Huang, X. 36. Zein, R.; Selting, W.; Hamblin, M.R. Review of light parameters and photobiomodulation efﬁcacy: Dive into complexity. J. Biomed. Opt. 2018, 23, 120901. [CrossRef] References Antimicrobial effects of photodynamic therapy with antiseptics on Staphylococcus aureus bioﬁlm on titanium surface. Photodiagn. Photodyn. Ther. 2019, 25, 382–388. [CrossRef] 34. Cai, Z.; Li, Y.; Wang, Y.; Chen, S.; Jiang, S.; Ge, H.; Lei, L.; Huang, X. Antimicrobial effects of photodynamic therapy with antiseptics on Staphylococcus aureus bioﬁlm on titanium surface. Photodiagn. Photodyn. Ther. 2019, 25, 382–388. [CrossRef] 35. McEwen, S.A.; Collignon, P.J. Antimicrobial Resistance: A One Health Perspective. Microbiol. Spectr. 2018, 6, 1–26. [CrossRef] [PubMed] 36. Zein, R.; Selting, W.; Hamblin, M.R. Review of light parameters and photobiomodulation efﬁcacy: Dive into complexity. J. Biomed. Opt. 2018, 23, 120901. [CrossRef]
https://openalex.org/W4249327250	https://www.qeios.com/read/XDE19O/pdf	English	null	Tilsotolimod Sodium	Definitions	2,020	cc-by	127	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Tilsotolimod Sodium National Cancer Institute National Cancer Institute Qeios ID: XDE19O · https://doi.org/10.32388/XDE19O Source National Cancer Institute. Tilsotolimod Sodium. NCI Thesaurus. Code C125080. National Cancer Institute. Tilsotolimod Sodium. NCI Thesaurus. Code C125080. A proprietary synthetic oligonucleotide-based agonist of toll-like receptor 9 (TLR9), with potential immunostimulating activity. Upon administration, TLR9 agonist IMO-2125 binds to and activates TLR9 expressed by plasmacytoid dendritic cells (pDCs) and B-cells. This initiates immune signaling pathways, activates B-cells and pDCs, and induces a cytotoxic T-lymphocyte (CTL)-mediated immune response against tumor cells. TLR9 is a member of the TLR family, which plays a fundamental role in pathogen recognition and activation of innate immunity. Qeios ID: XDE19O · https://doi.org/10.32388/XDE19O 1/1
https://openalex.org/W1534168101	https://hal.archives-ouvertes.fr/hal-01000975/file/2010_Pop_Notulae%20Botanicae%20Horti%20Agrobotanica%20Cluj-Napoca_1.pdf	English	null	Auxin Control in the Formation of Adventitious Roots	Notulae botanicae Horti Agrobotanici Cluj-Napoca	2,011	cc-by	9,111	Auxin Control in the Formation of Adventitious Roots Tiberia I. T. I. Pop, Doru D. Pamfil, Catherine C. Bellini To cite this version: Auxin Control in the Formation of Adventitious Roots Tiberia I. T. I. Pop, Doru D. Pamfil, Catherine C. Bellini Auxin Control in the Formation of Adventitious Roots Tiberia I. T. I. Pop, Doru D. Pamfil, Catherine C. Bellini To cite this version: Tiberia I. T. I. Pop, Doru D. Pamfil, Catherine C. Bellini. Auxin Control in the Formation of Adventitious Roots. Notulae Botanicae Horti Agrobotanica Cluj-Napoca, 2011, 39 (1), pp.307 - 316. hal-01000975 To cite this version: Tiberia I. T. I. Pop, Doru D. Pamfil, Catherine C. Bellini. Auxin Control in the Formation of Adventitious Roots. Notulae Botanicae Horti Agrobotanica Cluj-Napoca, 2011, 39 (1), pp.307 - 316. hal-01000975 Distributed under a Creative Commons Attribution 4.0 International License Introduction to adventitious roots maize are predominantly composed of adventitious roots (Hochholdinger et al., 2004; Osmont et al., 2007).i Vegetative propagation is extensively used in agricul ture, horticulture and forestry for multiplying elite plants selected from natural populations or obtained in breed ing programs (Hartmann et al., 1990). The formation of adventitious roots is an essential step in vegetative propa gation and therefore if cuttings do not form roots, losses occur (de Klerk et al., 1999). Researchers developed new rooting treatments, examining the effects of plant growth regulators (Haissig and Davis, 1994) by a short exposure to a solution with a high auxin concentration or by dipping in rooting powder (auxin with talc). Noticeable progress has been made recently in the research on rooting, which is not a single process but a progressive process consisting of different steps, each with its own requirements (Kevers et al., 1997). Definition. Adventitious roots can arise naturally from stem tissue under stressful environmental conditions; they may also be induced by mechanical damage or following tissue culture regeneration of shoots (Li et al., 2009). They are postembryonic roots which arise from the stem and leaves and from nonpericycle tissues in old roots (Geiss et al., 2009). There are at least two pathways by which ad ventitious roots form: by direct organogenesis from estab lished cell types such as the cambium or from callus tissue following mechanical damage (e.g. cuttings). g g g g Induction phases. Rooting phases, which can be dis tinguished in various ways, have different hormone re quirements (de Klerk et al., 1999). De Klerk et al. (1997) showed that apple microcuttings are not very sensitive to auxin and cytokinin during the first 24 h. Dedifferentia tion occurs during this lag period and cells can respond to auxin. The root primordia originates from the cells between the vascular bundles which accumulate starch during the initial 24 h. Between 72-96 h, activated cells become com mitted to the formation of root primordia by the rhizo genic action of auxin in the induction phase, when auxin pulses induce the highest number of roots. On the histo logical level the starch grains present at 24 h are degraded during the next 24 h, the first cell divisions occurring on the first 48 h and by 96 h meristemoids are present. Auxin Control in the Formation of Adventitious Roots Tiberia I. POP1, 2 , Doru PAMFIL1 , Catherine BELLINI2, 3 1University of Agricultural Sciences and Veterinary Medicine, 3-5 Mănăştur, Cluj-Napoca, Romania; tiberiapop@yahoo.com 2Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umeå, Sweden; Catherine.Bellini@plantphys.umu.se 3Institut Jean-Pierre Bourgin, UMR 1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon, F-78026 Versailles Cedex, France Abstract Adventitious rooting is a complex process and a key step in the vegetative propagation of economically important woody, horticultural and agricultural species, playing an important role in the successful production of elite clones. The formation of adventitious roots is a quantitative genetic trait regulated by both environmental and endogenous factors. Among phytohormones, auxin plays an essential role in regulating roots development and it has been shown to be intimately involved in the process of adventitious rooting. Great progress has been made in elucidating the auxin-induced genes and auxin signaling pathway, especially in auxin response Aux/IAA and Auxin Response Factor gene families. Although some important aspects of adventitious and lateral rooting signaling have been revealed, the intricate signaling network remains poorly understood. This review summarizes some of the current knowledge on the physiological aspects of adventitious root formation and highlights the recent progress made in the identification of putative molecular players involved in the control of adventitious rooting. Despite much has been discovered regarding the effects and regulation of auxins on plant growth since the Darwin experiments, there is much that remains unknown. Keywords: Aux/IAA, Auxin Response Factor, shoot-born roots, vegetative propagation HAL Id: hal-01000975 https://hal.science/hal-01000975v1 Submitted on 29 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Notulae Botanicae Horti Agrobotanici Cluj-Napoca Notulae Botanicae Horti Agrobotanici Cluj-Napoca Available online at www.notulaebotanicae.ro Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 Print ISSN 0255-965X; Electronic 1842-4309 The plant hormone auxin Auxins, cytokinins, gibberellins, abscisic acid and ethyl ene are the five classical groups of plant hormones (Kende and Zeevaart, 1997). Ideas about hormone function have evolved from numerous experiments in which the applica tion of the hormones have shown to affect cell division in the vascular cambium, cell expansion and control of dif ferentiation into different types of cambial derivates (Mel lerowicz et al., 2001).h The formation of adventitious roots is a process induced and regulated by environmental and endogenous factors, such as temperature, light, hormones (especially auxin), sugars, mineral salts and other molecules. Phytohormones have direct (involved in cell division or cell growth) or in direct (interacting with other hormones or molecules) ef fects on plants. Over the last years, a multitude of models have been proposed to show how plant hormones interact to control plant development (Jaillais and Chory, 2010; Nemhauser et al., 2006; Santner and Estelle, 2009). IBA is used for rooting in commercial operations, fol lowed by IAA and NAA and chemical analogues synthe sized and examined for auxin-like activity. Auxin enters cuttings mostly via the cut surface (Kenney et al., 1969), even in microcuttings that are known to have a poorly functioning epidermis (Guan and De Klerk, 2000) and is rapidly taken up in cells by pH trapping (Rubery and Sheldrake, 1973) and by influx carriers (Delbarre et al., 1996). Auxin metabolism studies on adventitious root ing have been done on cuttings exposed for a prolonged period to auxin, but in other studies cuttings have been exposed to auxin for short periods (Diaz-Sala et al., 1996; Liu and Reid, 1992) An optimal auxin concentration for one of the three phases may be supraoptimal or subopti mal for the next. It was observed that apple microcuttings cultured continuously on medium with auxin (IAA, IBA or NAA) show the best rooting performance (a large num ber of roots is formed over a broad range of auxin concen trations) when cultured with IAA (de Klerk et al., 1997). Although roots may be induced by auxin, wounding is usually required to achieve rooting and it was suggested that WRCs (wounding-related compounds) play a main role in the dedifferentiation phase (de Klerk et al., 1999). Auxins are a group of tryptophan-derived signals, which are involved in most aspects of plant development (Woodward and Bartel, 2005). Introduction to adventitious roots Auxin is not required after 96 h and the concentrations favorable Plant development is modulated by genetic and envi ronmental factors, which have effects on auxin biosynthe sis, metabolism, transport, and signaling pathway (Han et al., 2009). Recent advances in studying mutations of Arabidopsis and rice increased the understanding of the role of auxin in the regulation of rooting mechanisms and molecular studies are essential to reveal the basic mecha nisms operating in adventitious root formation (de Klerk et al., 1999). Many dicots like Arabidopsis thaliana have a primary root that branches to generate several orders of lateral roots, while the root systems of crops like rice and Pop, T.I. et al. / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 308 the active growth substance from oat coleoptile tips can diffuse into a block of agar and maintain its stimulative ef fect of the growth of coleoptiles. He developed a bioassay called Avena test to identify and quantify the biological activity of this growth substances he called AUXIN (from Greek auxein, to increase, to grow) (Went, 1935). In the 30s, Kögl et al. (1933) isolated from human urine various substances including IAA (indole-3-acetic acid). After few mistakes, the IAA was finally identified as the mol ecule that stimulates coleoptile growth (Jacobs, 1979) and some years later was isolated from plants. Auxin has been described by Van der Lek (1941) and Haissig and Davis (1994). Practical application of auxin for rooting became possible when it was descovered that it also acts when added on the cut surface of cuttings (Hitchcock and Zim merman, 1936). After the discovery of IAA, IBA (indolic- 3-butyric acid) and NAA (1-naphthalene acetic acid) were synthesized chemically, their capability to induce roots was discovered by Zimmerman and Wilcoxon, 1935, and talc powder was introduced as a carrier for auxin (Grace, 1937). for the meristemoids formation become inhibitory during this phase. The meristemoids develop into root primordia and further into roots during the differentiation phase (de Klerk et al., 1999). The plant hormone auxin Auxin plays a major role in controlling growth and development of plants, early stages of embryogenesis, organization of apical meristem (phyllotaxy) and branching of the plant aerial parts (apical dominance), formation of main root, lateral and adventi tious root initiation (Went and Thimann, 1937). Auxin is also involved in gravitropism and phototropism (Kepinski and Leyser, 2005). These multiple effects across the plant result from its control of cell division, cell elongation and certain stages of differentiation (Davies, 2004). Auxin is synthesized mainly in young leaves and is actively trans ported to other tissues to coordinate growth and facilitate responses to environmental variations.hi p Discovery and history. The first observations suggest ing the presence of a chemical substance modifying plant growth in response to unilateral illumination date from 1880, when Darwin published “The power of movement in plants”. Tips of plants exposed to light curved toward the light, which led Darwin to believe that a chemical mes senger transports a signal from the coleoptile to the rest of the plant (Darwin, 1880). In 1913, Peter Boysen-Jensen removed the coleoptiles and plants growth stopped and then he replaced the coleoptile with a piece of agar and placed the coleoptile on top of the agar block. He proved that a signal must be transported from the coleoptile to the rest of the plant, as Darwin had originally deduced (Moore et al., 1995). In 1918, Arpad Paal accumulated the first evidence showing that growth signal is chemical in nature (Moore et al., 1995). In 1926, Went showed that Auxin effect on adventitious rooting Auxin and ethylene are often described as activators, while cytokinins and gibberellins are seen as inhibitors of adventitious root formation, even when some positive effects have been observed. The widely used sources of growth hormones for cuttings rooting are the IBA, NAA, IAA and commercialization root promoters (root-growing powders). The successful formation of adventitious roots is an obligatory phase of vegetative propagation in many woody plants; this being related to the presence of auxin Pop, T.I. et al. / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 309 itself was active or that it modulated the activity of IAA (Van Der Kriken et al., 1992). Although many researchers thought growth hormone treatment would promote root ing of cuttings, improve rooting number and lower the rate of partial rooting, the rooting ratio decreased while the hormone concentration increased. High hormone concentrations have side effects on the root development (Edson et al., 1991; Mason, 1989). Auxin can increase the rate of ethylene biosynthesis (Riov and Yang, 1989) and stimulate the production of ethylene correlating with the fact that the ACC synthase4 gene has been found to be an early auxin-induced gene (Abel et al., 1995). Auxin and ethylene relationship in root development has been shown by a number of isolated mutants that have resistance to both hormones. The potential auxin efflux component, AtPIN2, allelic to the ethylene-insensitive root1 (EIR1) is an example (Muller et al., 1998). Analogously, axr2 is a dominant mutant that gives resistance to both ethylene and auxin (Wilson et al., 1990). IAA induced ethylene production may be a factor involved in the stimulation of adventitious rooting (Pan et al., 2002). (Kim et al., 1998; McClelland et al., 1990). IAA was the first used to stimulate rooting of cuttings (Cooper, 1935) and soon after another auxin which also promoted rooting, IBA, was discovered and was considered even more effec tive (Zimmerman and Wilcoxon, 1935). Nowadays IBA is used commercially to root microcuttings and is more ef ficient than IAA (Epstein and Ludwig-Müller, 1993). i ( p g ) Auxin is one of the major endogenous hormones known to be intimately involved in the process of adventi tious rooting (Wiesman et al., 1988) and the physiological stages of rooting are correlated with changes in endoge nous auxin concentrations (Heloir et al., 1996). Genes asociated with the adventitious root formation Some mutants affected in hormone homeostasis or signaling are also affected in adventitious root formation. The ABA-deficient tomato mutants flacca and notabilis produce an excess of adventitious roots on the stems (Tal, 1966). Recently, it has been shown using notabilis mutant that adventitious root phenotype can be restored to wild type by expressing the LeNCED1 gene involved in ABA biosynthesis, suggesting that ABA can be a negative regula tor of adventitious roots (Thompson et al., 2004). Mutants overproducing auxin in Arabidopsis, like sur1 and sur2 (Boerjan et al., 1995; Delarue et al., 1998) or yucca (Zhao et al., 2001) produce adventitious roots on hypocotyls of light grown seedlings. SUR1 and SUR2 genes encode a C-S-lyase protein and the cytochrome P450 Cyp83B1, both involved in the indole glucosinolate pathway (Bak et al., 2001; Barlier et al., 2000; Mikkelsen et al., 2004). YUCCA1 gene encodes a flavin monooxygenase suitable for converting tryptamine in N-hydroxyl tryptamine in vitro (Zhao et al., 2001, 2002) and belongs to a family of YUCflavin mono-oxigenases from which 4 have a role in auxin biosynthesis (Cheng et al., 2006). The pattern of auxin action, despite its crucial role in adventitious root development, is still poorly understood. In Arabidopsis, the superroot (sur1 and sur2) mutants ac cumulate IAA and develop numerous adventitious roots on the hypocotyl and cuttings of different organs in the case of sur1 (Boerjan et al., 1995; Delarue et al., 1998). Re cently, differential roles for IAA and IBA have been found in the regulation of adventitious root formation from stem segments of Arabidopsis (Ludwig-Muller et al., 2005). f g p g Several gain-of-function iaa mutations affect produc tion of lateral or adventitious roots (Fukaki et al., 2002; Rogg et al., 2001; Tatematsu et al., 2004). TIBA, an auxin polar transport inhibitor, applied to the top of the hypo cotyls lowered the rate of root formation (Fabijan et al., 1981). It was suggested that endogenous IAA and exog enous IBA might interact to promote adventitious rooting in Arabidopsis stem segments. The performance of IBA versus IAA can be explained by several possibilities: higher stability, differences in metabolism, differences in transport and IBA as a slow release source of IAA. The conversion of IBA to IAA occurs in many plant species (Ludwig-Muller et al., 2005). Auxin effect on adventitious rooting High en dogenous auxin concentration is normally associated with a high rooting rate at the beginning of the rooting process (Blažková et al., 1997; Caboni et al., 1997). Auxins have been shown to be effective inducers of adventitious roots in many woody species (de Klerk et al., 1999; Diaz-Sala et al., 1996; Goldfarb et al., 1998; Selby et al., 1992) and are usually synthesized in the stem tip and tender leaves of aerial parts of plants and then transportted to the action site (Ljung et al., 2001). When applying exogenous auxin on cuttings, the endogenous auxin concentration reaches a peak after wounding (Gaspar et al., 1996; Gatineau et al., 1997) coinciding with the initiation of the rooting pro cess. The importance of the auxin during the expression phase was demonstrated in Populus sp. (Bellamine et al., 1998). Rice mutants affected in the expression of PIN FORMEDl (OsPIN1) gene, potentially involved in auxin polar transport, are affected in adventitious root emer gence and tillering confirming that the auxin concentra tion and distribution in the different tissues is important (Xu et al., 2005).h Studies have emphasized that polyamines play a role in adventitious rooting (Biondi et al., 1990; Hausman et al., 1994; Heloir et al., 1996) and a possible interrelationship between polyamines and auxin controlling rooting induc tion was suggested (Hausman et al., 1995). Genes asociated with the adventitious root formation However, in microcuttings of Malus sp., IBA induced more roots than IAA although it was converted to IAA only at very low levels, suggesting that either IBA Temperature-sensitive Arabidopsis mutants (rrd1, rrd2 and rrd4) affected in root redifferentiation were identified (Sugiyama, 2003) and it was suggested that RRD1 and RRD2 genes have a role in fundamental processes for active cell proliferation. RRD4 is involved in the acquisition step of cell proliferation during callus initiation in hypocotyl Pop, T.I. et al. / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 310 related transcription factors belonging to the GRAS gene family (DiLaurenzio et al., 1996; Helariutta et al., 2000). SHR has been demonstrated to directly regulate the ex pression of genes including SCR (Levesque et al., 2006) and a number of cell cycle components including the D- type cyclin, CYCD6; 1 (Sozzani et al., 2010). explants. Other temperature-sensitive mutants defective in various stages of adventitious root formation were iso lated (Konishi and Sugiyama, 2003). The root growth de fective mutants rgd1, rgd2 and rgd3 became defective after the establishment of the root apical meristem. The ROOT INITIATION DEFECTIVE S (RIDS) gene was identi fied as the MOR1 / GEM gene encoding a microtubule- associated protein (Konishi and Sugiyama, 2006). The rid 2-1 mutant is recessive temperature-sensitive mutant of Arabidopsis that was isolated by screening using adventi tious root formation as an index phenotype. RID2 gene encodes an evolutionarily conserved methyltransferase- like protein, which was localized in the nucleus, contrib uting to the nucleolar activity for pre-rRNA processing (Ohbayashi et al., 2011).h explants. Other temperature-sensitive mutants defective in various stages of adventitious root formation were iso lated (Konishi and Sugiyama, 2003). The root growth de fective mutants rgd1, rgd2 and rgd3 became defective after the establishment of the root apical meristem. The ROOT INITIATION DEFECTIVE S (RIDS) gene was identi fied as the MOR1 / GEM gene encoding a microtubule- associated protein (Konishi and Sugiyama, 2006). The rid 2-1 mutant is recessive temperature-sensitive mutant of Arabidopsis that was isolated by screening using adventi tious root formation as an index phenotype. RID2 gene encodes an evolutionarily conserved methyltransferase- like protein, which was localized in the nucleus, contrib uting to the nucleolar activity for pre-rRNA processing (Ohbayashi et al., 2011). Genes asociated with the adventitious root formation yp y ( ) Genes related to the induction of adventitious root ing in forest species have been described (Goldfarb et al., 2003; Lindroth et al., 2001a, 2001b; Sanchez et al., 2007). Recently, two genes in pine were characterized, a P. ra diata SCARECROW-LIKE1 gene (PrSCL1)(Sanchez et al., 2007) and P. radiata SHORT-ROOT (PrSHR)(Sole et al., 2008), both genes may play a role during the earliest stages of adventitious root formation. The expression of PrSHR gene during adventitious rooting is also affected by the presence of MDPUs (methylenedioxyphenyl urea) which could interact, directly or indirectly, with the auxin- signalling pathways in rooting-competent cuttings during adventitious rooting (Ricci et al., 2008). SCARECROW (SCR) is a putative transcription factor, expressed in cor tical and endodermal initials, and it is required for the asymmetric cell division that gives rise to cortex and endo dermis and to other tissues in aerial organs of Arabidopsis thaliana (DiLaurenzio et al., 1996; Heidstra et al., 2004; Wysocka-Diller et al., 2000). Arabidospis SCR (AtSCR) is also involved in the establishment of quiescent center identity and in the maintenance of the stem cell status of the surrounding initial cells during embryonic pattern for mation and postembryonal development (Sabatini et al., 2003), and its expression is associated with auxin distribu tion in the root apical meristem (DiLaurenzio et al., 1996; Sabatini et al., 1999). y The auxin insensitive rice mutant crl1/arl1 (crown root less1/adventitious rootless1) defective in adventitious roots formation was identified (Inukai et al., 2005; Liu et al., 2005). CRL1/ARL1, an auxin-responsive gene, encodes a nuclear protein containing ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) do main (Inukai et al., 2005; Liu et al., 2005). CRL1/ARL1 can be considered as a positive regulator for crown root for mation in rice. Phylogenetic reconstructions revealed that the allelic rice genes CRL1 and ARL1 (Liu et al., 2005), the maize gene RTCS (Taramino et al., 2007) and the Ara bidopsis genes LBD16 and LBD29 (Shuai et al., 2002) are closely related. The genes have been involved in different aspects of root formation, CRL1/ARL1 and RTCS genes are localized in synthenic regions of the genomes and their loss of function results in similar phenotypes indicating orthologous functions during shoot-borne root forma tion (Hochholdinger and Zimmermann, 2008; Inukai et al., 2005). ARF and AUX/IAA involved in adventitious rooting Great progress has been made in recent years in un derstanding the auxin response genes and auxin signaling (Parry and Estelle, 2006; Quint et al., 2009). The response to auxin includes a rapid initial cell growth response that may involve auxin-induced changes in pH, calcium and gene expression. Auxin response is regulated by AUX/ IAA proteins and the ARF (Auxin Response Factor) pro teins (Overvoorde et al., 2005). The Aux/IAA genes are induced in response to auxin, encoding small nuclear pro teins that share four domains (I, II, III and IV) and func tion as transcription factors that regulate downstream auxin responses (Guilfoyle and Hagen, 2007; Reed, 2001). Aux/IAA genes were identified in screens for mRNA transcripts induced rapidly by auxin. Aux/IAA proteins likely function as homodimers and/or heterodi mers and have been found to interact in homotypic and heterotypic associations in yeast two-hybrid experiments, and these interactions were dependent on the presence of domains III and IV (Kim et al., 1997; Rouse et al., 1998). Arabidopsis has 29 Aux/IAA proteins which have four conserved domains called I–IV. Domain I is a transcrip tional repression domain and can repress auxin gene in duction responses (Kim et al., 1997; Tiwari et al., 2003). Domanin II is recognized by SCFTIR1 and probably other closely related E3 ubiquitin ligases (Kepinski and Leyser, 2005). Domains III and IV constitute a dimerization do main and can interact with similar motifs in ARF proteins (Ulmasov et al., 1999). Gain-of-function mutations in motif II of several IAA genes stabilize the corresponding protein and affect developmental responses to auxin. In several cases these mutations decrease auxin-induced gene expression (Tatematsu et al., 2004). Aux/IAA proteins have short half-lives, suggesting a primary role for protein degradation in the regulation of their activity. Guilfoyle et al. (1998) assumed that domain II was responsible for the rapid turnover of these proteins, because mutations in this domain (like axr3 mutants) led to a semidominant gain- of-function phenotype (Guilfoyle et al., 1998). Worley et al. (2000) suggested that rapid degradation of Aux/IAA proteins is essential for a normal auxin response. It was also found that overexpression of the IAA17 protein in Ara bidopsis resulted in plants with an axr3-like phenotype (Worley et al., 2000). Aux/IAA proteins do not appear to bind DNA themselves but can affect the transcription of ARF-regulated genes by dimerising with ARFs (Guilfoyle and Hagen, 2007; Tiwari et al., 2003). Genes asociated with the adventitious root formation In Arabidopsis LBD16 and LBD29 genes are involved in lateral root formation (Okushima et al., 2007) and are activated by ARF7 and ARF19 which indicates that these LOB domain genes are early auxin responsive genes. Therefore, these closely related monocot and dicot LOB domain proteins all probably act early in auxin sig naling in the root, yet in different developmental contexts: The rice gene is involved in shoot-borne and lateral root formation, the maize gene in shoot-borne root formation and the Arabidopsis genes in lateral root initiation (Hoch holdinger and Zimmermann, 2008). i VvPRP1 and VvPRP2, induced in stem cuttings of Vi tis vinifera L. during rooting, encode proline-rich proteins. Induction of these genes is not enhanced by IAA treatment and the expression of the VvPRP1 is wound-inducible. The results suggest that the genes have an important role in the initiation of new roots by altering the cell wall mechanical properties to enable root emergence increasing the plastic ity of the cell wall (Thomas et al., 2003).h h The gene expression pattern during adventitious root development of Pinus contorta was investigated. During the root initiation phase, genes involved in cell replica tion and cell wall weakening and a transcript encoding a PINHEAD/ZWILLE-like protein were upregulated, while genes related to auxin transport, photosynthesis and cell wall synthesis were downregulated. During the root elongation phase downregulation of transcripts encoding proteins involved in cell replication and stress occurred (Brinker et al., 2004). Transgenic lines expressing an ac tive form of the Populus type-B cytokinin response regu lator PtRR13 (ΔDDKPtRR13) have a delayed rooting phenotype and cause misregulation of CONTINUOUS VASCULAR RING1, a negative regulator of vasculariza tion. Inappropriate cytokinin action via ΔDDKPtRR13 expression appeared to disrupt adventitious root develop Researchers have identified several genes that regulate indeterminate root growth in Arabidopsis. SHORT-ROOT (SHR) is a regulator of radial patterning and indetermi nacy of Arabidopsis thaliana primary root. SHR mutant fails to initiate cell division following germination. SHR is also required for the initiation and patterning of lateral root primordia, to maintain the indeterminate growth of lateral and anchor roots, regulating root-related develop mental processes (Lucas et al., 2011). p Another related protein, SCARECROW (SCR), has a similar role alongside SHR (Benfey et al., 1993; DiLau renzio et al., 1996). SHR and SCR genes encode closely Pop, T.I. et al. Genes asociated with the adventitious root formation / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 311 ment 24 h after shoot excision, when root founder cells are hypothesized to be sensitive to the negative effects of cytokinin (Ramirez-Carvajal et al., 2009). transition in crown root meristem and the emergence of crown root, providing new information about the molecu lar regulation of the emergence of crown root (Wang et al., 2011). An Arabidopsis transgenic line overexpressing ARF17 developed fewer adventitious roots than wild-type plants, confirming the potential role of ARF genes in the regula tion of adventitious root development by auxin (Sorin et al., 2005). It was shown that ARF17, a target of miR160, is a negative regulator, and ARF6 and ARF8, targets of miR167, are positive regulators of adventitious rooting. These results provide evidence of microRNA control of phenotypic variability and are an important step in under standing the molecular mechanisms regulating adventi tious rooting (Gutierrez et al., 2009). The proteomic anal ysis of ago1-3, sur1-3, sur2-1 and the sur2-1 ago1-3 double mutant led to the identification of 11 proteins, including three auxin-inducible GH3-like proteins, whose expres sion was altered by mutations, particularly in adventitious rooting formation. The results strongly suggest that those proteins will be valuable markers for quantitative genetic analysis of adventitious root development (Sorin et al., 2006). ARF and AUX/IAA involved in adventitious rooting Auxin response factors (ARF) are transcription fac tors that regulate the expression of auxin response genes MsAPK1, member of the plant kinases family con taining the Ankyrin-Protein Kinases (APKs), is induced by osmotic stress in roots of Medicago sativa and is related to two APK genes in Arabidopsis thaliana, AtAPK1 and AtAPK2. Promoter-GUS fusions assays revealed that Ara bidopsis APK genes show distinct expression patterns in roots and hypocotyls. The DN mutant lines showed in creased capacity to develop adventitious roots when com pared with control or MsAPK1-expressing plants (Del phine et al., 2008). p ) In a study made on tomato mutants, the epi (epinastic) mutation increased adventitious root formation and the Nr (Never ripe) mutation reduced the number of adven titious roots (Negi et al., 2010). This indicates a negative role for ethylene in lateral root formation of tomatoes and a positive role in adventitious root formation with modu lation of auxin transport as a central point of ethylene-aux in crosstalk (Negi et al., 2010). The treatment of tomatoes with AVG (aminoethoxyvinyglycine) and NPA (1-N- Naphthylphthalamic acid) resulted in a reduction of ad ventitious roots in waterlogged plants. Ethylene, perceived by the Nr receptor, stimulated auxin transport. Auxin accumulation in the base of the plant induces growth of adventitious roots, forming a new root system capable of replacing the one damaged by submergence (Vidoz et al., 2010).fl Auxin efflux carrier, PIN1, is a highly conserved gene family, which may play a key role in polar auxin transport (Friml and Palme, 2002).The finding that OsPIN1 is in volved in auxin-dependent adventitious root emergence and tillering provides a new insight into the function of the PIN1 family in rice (Xu et al., 2005). The rice gene OsCAND1, the homolog of Arabidopsis CAND1, is in volved in auxin signaling to maintain the G2/M cell cycle g Auxin response factors (ARF) are transcription fac tors that regulate the expression of auxin response genes (Guilfoyle and Hagen, 2007; Tiwari et al., 2003). Arabi Pop, T.I. et al. / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 312 dopsis has 22 ARF proteins encoded by 22 ARF genes, each containing a conserved DNA binding domain near their N terminus and a dimerization domain near their C terminus that can interact with a corresponding domain in Aux/IAA proteins (Ulmasov et al., 1999). References Abel S, Nguyen MD, Chow W, Theologis A (1995). ACS4, a primary indoleacetic acid-responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase in Arabidopsis thaliana. Structural characterization, expression in Escherichia coli, and expression characteristics in response to auxin [corrected]. J Biol Chem 270(32):19093-19099. Bak S, Tax FE, Feldmann KA, Galbraith DW, Feyereisen R (2001). CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis thaliana. Plant Cell 12:101-111. Barlier I, Kowalczyk M, Marchant A, Ljung K, Bhalerao R, Bennett M, Sandberg G, Bellini C (2000). The SUR2 gene of Arabidopsis thaliana encodes the cytochrome P450 CYP83B1, a modulator of auxin homeostasis. Proc Natl Acad Sci USA 97(26):14819-14824. Bellamine J, Penel C, Greppin H, Gaspar T (1998). Confirmation of the role of auxin and calcium in the late phases of adventitious root formation. Plant Grow Regul 26(3):191-194. Genetic studies have implicated different ARFs in di verse growth processes. Highly specific and/or dynamic patterns of gene expression have been observed for ARF7 in seedlings, roots and embryos development (Hardtke et al., 2004; Okushima et al., 2005; Wilmoth et al., 2005), ARF10 in the root caps development, vascular tissue of roots and leaves (Wang et al., 2005), and ARF19 in seed lings and roots development (Li et al., 2006; Okushima et al., 2005; Wilmoth et al., 2005). Mutations in ARF19 in combination with mutations in NPH4/ARF7, encod ing the most closely related adventitious root, cause phe notypes like a drastic decrease in lateral and adventitious roots. Benfey PN, Linstead PJ, Roberts K, Schiefelbein JW, Hauser MT, Aeschbacher RA (1993). Root Development in Arabidopsis-4 Mutants with Dramatically Altered Root Morphogenesis. Develop 119(1):57-70. Biondi S, Diaz T, Iglesias I, Gamberini G, Bagni N (1990). Polyamines and ethylene in relation to adventitious root formation in Prunus avium shoot cultures. Physiol Plant 78(3):474-483. Blažková A, Sotta B, Tranvan H, Maldiney R, Bonnet M, Einhorn J, Kerhoas L, Miginiac E (1997). Auxin metabolism and rooting in young and mature clones of Sequoia sempervirens. Physiol Plant 99(1):73-80. ARF and AUX/IAA involved in adventitious rooting The role of ARFs in plant development and growth has been revealed by studies on arf mutants. Mutations were identified: arf7/ nonphototropic hypocotyl 4 (nph4), which present defects in hypocotyls tropisms and resistance to auxin and ethylene (Harper et al., 2000; Waller et al., 2002) and arf19, which show insensitivity to auxin and ethylene (Li et al., 2006). arf double mutants (Remington et al., 2004) have stron ger phenotypes than the single mutants, suggesting that related ARFs might have redundant roles in Arabidopsis. Single mutants of arf7 and arf19 have reduced lateral and adventitious root numbers, but arf7 arf19 double mutants have extremely reduced numbers of adventitious and lat eral roots (Okushima et al., 2007; Wilmoth et al., 2005). While no phenotypic defects were reported for single mu tants arf10 or arf16, arf10 arf16 double mutants have root cap defects and abnormal root gravitropism (Wang et al., 2005).f from data mining. Unfortunately, the adventitious root response genes are still not entirely identified, therefore this process represents an area open for research. on stem cuttings. Plant Physiol 10:789-794. Darwin C (1880). The Power of Movement in Plants. London: John Murray. Gatineau F, Fouche JG, Kevers C, Hausman JF, Gaspar T (1997). Quantitative variations of indolyl compounds including IAA, IAA aspartate and serotonin in walnut microcuttings during root induction. Biol Plant 39:131-137. Davies PJ (2004). Plant hormones: Biosynthesis, Signal Transduction, Action! Dordrecht, Kluwer Academic Publishers, The Netherlands. De Klerk GJ, van der Krieken W, de Jong J (1999). Review the formation of adventitious roots: New concepts, new possibilities. In Vitro Cell Develop Biol Plant 35(3):189- 199. Geiss G, Gutierrez L, Bellini C (2009). Adventitious Root Formation: New Insights and Perspectives, Wiley- Blackwell. Goldfarb B, Hackett WP, Furnier GR, Mohn CA, Plietzsch A (1998). Adventitious root initiation in hypocotyl and epicotyl cuttings of eastern white pine (Pinus strobus) seedlings. Physiol Plant 102(4):513-522. De Klerk GJ, Ter Bruge J, Marinova S (1997). Effectiveness of indoleacetic acid, indolebutyric acid and naphthalene acetic acid during adventitious root formation in vitro in Malus ‘Jork 9’. Plant Cell Tiss Organ Cult 49:39-44. Goldfarb B, Lanz-Garcia C, Lian ZG, Whetten R (2003). Aux/ IAA gene family is conserved in the gymnosperm, loblolly pine (Pinus taeda). Tree Physiol 23(17):1181-1192. Delarue M, Prinsen E, Onckelen HV, Caboche M, Bellini C (1998). Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis. Plant J 14(5):603-611. Grace NH (1937). Physiologic curve of response to phytohormones by seeds, growing plants, cuttings and lower plant forms. Can J Res C 15:538-546. Delbarre A, Müller P, Imhoff V, Guern J (1996). Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells. Planta 198:532-541. Guan H, De Klerk GJ (2000). Stem segments of apple microcuttings take up auxin predominantly via the cut surface and not via the epidermal surface. Sci Horti 86: 23- 32 Delphine C, Frugier F, Raices M, Merchan F, Giammaria V, Gargantini P, Gonzalez-Rizzo S, Crespi M, Ulloa R (2008). A mutant ankyrin protein kinase from Medicago sativa affects Arabidopsis adventitious roots. Funct Plant Biol 35(1):92-101. Guilfoyle TJ, Hagen G (2007). Auxin response factors. Current Opin Plant Biol 10(5):453-460. Guilfoyle TJ, Ulmasov T, Hagen G (1998). The ARF family of transcription factors and their role in plant hormone- responsive transcription. Cell Mol Life Sci 54(7):619-627. Diaz-Sala C, Hutchison KW, Goldfarb B, Greenwood MS (1996). Conclusions Boerjan W, Cervera MT, Delarue M, Beeckman T, Dewitte W, Bellini C, Caboche M, Onckelen HV, Montagu MV, Inze D (1995). Superroot, a Recessive Mutation in Arabidopsis, Confers Auxin Overproduction. Plant Cell Online 7(9):1405-1419. The process of adventitious root formation is very complex and plays a key role in the vegetative propagation of difficult-to-root genotypes. Although the importance of auxin in root development is well known, this plant hormone is not always efficient and the molecular mecha nisms involved in the formation of adventitious roots are still partly unknown, despite the efforts done by research ers all over the world. For improving rooting conditions of economically important genotypes, scientists need to elu cidate these molecular mechanisms through which auxin regulate adventitious rooting. Characterizing adventitious rooting mutants and identifying the genes responsible for the mutations leads to a better understanding of the regulation of the adventitious rooting mechanisms. New information on ARFs and AUX/IAA roles in plant growth and development has been revealed in the past years by forward and reverse genetic studies. Candidate genes regulated by ARFs, which may function in these mecha nisms, have been identified experimentally or predicted Brinker M, van Zyl L, Liu WB, Craig D, Sederoff RR, Clapham DH, von Arnold S (2004). Microarray analyses of gene expression during adventitious root development in Pinus contorta. Plant Physiol 135(3):1526-1539. Caboni E, Tonelli MG, Lauri P, Iacovacci P, Kevers C, Damiano C, Gaspar T (1997). Biochemical aspects of almond microcuttings related to in vitro rooting ability. Biol Plant 39:91-97. Cheng Y, Dai X, Zhao Y (2006). Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Dev 20(13):1790-1799. Cooper WC (1935). Hormones in relation to root formation Pop, T.I. et al. / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 313 313 (1996). Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell Develop Biol Plant 32(4):272- 289. on stem cuttings. Plant Physiol 10:789-794. Acad Sci USA 94(22):11786-11791. 3 Hartmann HT, Kester DE, Davies FT (1990). Plant Propagation: Principles and Practices. Prentice-Hall, Englewood Cliffs, NJ 5th ed., p. 647. Kim MS, Klopfenstein NB, Cregg BM (1998). In vitro and ex vitro rooting of micropropagated shoots using three green ash (Fraxinus pennsylvanica) clones. New For 16:43-57. Hausman JF, Gevers C, Gaspar T (1994). Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro. Physiol Plant 92(2):201-206. Kögl F, Haagen-Smit AJ, Erxleben H (1933). Z Physiol Chem, 241. Hausman JF, Kevers C, Gaspar T (1995). Auxin-polyamine interaction in the control of the rooting inductive phase of poplar shoots in vitro. Plant Sci 110(1): 63-71. Konishi M, Sugiyama M (2003). Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana. Develop 130(23):5637- 5647. Heidstra R, Welch D, Scheres B (2004). Mosaic analyses using marked activation and deletion clones dissect Arabidopsis SCARECROW action in asymmetric cell division. Genes Dev 18:1964-1969. Konishi M, Sugiyama M (2006). A Novel Plant-Specific Family Gene, Root Primordium Defective 1, is Required for the Maintenance of Active Cell Proliferation. Plant Physiol 140(2):591-602. Helariutta Y, Fukaki H, Wysocka-Diller J, Nakajima K, Jung J, Sena G, Hauser MT, Benfey PN (2000). The SHORT- ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101(5):555-567. Levesque MP, Vernoux T, Busch W, Cui HC, Wang JY, Blilou I, Hassan H, Nakajima K, Matsumoto N, Lohmann JU, Scheres B, Benfey PN (2006). Whole-genome analysis of the SHORT-ROOT developmental pathway in Arabidopsis. Plos Biol 4(5):739-752. Heloir MC, Kevers C, Hausman JF, Gaspar T (1996). Changes in the concentrations of auxins and polyamines during rooting of in-vitro-propagated walnut shoots. Tree Physiol 16(5):515-519. Li J, Dai X, Zhao Y (2006). A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis. Plant Physiol 140(3):899-908. Hitchcock AE, Zimmerman PW (1936). Effect of the use of growth substances on the rooting response of cuttings. Contrib. Boyce Thompson Inst 8:63-79. Li SW, Xue L, Xu S, Feng H, An L (2009). Mediators, Genes and Signaling in Adventitious Rooting. The Bot Rev 75(2):230- 247. Hochholdinger F, Park WJ, Sauer M, Woll K (2004). From weeds to crops: genetic analysis of root development in cereals. Trends Plant Sci 9(1):42-48. Lindroth AM, Kvarnheden A, von Arnold S (2001a). Isolation of a PSTAIRE CDC2 cDNA from Pinus contorta and its expression during adventitious root development. on stem cuttings. Plant Physiol 10:789-794. Maturation-related loss in rooting competence by loblolly pine stem cuttings: The role of auxin transport, metabolism and tissue sensitivity. Physiol Plant 97(3):481- 490. Gutierrez L, Bussell JD, Pacurar DI, Schwambach J, Pacurar M, Bellini C (2009). Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of auxin response factor transcripts and MicroRNA Abundance. Plant Cell 21(10):3119-3132. DiLaurenzio L, WysockaDiller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann KA, Benfey PN (1996). The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86(3):423-433. Haissig BE, Davis TD (1994). An historical evaluation of adventitious rooting research to 1993, p. 275-331. In: Davis TD, Haissig BE (Eds.). Biology of adventitious root formation. New York, London: Plenum Publishing Corporation. Edson JL, Wenny D, Fins L (1991). Propagation of Western Larch by Stem Cuttings. West J Appl Forest 6:47-49. Han H, Zhang S, Sun X (2009). A review on the molecular mechanism of plants rooting modulated by auxin. Afr J Biotech 8(3):348-353 Epstein E, Ludwig-Müller J (1993). Indole-3-butyric acid in plants: occurrence, synthesis, metabolism and transport. Physiol Plant 88:382-389. Hardtke CS, Ckurshumova W, Vidaurre DP, Singh SA, Stamatiou G, Tiwari SB, Hagen G, Guilfoyle TJ, Berleth T (2004). Overlapping and non-redundant functions of the Arabidopsis Auxin Response Factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Develop 131(5):1089-1100. Fabijan D, Taylor JS, Reid DM (1981). Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. Physiol Plant 53(4):589-597. Friml J, Palme K (2002). Polar auxin transport-old questions and new concepts? Plant Mol Biol 49(3-4):273-284. Harper RM, Stowe-Evans EL, Luesse DR, Muto H, Tatematsu K, Watahiki MK, Yamamoto K, Liscum E (2000). The NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue. Plant Cell 12(5):757-770. Fukaki H, Tameda S, Masuda H, Tasaka M (2002). Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant J 29(2):153-168. Gaspar T, Kevers C, Penel C, Greppin H, Reid D, Thorpe T Pop, T.I. et al. / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 314 cuttings. Plant Grow Regul 36(2):135-139. 2105. Mason WL (1989). Vegetative propagation of hybrid larch (Larix × eurolepis Henry) using winter cuttings. For Suppl 62:189-199. Parry G, Estelle M (2006). Auxin receptors: a new role for F-box proteins. Curr Opin Cell Biol 18(2):152-156. Quint M, Barkawi LS, Fan KT, Cohen JD, Gray WM (2009). Arabidopsis IAR4 modulates auxin response by regulating auxin homeostasis. Plant Physiol 150(2):748-758. McClelland MT, Smith MAL, Carothers ZB (1990). The effects of in vitro and ex vitro root initiation on subsequent microcutting root quality in three woody plants. Plant Cell Tissue Org Cult (Historical Archive) 23:115-123. Ramirez-Carvajal GA, Morse AM, Dervinis C, Davis JM (2009). The Cytokinin Type-B Response Regulator PtRR13 Is a Negative Regulator of Adventitious Root Development in Populus. Plant Physiol 150(2):759-771. Mellerowicz EJ, Baucher M, Sundberg B, Boerjan W (2001). Unravelling cell wall formation in the woody dicot stem. Plant Mol Biol 47(1-2):239-274. Reed JW (2001). Roles and activities of Aux/IAA proteins in Arabidopsis. Trends in Plant Sci 6(9):420-425. Mikkelsen MD, Naur P, Halkier BA (2004). Arabidopsis mutants in the C-S lyase of glucosinolate biosynthesis establish a critical role for indole-3-acetaldoxime in auxin homeostasis. Plant J 37(5):770-777. Remington DL, Vision TJ, Guilfoyle TJ, Reed JW (2004). Contrasting modes of diversification in the Aux/IAA and ARF gene families. Plant Physiol 135(3):1738-1752. Moore R, Clark DW, Stern KR, Vodopich D (1995). Botany. Wm. C. Brown Communications Inc., Dubuque, USA. Ricci A, Rolli E, Dramis L, Diaz-Sala C (2008). N,N ‘-bis- (2,3-methylenedioxyphenyl)urea and N,N ‘-bis-(3,4- methylenedioxyphenyl)urea enhance adventitious rooting in Pinus radiata and affect expression of genes induced during adventitious rooting in the presence of exogenous auxin. Plant Sci 175(3):356-363. Muller A, Guan C, Galweiler L, Tanzler P, Huijser P, Marchant A, Parry G, Bennett M, Wisman E, Palme K (1998). AtPIN2 defines a locus of Arabidopsis for root gravitropism control. Embo J 17(23):6903-6911. Riov J, Yang S (1989). Ethylene and auxin-ethylene interaction in adventitious root formation in mung bean (Vigna radiata) cuttings. J Plant Grow Regul 8(2):131-141. Negi S, Sukumar P, Liu X, Cohen JD, Muday GK (2010). Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato. Plant J 61(1):3-15. Rogg LE, Lasswell J, Bartel B (2001). A gain-of-function mutation in IAA28 suppresses lateral root development. Plant Cell 13(3):465-480. Nemhauser JL, Hong F, Chory J (2006). Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses. Cell 126(3):467- 475. Acad Sci USA 94(22):11786-11791. Plant Physiol Biochem 39(2):107-114. Hochholdinger F, Zimmermann R (2008). Conserved and diverse mechanisms in root development. Current Opin Plant Biol 11(1):70-74. Lindroth AM, Saarikoski P, Flygh G, Clapham D, Gronroos R, Thelander M, Ronne H, von Arnold S (2001b). Two S-adenosylmethionine synthetase-encoding genes differentially expressed during adventitious root development in Pinus contorta. Plant Mol Biol 46(3):335-346. Inukai Y, Sakamoto T, Ueguchi-Tanaka M, Shibata Y, Gomi K, Umemura I, Hasegawa Y, Ashikari M, Kitano H, Matsuoka M (2005). Crown rootless1, which is essential for crown root formation in rice, is a target of an auxin response factor in auxin signaling. Plant Cell 17(5):1387-1396. Liu HJ, Wang SF, Yu XB, Yu J, He XW, Zhang SL, Shou HX, Wu P (2005). ARL1, a LOB-domain protein required for adventitious root formation in rice. Plant J 43(1):47-56. Jacobs WP (1979). Plant hormones and plant development. Cambridge, UK: Cambridge University Press. Liu HJ, Reid DM (1992). Auxin and ethylene-stimulated adventitious rooting in relation to tissue sensitivity to auxin and ethylene production in sunflower hypocotyls. J Exp Bot 43:1191-1198. Jaillais Y, Chory J (2010). Unraveling the paradoxes of plant hormone signaling integration. Nat Struct Mol Biol 17(6):642-645. Kende H, Zeevaart J (1997). The Five “Classical” Plant Hormones. Plant Cell 9(7):1197-1210. Ljung K, Bhalerao RP, Sandberg G (2001). Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J 28(4):465-474. Kenney G, Sudi J, Blackman GE (1969). The uptake of growth substances XIII. Differential uptake of indole-3yl-acetic acid through the epidermal and cut surfaces of etiolated stem segments. J Exp Bot 20:820-840. Lucas M, Swarup R, Paponov IA, Swarup K, Casimiro I, Lake D, Peret B, Zappala S, Mairhofer S, Whitworth M, Wang JH, Ljung K, Marchant A, Sandberg G, Holdsworth MJ, Palme K, Pridmore T, Mooney S, Bennett MJ (2011). SHORT- ROOT Regulates Primary, Lateral, and Adventitious Root Development in Arabidopsis. Plant Physiol 155(1):384- 398. Kepinski S, Leyser O (2005). Plant development: auxin in loops. Curr Biol 15(6):208-210. Kevers C, Hausman JE, Faivre-Rampant O, Evers D, Gaspar T (1997). Hormonal control of adventitious rooting: progress and questions. J Appl Bot Angew Bot 71:71-79. Ludwig-Muller J, Vertocnik A, Town CD (2005). Analysis of indole-3-butyric acid-induced adventitious root formation on Arabidopsis stem segments. J Exp Bot 56(418):2095- Kim J, Harter K, Theologis A (1997). Protein-Protein Interactions among the Aux/IAA Proteins. Proceed Nati Pop, T.I. et al. Acad Sci USA 94(22):11786-11791. / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 315 cuttings. Plant Grow Regul 36(2):135-139. cuttings. Plant Grow Regul 36(2):135-139. Rouse D, Mackay P, Stirnberg P, Estelle M, Leyser O (1998). Changes in auxin response from mutations in an AUX/IAA gene. Sci 279(5355):1371-1373. Ohbayashi I, Konishi M, Ebine K, Sugiyama M (2011). Genetic identification of Arabidopsis RID2 as an essential factor involved in pre-rRNA processing. The Plant J doi: 10.1111/j.1365-313X.2011.04574.x. Rubery PH, Sheldrake AR (1973). Effect of pH and surface charge on cell uptake of auxin. Nat New Biol 244(139):285- 288. Okushima Y, Fukaki H, Onoda M, Theologis A, Tasaka M (2007). ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. Plant Cell 19(1):118-130. Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold N, Weisbeek P, Scheres B (1999). An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 99(5):463-472. Okushima Y, Overvoorde PJ, Arima K, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Lui A, Nguyen D, Onodera C, Quach H, Smith A, Yu G, Theologis A (2005). Functional genomic analysis of the auxin response factor gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19. Plant Cell 17(2):444-463. Sabatini S, Heidstra R, Wildwater M, Scheres B (2003). SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem. Genes Dev 17(3):354-358. Sanchez C, Vielba JM, Ferro E, Covelo G, Sole A, Abarca D, De Mier BS, Diaz-Sala C (2007). Two SCARECROW-LIKE genes are induced in response to exogenous auxin in rooting- competent cuttings of distantly related forest species. Tree Physiol 27(10):1459-1470. Osmont KS, Sibout R, Hardtke CS (2007). Hidden branches: Developments in root system architecture. Ann Rev Plant Biol 58:93-113. Overvoorde PJ, Okushima Y, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Liu A, Onodera C, Quach H, Smith A, Yu G, Theologis A (2005). Functional genomic analysis of the auxin/indole-3-acetic acid gene family members in Arabidopsis thaliana. Plant Cell 17(12):3282-3300. Santner A, Estelle M (2009). Recent advances and emerging trends in plant hormone signalling. Nature 459(7250):1071- 1078. Shuai B, Reynaga-Pena CG, Springer PS (2002). The lateral organ boundaries gene defines a novel, plant-specific gene family. Plant Physiol 129(2):747-761. Pan R, Wang J, Tian X (2002). Influence of ethylene on adventitious root formation in mung bean hypocotyl Sole A, Sanchez C, Vielba JM, Valladares S, Abarca D, Diaz-Sala Pop, T.I. et al. 22:29-35. 22:29-35. Vidoz ML, Loreti E, Mensuali A, Alpi A, Perata P (2010). Hormonal interplay during adventitious root formation in flooded tomato plants. Plant J 63(4):551-562. Sorin C, Bussell JD, Camus I, Ljung K, Kowalczyk M, Geiss G, McKhann H, Garcion C, Vaucheret H, Sandberg G, Bellini C (2005). Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell 17(5):1343-1359. Waller F, Furuya M, Nick P (2002). OsARF1, an auxin response factor from rice, is auxin-regulated and classifies as a primary auxin responsive gene. Plant Mol Biol 50(3):415-425. Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY (2005). Control of root cap formation by MicroRNA- targeted auxin response factors in Arabidopsis. Plant Cell 17(8):2204-2216. Sorin C, Negroni L, Balliau T, Corti H, Jacquemot MP, Davanture M, Sandberg G, Zivy M, Bellini C (2006). Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development. Plant Physiol 140(1):349-364. Wang XF, He FF, Ma XX, Mao CZ, Hodgman C, Lu CG, Wu P (2011). OsCAND1 Is Required for Crown Root Emergence in Rice. Mol Plant 4(2):289-299. Sozzani R, Cui H, Moreno-Risueno MA, Busch W, Van Norman JM, Vernoux T, Brady SM, Dewitte W, Murray JAH, Benfey PN (2010). Spatiotemporal regulation of cell- cycle genes by shortroot links patterning and growth. Nature 466(7302):128-149. Went FW (1935). Auxin, the plant growth hormone. Bot Rev 1:162-182. Went FW, Thimann KV (1937). Phytohormones. New York, The Macmillan Company. Sugiyama M (2003). Isolation and initial characterization of temperature-sensitive mutants of Arabidopsis thaliana that are impaired in root redifferentiation. Plant Cell Physiol 44(6):588-596. Wiesman Z, Riov J, Epstein E (1988). Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings. Physiol Plant 74(3):556-560. Wilmoth JC, Wang S, Tiwari SB, Joshi AD, Hagen G, Guilfoyle TJ, Alonso JM, Ecker JR, Reed JW (2005). NPH4/ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root formation. Plant J 43(1):118-130. Tal M (1966). Abnormal stomatal behavior in wilty mutants of tomato. Plant Physiol 41:1387-1391. Taramino G, Sauer M, Stauffer JL, Multani D, Niu XM, Sakai H, Hochholdinger F (2007). The maize (Zea mays L.) RTCS gene encodes a LOB domain protein that is a key regulator of embryonic seminal and post-embryonic shoot-borne root initiation. Plant J 50(4):649-659. Wilson AK, Pickett FB, Turner JC, Estelle M (1990). cuttings. Plant Grow Regul 36(2):135-139. / Not Bot Hort Agrobot Cluj, 2011, 39(1):307-316 316 C (2008). Characterization and expression of a Pinus radiata putative ortholog to the Arabidopsis SHORT-ROOT gene. Tree Physiol 28(11):1629-1639. 22:29-35. A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid. Mol Gen Genetics 222(2):377-383. Tatematsu K, Kumagai S, Muto H, Sato A, Watahiki MK, Harper RM, Liscum E, Yamamoto KT (2004). MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana. Plant Cell 16(2):379-393. Woodward AW, Bartel B (2005). Auxin: regulation, action, and interaction. Ann Bot 95(5):707-735. Worley CK, Zenser N, Ramos J, Rouse D, Leyser O, Theologis A, Callis J (2000). Degradation of Aux/IAA proteins is essential for normal auxin signalling. Plant J 21:553-562. Wysocka-Diller JW, Helariutta Y, Fukaki H, Malamy JE, Benfey PN (2000). Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot. Develop 127(3):595-603. Thomas P, Lee MM, Schiefelbein J (2003). Molecular identification of proline-rich protein genes induced during root formation in grape (Vitis vinifera L.) stem cuttings. Plant Cell Environ 26(9):1497-1504. Xu M, Zhu L, Shou H, Wu P (2005). A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. Plant Cell Physiol 46(10):1674-1681. Thompson AJ, Thorne ET, Burbidge A, Jackson AC, Sharp RE, Taylor IB (2004). Complementation of notabilis, an abscisic acid-deficient mutant of tomato: importance of sequence context and utility of partial complementation. Plant Cell Environ 27(4):459-471. Zhao Y, Christensen SK, Fankhauser C, Cashman JR, Cohen JD, Weigel D, Chory J (2001). A Role for Flavin Monooxygenase-Like Enzymes in Auxin Biosynthesis. Sci 291(5502):306-309. Tiwari SB, Hagen G, Guilfoyle T (2003). The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell 15(2):533-543. Zhao Y, Hull AK, Gupta NR, Goss KA, Alonso J, Ecker JR, Normanly J, Chory J, Celenza JL (2002). Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3. Genes Dev 16(23):3100-3112. Ulmasov T, Hagen G, Guilfoyle TJ (1999). Activation and repression of transcription by auxin-response factors. Proc Natl Acad Sci USA 96(10):5844-5849. Van Der Kriken WM, Breteler H, Visser MHM (1992). Uptake and metabolism of indolebutyric acid during root formation on Malus microcuttings. Acta Bot Neerlandica 41:435-442. Zimmerman PW, Wilcoxon F (1935). Several chemical growth substances which cause initiation of roots and other responses in plants. Contrib Boyce Thompson Inst 7:209-229. Zimmerman PW, Wilcoxon F (1935). Several chemical growth substances which cause initiation of roots and other responses h in plants. 22:29-35. Contrib Boyce Thompson Inst 7:209-229. Van der Lek HAA (1941). Over eenige toepasingen van ‘groeistoffen’ in de practijk van de plantenteelt. Vakbl Biol
https://openalex.org/W3194497374	https://wes.copernicus.org/articles/6/1413/2021/wes-6-1413-2021.pdf	English	null	Response to referee 2 wes-2021-52	null	2,021	cc-by	12,358	Some effects of ﬂow expansion on the aerodynamics of horizontal-axis wind turbines David H. Wood1 and Eric J. Limacher2 1Department of Mechanical and Manufacturing Engineering, University of Calgary, Calgary T2N 1N4, AB, Canada 2Department of Mechanical Engineering, Federal University of Pará, Belém, Brazil Correspondence: David H. Wood (dhwood@ucalgary.ca) Received: 27 May 2021 – Discussion started: 7 June 2021 Revised: 25 August 2021 – Accepted: 9 October 2021 – Published: 9 November 2021 David H. Wood1 and Eric J. Limacher2 1Department of Mechanical and Manufacturing Engineering, University of Calgary, Calgary T2N 1N4, AB, Canada 2Department of Mechanical Engineering, Federal University of Pará, Belém, Brazil Correspondence: David H. Wood (dhwood@ucalgary.ca) Received: 27 May 2021 – Discussion started: 7 June 2021 Revised: 25 August 2021 – Accepted: 9 October 2021 – Published: 9 November 2021 Abstract. The ﬂow upwind of an energy-extracting horizontal-axis wind turbine expands as it approaches the rotor, and the expansion continues in the vorticity-bearing wake behind the rotor. The upwind expansion has long been known to inﬂuence the axial momentum equation through the axial component of the pressure, although the extent of the inﬂuence has not been quantiﬁed. Starting with the impulse analysis of Limacher and Wood (2020), but making no further use of impulse techniques, we derive its exact expression when the rotor is a circumferentially uniform disc. This expression, which depends on the radial velocity and the axial induction factor, is added to the thrust equation containing the pressure on the back of the disc. Removing the pressure to obtain a practically useful equation shows the axial induction in the far wake is twice the value at the rotor only at high tip speed ratio and only if the relationship between vortex pitch and axial induction in non-expanding ﬂow carries over to the expanding case. At high tip speed ratio, we assume that the expanding wake approaches the Joukowsky model of a hub vortex on the axis of rotation and tip vortices originating from each blade. The additional assumption that the helical tip vortices have constant pitch allows a semi-analytic treatment of their effect on the rotor ﬂow. Expansion modiﬁes the relation between the pitch and induced axial velocity so that the far-wake area and induction are signiﬁcantly less than twice the values at the rotor. There is a moderate decrease – about 6 % – in the power production, and a similar size error occurs in the familiar axial momentum equation involving the axial velocity. Some effects of ﬂow expansion on the aerodynamics of horizontal-axis wind turbines When a turbine extracts kinetic energy from the wind, the ﬂow must expand both upwind and downwind of the ro- tor. As noted on p. 185 of Glauert (1935) and by Goorjian (1972), the axial momentum equation may receive contri- butions from the pressure in the expanding ﬂow upwind of the rotor. The expansion causes the pressure force to have an axial component which alters the rotor thrust by an amount equal to the momentum ﬂux external to the rotor. This is be- cause the pressure forces acting on the cylindrical control surfaces at radius RCV in Fig. 1 are entirely radial. Although the role of pressure has been recognized for a long time and is discussed by Sørensen (2016) and van Kuik (2018) among others, a satisfactory analysis of it is lacking. The ﬁrst main Wind Energ. Sci., 6, 1413–1425, 2021 https://doi.org/10.5194/wes-6-1413-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics Figure 1. Control volumes (CVs) to be used in the present analysis. In both variants, the upstream face extends in z to −∞, where the velocity is the wind speed, and RCV ≫R. The downstream control surface is just downstream and just upstream of the rotor plane in CV1 and CV2, respectively, and the corresponding downstream control surfaces (CS) are labelled SD and SU. Taken from LW. Figure 1. Control volumes (CVs) to be used in the present analysis. In both variants, the upstream face extends in z to −∞, where the velocity is the wind speed, and RCV ≫R. The downstream control surface is just downstream and just upstream of the rotor plane in CV1 and CV2, respectively, and the corresponding downstream control surfaces (CS) are labelled SD and SU. Taken from LW. 3. viscous and/or Reynolds stresses can be neglected on the CV surfaces; result of the present analysis is a closed-form expression for the pressure force for a circumferentially uniform rotor. Limacher and Wood (2020) (hereinafter “LW”) investi- gated steady wind turbine thrust, T , using an impulse anal- ysis, whereby the pressure in the axial momentum equa- tion for any CV is replaced by terms that include vorticity ﬂuxes across the CV boundaries. This removal of pressure is achieved by the substitution of various integral identities into a standard momentum-based control volume analysis, as demonstrated by Noca (1997). We will use what we call the “impulse perspective” as explained below but not impulse techniques in this paper; the interested reader is referred to LW for a short history and more details. LW showed that by approximating a rotor as an actuator disc, T is given exactly by integration over the face SD of CV1, situated just down- wind of the rotor on the left of Fig. 1: 4. the axial, u, and radial velocity, v, are continuous through the rotor disc; 5. viscous drag is negligible; 6. w is zero in the upwind ﬂow and outside the wake; 7. the vorticity in the wake is concentrated in line vortices or vortex sheets aligned with the local streamlines in the rotating frame of reference – in other words, the wake vortices rotate rigidly with the blades and vortex lines and streamlines coincide; 8. to derive the local or differential form of Eq. 1 Introduction Conservation of axial and angular momentum are fundamen- tal principles for wind turbine analysis. They are applied us- ing control volumes (CVs) such as those in Fig. 1 or, more commonly, to a CV coinciding with a mean streamtube and extending into the far wake, the hypothetical region of no fur- ther wake development. For blade-element momentum the- ory, the CVs become expanding annular streamtubes inter- secting the elements. The change in axial or angular momen- tum of the ﬂow determines the net thrust or torque, respec- tively, acting on the rotor or blade elements (e.g. Burton et al., 2011; Hansen, 2015; Sørensen, 2016). Angular momen- tum is easier to analyze because in most cases it is generated only at the blades. Published by Copernicus Publications on behalf of the European Academy of Wind Energy e.V. 1414 1414 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics (1), the vor- ticity piercing the lateral boundaries of the annular CVs intersecting the blade elements must have no effect on the element’s thrust. T ρ = Z SD 1 2w2 + λwx dS, (1) (1) where ρ is the air density, and w is the circumferential veloc- ity (in the direction of θ in Fig. 1) on SD; in LW, w denoted the circumferential velocity at the rotor plane, which was as- sumed to be one-half that on SD. This assumption is also used in the present analysis. λ is the tip speed ratio (λ > 0 for clockwise rotation, as viewed from the positive z axis), and x is the radius normalized by the tip radius so that x ≤1 for the rotor. The downwind face of the second CV in the ﬁgure, SU, is just upwind of the rotor. The term “exact” will be used throughout this paper to indicate that no assump- tions beyond those listed below have been invoked. Taking the “wake” to be the ﬂow that has passed through the rotor, which rotates with a constant angular velocity, these assump- tions are as follows: where ρ is the air density, and w is the circumferential veloc- ity (in the direction of θ in Fig. 1) on SD; in LW, w denoted the circumferential velocity at the rotor plane, which was as- sumed to be one-half that on SD. This assumption is also used in the present analysis. λ is the tip speed ratio (λ > 0 for clockwise rotation, as viewed from the positive z axis), and x is the radius normalized by the tip radius so that x ≤1 for the rotor. The downwind face of the second CV in the ﬁgure, SU, is just upwind of the rotor. The term “exact” will be used throughout this paper to indicate that no assump- tions beyond those listed below have been invoked. Taking the “wake” to be the ﬂow that has passed through the rotor, which rotates with a constant angular velocity, these assump- tions are as follows: Assumption no. 7 simpliﬁes the terms involving the trailing vorticity crossing SD in the impulse derivation. Assumptions no. 3, no. 5, and no. 8 are likewise embedded in the equations derived by LW and are not explicitly required in the analysis to follow. As LW note, a thorough investigation of assump- tion no. https://doi.org/10.5194/wes-6-1413-2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics (3) thus becomes 1 ρ dT dx λ→∞ ≈2πλwx2 ≈4a(1 −a)πx, (6) (6) 1 ρ dT dx = 2π Z 0 1 2w2 + λwx xdθ = 2πwx w 2 + λx , (3) (3) recovering the familiar 4a(1 −a) integrand from classical momentum theory. At smaller λ, the relationship between the momentum- and impulse-based thrust expressions has not been fully investigated. where θ is the circumferential co-ordinate, deﬁned in Fig. 1. where θ is the circumferential co-ordinate, deﬁned in Fig. 1. This result is also not new: it is, for example, Eq. (4.24) of van Kuik (2018). It is often referred to as the Kutta– Joukowsky theorem for blade-element thrust because it gives the axial force, dT/dx, as the product of the circumferential velocity at the rotor, w/2 + λx, and the sum of the circu- lation on all blades, 2πwx. Impulse analysis, however, can also be applied if the CV outlet is moved to the far wake to give dT /dx in terms of the w in the far wake. The Glauert (1935) original derivation of Eq. (1) – based on the Bernoulli equation – also suggests the exactness of Eq. (3). In the next section, we express the contribution of pres- sure on the expanding upstream streamtube to actuator disc thrust. The section thereafter analyzes the local form of the thrust equation. It contains our second main result about the behaviour of a – that it is negligible at λ = 0 and a∞≈2a is possible at high λ only if Eq. (4) remains valid for ex- panding ﬂow. In Sect. 4, we apply the Biot–Savart law to an expanding Joukowsky wake, which contains only hub and tip vortices. On the further assumption of constant p, we show, again for the ﬁrst time, that a ≤a∞≤2a depending on the extent of the vortex expansion; the larger the expansion, the closer a approaches a∞. Not surprisingly, the far-wake radius is reduced as is the power extracted by the turbine. The ﬁnal two sections contain the general discussion and conclusions, respectively. Equation (2) can be derived using standard CV momen- tum analysis, but the authors are unaware of it appearing in the literature prior to LW. It is a natural outcome of the im- pulse perspective which we use to investigate the effects of ﬂow expansion on the conventional axial momentum equa- tion. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1415 main ﬁnding for circumferentially uniform, expanding ﬂow is main ﬁnding for circumferentially uniform, expanding ﬂow is For context, we now examine the connection between the impulse- and momentum-based approaches to turbine thrust, which requires a relationship between a and w. As explained by, for example, Wood et al. (2021), the Kawada– Hardin (KH) equations for the velocity ﬁeld of a constant pitch, p, constant radius helical vortex, Kawada (1936) and Hardin (1982), yield 0 = Z SU v2 −a2 dS = Z SD v2 −a2 dS, (2) (2) (2) where a = 1 −u (when u is normalized by the wind speed, U0) is the usual axial induction factor. To maintain consistency we use only normalized velocities from here on. The easiest was to do this is to mentally replace the density ρ by ρU2 0 . p x = w/2 a , (4) (4) as only half of the near-wake azimuthal velocity is induced by the wake (the other half is due to the blades). Pitch can also be related geometrically to a and λ by treating the wake as a non-expanding rigid helicoidal surface, as done by Okulov and Sørensen (2008). In the limit where λx ≫w, we have 0 van Kuik (2020) found Eq. (2) was satisﬁed by his model of the expanding ﬂow through a wind turbine rotor. When a and v are further assumed to be C0 continuous on SU and SD, Eq. (2) tells us that \|a\| = \|v\| at some radial location, and LW cite three simulations that show \|a\| ≈\|v\| near the rotor tip. The vanishing of the ﬁrst integral on SU in Eq. (2) is the more general result; the vanishing of the second inte- gral on SD follows from assumption no. 4 above. Until the end of Sect. 3, we treat the rotor as circumferentially uni- form. Since Eq. (1) contains no terms representing pressure redistribution, LW assert that its local version giving the con- tribution to the thrust at radius x is also exact: p x ≈1 −a λx , (5) (5) and the preceding two equations can be combined to give λwx ≈2a(1 −a). The high-λ limit of Eq. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics It will be shown that Eq. (2) is closely related to the ef- fects of pressure in the upwind ﬂow on the conventional axial momentum equation and the general relationship between a and the far-wake induction, a∞. T is derived in Sect. 4 of Sørensen (2016) and Sect. 5.2.4 of van Kuik (2018) using a CV ending in the far wake. We take the different approach of using the CVs shown in Fig. 1 because that choice clari- ﬁes the effects of expansion. We also make further use of the impulse form of the T equation. The derivations of the re- maining equations in this paper are straightforward and could have been easily done in the past if the impulse perspective had been available. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 8 remains an important area of future research, but as yet the assumption remains necessary to recover the Kutta– Joukowsky expression for local thrust that is conventionally employed in blade-element momentum (BEM) analyses. As such, we perpetuate the use of assumption no. 8 for the time being. We also note, emphatically, that none of the eight as- sumptions places any restrictions on ﬂow expansion. Since the impulse derivation of Eq. (1) is likewise unrestricted, the equation is exact in the presence of ﬂow expansion and for any distribution of w(x). Assumption no. 7 simpliﬁes the terms involving the trailing vorticity crossing SD in the impulse derivation. Assumptions no. 3, no. 5, and no. 8 are likewise embedded in the equations derived by LW and are not explicitly required in the analysis to follow. As LW note, a thorough investigation of assump- tion no. 8 remains an important area of future research, but as yet the assumption remains necessary to recover the Kutta– Joukowsky expression for local thrust that is conventionally employed in blade-element momentum (BEM) analyses. As such, we perpetuate the use of assumption no. 8 for the time being. We also note, emphatically, that none of the eight as- sumptions places any restrictions on ﬂow expansion. Since the impulse derivation of Eq. (1) is likewise unrestricted, the equation is exact in the presence of ﬂow expansion and for any distribution of w(x). 1. the ﬂow upwind of the rotor and outside the wake is inviscid, steady, and spatially uniform; Although Eq. (1) has been known since Glauert (1935) and appears in modern texts, such as Eq. (4.6) in van Kuik (2018), LW’s analysis provides the ﬁrst proof of its exactness when the trailing vortex sheets have ﬁnite thickness. LW’s second 2. the total energy of the wake is reduced instantaneously at the rotor, after which it is conserved; Wind Energ. Sci., 6, 1413–1425, 2021 https://doi.org/10.5194/wes-6-1413-2021 2 Actuator disc thrust for expanding ﬂow Some results of the impulse analysis can be converted easily to conventional equations containing the axial velocity and the pressure on the CV surface even when the ﬂow expands through the rotor. For example, Bernoulli’s equation for PU, the pressure on SU, is 2PU ρ = 1 −v2 −u2. (7) (7) https://doi.org/10.5194/wes-6-1413-2021 Wind Energ. Sci., 6, 1413–1425, 2021 Wind Energ. Sci., 6, 1413–1425, 2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1416 PU and all pressures considered herein are gauge pressures relative to the free-stream pressure in the wind. Equation (7) allows the removal of v2 from Eq. (2) to give where the last integrand is evaluated on BS. dx/dz gives the local slope of BS, so P dx/dz is the axial component of the pressure acting on BS. It follows immediately from Eqs. (12) and (13) that ∞ Z 0 PU ρ xdx = ∞ Z 0 u(1 −u)xdx, (8) 0 Z −∞ P dx dz BS xdz = 1 2 1 Z 0 a2 −v2 xdx, (14) (8) (14) which is also the outcome of a conventional momentum bal- ance on CV2. The momentum balance on CV1 yields which gives the ﬁrst quantiﬁcation known to the authors of the axial force due to the expanding ﬂow through a wind tur- bine rotor. It is easy to generalize this equation because there is no thrust extracted in the upwind ﬂow. For any x and z ≤0, which gives the ﬁrst quantiﬁcation known to the authors of the axial force due to the expanding ﬂow through a wind tur- bine rotor. It is easy to generalize this equation because there is no thrust extracted in the upwind ﬂow. For any x and z ≤0, T 2πρ = ∞ Z 0 u(1 −u)xdx − ∞ Z 0 PD ρ xdx, (9) (9) (9) z Z −∞ P dx dz S(x,z) xdz = 1 2 x Z 0 a2 −v2 xdx, (15) (15) where PD is the pressure on SD. It is important to note that the effective upper limit on the integrals in Eq. (9) is outside the rotor. Nevertheless, where S(x,z) is the streamsurface passing through (x,z) so that BS = S(1,0). The second integral is evaluated at z ∈[−∞,0]. 2 Actuator disc thrust for expanding ﬂow T 2πρ = 1 Z 0 PU −PD ρ xdx = 1 Z 0 1P ρ xdx, (10) (10) PD in Eq. (12) can be evaluated in the standard manner by assuming that the unsteady Bernoulli equation is valid from immediately behind the rotor to the far wake: since PD = PU for x > 1. In other words, there is no pressure jump at z = 0 outside the rotor. The thrust equation with inte- gration only over the rotor can be found by rewriting Eq. (9) as since PD = PU for x > 1. In other words, there is no pressure jump at z = 0 outside the rotor. The thrust equation with inte- gration only over the rotor can be found by rewriting Eq. (9) as −2PD ρ = u2 + v2 + w2 −u2 ∞−w2 ∞−2P∞ ρ −2λx∞w∞+ 2λxw, (16 −2PD ρ = u2 + v2 + w2 −u2 ∞−w2 ∞−2P∞ ρ −2λx∞w∞+ 2λxw, (16) (16) −2λx∞w∞+ 2λxw, T 2πρ = 1 Z 0 a(1 −a)xdx − 1 Z 0 PD ρ xdx + ∞ Z 1 a(1 −a)xdx − ∞ Z 1 PD ρ xdx. (11) where the far-wake terms have the subscript “∞”. The last two terms arise from the unsteady potential terms, evaluated by assuming rigid wake rotation (see appendix B of LW). Conveniently, these terms cancel due to conservation of an- gular momentum, yielding (11) −2PD ρ = u2 + v2 + w2 −u2 ∞−w2 ∞−2P∞ ρ . (17) (17) To remove the last two integrals for x ≥1, we use Eq. (7) for PD = PU and then Eq. (2) to arrive at To remove the last two integrals for x ≥1, we use Eq. (7) for PD = PU and then Eq. (2) to arrive at Combining Eqs. (12) and (17) we get Combining Eqs. (12) and (17) we get T πρ = 1 Z 0 1 −u2 ∞ xdx − 1 Z 0 2P∞ ρ −w2 + w2 ∞ xdx, (18) T πρ = 2 1 Z 0 a(1 −a)xdx −2 1 Z 0 PD ρ xdx + 1 Z 0 a2 −v2 xdx. (12) (18) where w∞and P∞are evaluated at x∞in the wake, con- nected to x at the rotor by a mean streamsurface. The ﬁrst integral in Eq. (12) contributes half of the conven- tional thrust. Wind Energ. Sci., 6, 1413–1425, 2021 3 Local thrust in expanding ﬂow (28) (28) In considering the local equation for dT/dx in the next section, it is useful to have the alternative form of Eq. (23) from the impulse analysis of LW. The direct application of LW’s Eq. (22), The exactness of the local form of Eq. (23) is not easy to establish in general because all three velocity components can be important in the wake and the total pressure is not constant. This is the ﬁrst reason we based our analysis on the CVs shown in Fig. 1 rather than one extending to the far wake. We note, however, that there is no interchange be- tween pressure on BS and axial momentum in the ﬂow out- side the far wake where v2 = a2 = 0. In other words, the in- terchange is completed before the far wake is reached. This is the second reason we used the CVs in Fig. 1. The local form of Eq. (23) will have a term corresponding to the bracketed term in Eq. (26) of 2u(1−u∞). Combining with Eq. (28), we retrieve the standard result that u = (1 + u∞)/2 or a∞= 2a which can be accurate only at high λ; note that the discussion The exactness of the local form of Eq. (23) is not easy to establish in general because all three velocity components can be important in the wake and the total pressure is not constant. This is the ﬁrst reason we based our analysis on the CVs shown in Fig. 1 rather than one extending to the far wake. We note, however, that there is no interchange be- tween pressure on BS and axial momentum in the ﬂow out- side the far wake where v2 = a2 = 0. In other words, the in- terchange is completed before the far wake is reached. This is the second reason we used the CVs in Fig. 1. The local form of Eq. (23) will have a term corresponding to the bracketed term in Eq. (26) of 2u(1−u∞). Combining with Eq. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics Note that Eq. (24) holds anywhere behind the rotor, i.e. for z > 0 with the second integral approaching zero as z ↓+0. As pointed out by van Kuik (2018) in conjunction with his Eq. (6.8), any swirl at the edge of the wake makes P∞(x∞) ̸= 0. The present analysis can accommodate this behaviour, but for the present we take the simpler path of assuming P∞(x∞) = 0. The main justiﬁcation for this assumption is that we expect the magnitude of the swirl to become negli- gible everywhere at the edge of the wake at high λ. When P∞(x∞) = 0, Eq. (18) reduces to The results for the far wake can be used to estimate the conventional thrust when λ = 0, and the expansion is neg- ligible by application to SD. Equation (20) will then be ap- proximately valid for a and PD replacing a∞and P∞, and comparison with Eq. (1) shows that the momentum ﬂux term 2a(1 −a) will be negligible. In other words, the thrust on a stationary disc occurs predominately through the pressure on its back face associated with w. T πρ ≈ 1 Z 0 1 −u2 ∞ xdx + 1 Z 0 w2xdx. (21) (21) 3 Local thrust in expanding ﬂow Deﬁning the axial induction in the far wake as a∞= 1−u∞, we obtain Deﬁning the axial induction in the far wake as a∞= 1−u∞, we obtain Having considered the thrust for the complete rotor, we now consider the local contribution at radius x. We continue to use a circumferentially uniform disc. It is easy to show that the local form of Eq. (12), T πρ ≈ 1 Z 0 a∞(2 −a∞)xdx + 1 Z 0 w2xdx, (22) (22) 1 πρ dT dx = 2a(1 −a) −2PD ρ + a2 −v2 x, (26) (26) and the standard thrust equation is recovered if a∞≈2a and w2 ≈0, which is typically the case at high λ but may not be generally correct. Note that Eq. (22) is accurate at λ = 0, where the ﬁrst integral is negligible but a∞̸= 2a. is exact for a circumferentially uniform disc in expanding ﬂow. This can be done in at least two ways. First, using Eq. (7) and simple manipulation, the bracketed terms can be written as To recover the classical thrust equation and to provide a comparison to the analyses of Sørensen (2016) and van Kuik (2018), we now move the downwind face of the CV to the far wake and use Eq. (20). This results in 1 −u2 −v2 −2PD ρ = 21P ρ , (27) (27) T 2πρ = R∞ Z 0 a∞(1 −a∞)xdx − R∞ Z 0 P∞ ρ xdx, = R∞ Z 0 a∞(1 −a∞)xdx + 1 2 R∞ Z 0 w2 ∞xdx. (23) and the pressure difference across the annulus containing the blade elements must give the exact thrust by assumption no. 4. Secondly, starting from Eq. (15) it is easy to prove that a2 −v2 in Eq. (26) accounts for the difference in pres- sure acting on the top and bottom of the expanding annular streamtube that intersects the blade elements. (23) We now consider the consequences of the exact Eq. (26) for the far wake. If the w and P∞terms in Eq. (17) are neg- ligible at high λ, the bracketed term in Eq. (26) becomes If we ignore the second integral in Eq. (18) and the integrals in Eq. (23) containing P and w, and assume a and a∞are constant with x, we again recover the conventional relation a∞≈2a by invoking conservation of mass. 2a(1 −a) −2PD ρ + a2 −v2 ≈1 −u2 ∞. 2 Actuator disc thrust for expanding ﬂow The ﬁrst and second integrals are components of conventional CV analysis, whereas the third integral is new. It makes Eq. (12) exact for an actuator disc when the ﬂow expands and will be shown below to be generally positive. We now change the CV from that shown in Fig. 1 to the more commonly used one formed by the bounding stream- surface (BS) dividing the ﬂow passing through the rotor from the external ﬂow. BS begins at z = −∞, where z is the ax- ial co-ordinate with origin at the rotor (Fig. 1). The vertical faces of the new CV are, therefore, subsets of those shown in Fig. 1. A straightforward momentum balance gives In the far wake, the pressure and circumferential velocity are related by dP∞/ρ dx = w2 ∞ x . (19) (19) p g y p We now change the CV from that shown in Fig. 1 to the more commonly used one formed by the bounding stream- surface (BS) dividing the ﬂow passing through the rotor from the external ﬂow. BS begins at z = −∞, where z is the ax- ial co-ordinate with origin at the rotor (Fig. 1). The vertical faces of the new CV are, therefore, subsets of those shown in Fig. 1. A straightforward momentum balance gives The relationship between the area integrals of P and w can be found using the technique introduced by McCutchen (1985) and rediscovered by Wood (2007): multiply both sides by x2 and integrate by parts for the left side. If P∞x2 →0 as x ↓0, and is zero at the edge of the far wake, then R∞ Z 0 P∞ ρ xdx = −1 2 R∞ Z 0 w2 ∞xdx. (20) T πρ = 2 1 Z 0 a(1 −a)xdx −2 1 Z 0 PD ρ xdx + 2 0 Z −∞ P dx dz BS xdz, (13) (20) (13) https://doi.org/10.5194/wes-6-1413-2021 https://doi.org/10.5194/wes-6-1413-2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1417 3 Local thrust in expanding ﬂow (28), we retrieve the standard result that u = (1 + u∞)/2 or a∞= 2a which can be accurate only at high λ; note that the discussion T 2πρ = ∞ Z 0 1 2w2 + λwx xdx + 1 2 ∞ Z 0 v2 −a2 xdx, (24) (24) together with v = 0 everywhere in the far wake and a∞= 0 for r > R∞gives T 2πρ = R∞ Z 0 1 2w2 ∞+ λw∞x xdx −1 2 R∞ Z 0 a2 ∞xdx. (25) (25) https://doi.org/10.5194/wes-6-1413-2021 Wind Energ. Sci., 6, 1413–1425, 2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1418 immediately below Eq. (22) shows the result does not hold at λ = 0. Further, from Eq. (7), sure is constant for all streamsurfaces. In addition, indepen- dence will hold in the sense that the integrands in Eqs. (23) and (25) must be equal. Thus, 2PU ρ = 1 −u2 −v2 = 2a(1 −a) + a2 −v2. (29) (29) 1 2w2 ∞+ λw∞x −1 2a2 ∞= a∞(1 −a∞) + 1 2w2 ∞, (31) (31) If Eq. (6) is valid, then Eq. (26) becomes If Eq. (6) is valid, then Eq. (26) becomes without making any assumption about the relationship be- tween a and a∞. p∞, the constant pitch of the constant ra- dius tip vortices, is related to the velocities by the equivalent of Eq. (4): p∞/x∞= w∞/a∞. Equation (31) can be rewrit- ten as without making any assumption about the relationship be- tween a and a∞. p∞, the constant pitch of the constant ra- dius tip vortices, is related to the velocities by the equivalent of Eq. (4): p∞/x∞= w∞/a∞. Equation (31) can be rewrit- ten as −2PD ρ ≈2a(1 −a) −a2 + v2 (30) −2PD ρ ≈2a(1 −a) −a2 + v2 −2PD ρ ≈2a(1 −a) −a2 + v2 (30) (30) for x ≤1. The preceding analysis shows that PU ̸= −PD in general, in contrast to the familiar results of one-dimensional momen- tum theory. PU = −PD would require a = v, which cannot hold everywhere for several reasons. First, v →0 as x ↓0, whereas there is no similar constraint on a. Secondly, we argued above that the ﬂow outside the wake has an axial momentum deﬁcit so a ≥0 but not necessarily equal to v for x > 1. 3 Local thrust in expanding ﬂow Equation (2) would then be violated if a = v for x ≤1. Thirdly, van Kuik (2018) Sect. 5.4.4 points out that there is no theoretical requirement that PU = −PD. They are unlikely, however, to differ greatly in general. This suggests v →a as x →1, as argued by LW and shown by the model calculations of van Kuik (2020), who found also that v was signiﬁcantly larger than a outside the wake until at least x ≈1.2. If a > v over most of the rotor, then the positive a2 −v2 in Eq. (26) corresponds to a positive pressure exerted by the external ﬂow on the wake. p∞= 1 −a∞/2 λ . (32) p∞= 1 −a∞/2 λ . (32) In the next section, λ will be calculated using Eq. (32) for a given p∞and the corresponding calculated value of a∞. Equation (32) is the high−λ equivalent of Eq. (22) of Okulov and Sørensen (2008) for vortex pitch provided the convection velocity of the vortex – w in their notation but wv here – is equal to a∞/2. Table 1 of Wood and Okulov (2017) shows that wv →a for ideal Betz–Goldstein rotors as λ →∞, and so Eq. (32) is recovered since a∞→2a in the same limit. Another way to view this result is that the axial velocity in the Joukowsky far wake is constant and equal to 1 −a∞outside the vortex cores, so the tip vortices must travel downwind at a velocity of 1 −a∞/2 to be force-free. The KH equations for a doubly inﬁnite helical vortex of constant radius and pitch lead to A more deﬁnite statement about PU and PD can be made for stationary rotors (λ = 0) following the last paragraph of the previous section. It is shown there that a is negligible at λ = 0 so that PU ≈0 and PD is associated with w behind the rotor. The inequality reduces as λ increases but is always present because of non-zero a2 −v2. a∞= N0/(2πp∞). (33) (33) a∞= N0/(2πp∞). If we ignore wake expansion, then a ≈N0/(4πp) for the singly inﬁnite near-wake, and if p∞≈p, then a∞≈2a. This result suggests the strategy for the next Section, where we analyze the ﬂow associated with expanding tip vortices by assuming they have constant pitch everywhere. This allows a semi-analytic determination of their inﬂuence on the ﬂow through the rotor. 3 Local thrust in expanding ﬂow In other words, we relax one of the lim- itations of the KH equations, that of constant radius, which must be relaxed, but keep the limitation on p, which, hope- fully, leads to results of sufﬁcient generality. We now consider the far wake in more detail to determine the vortex pitch and its relation to a∞, which are required in the next section. Equation (19) requires P∞/ρ = −w2 ∞/2 when w∞∼1/x∞. We assume that at sufﬁciently high λ, the wake approximates a Joukowsky wake with the hub vortex lying along the axis of rotation and the tip vortices at radius R∞in the far wake, with no vorticity in between. The main justiﬁcation for this assumption comes from Eq. (1). When λ = 0, the ﬁrst term implies that the bound vorticity, 0, can- not be constant; Wood (2015) showed that 0 ∼x2. At high λ, however, the ﬁrst term becomes negligible in comparison to the second for most x. The simplest wake for which the thrust remains bounded on a turbine with N blades occurs when N0λ ∼λwx is constant in x and λ; this is the Joukowsky wake in which assumption no. 7 of Sect. 1 becomes irrele- vant to the ﬂow between the tip and hub vortices. Further, the tip vortices now separate the wake and the external ﬂow, which may have very different velocities. The vortex velocity should then be the average of these two, and the vortex lines need not align with the wake streamlines. 4 The expanding Joukowsky wake with constant pitch We assume p remains constant and use the results of the previous section and the Biot–Savart law to investigate the ﬂow immediately behind the rotor to determine the thrust and power coefﬁcients. The circumferentially averaged velocities are due entirely to the trailing vorticity: w is due to the hub vortex only, whereas u and v result from the expanding tip vortices only. 4.1 Biot–Savart analysis of expanding tip vortices Without loss of generality, let the lifting line representing one blade lie instantaneously along the x axis in Fig. 1 and con- Outside the hub vortex core of a Joukowsky wake, w∞∼ 1/x∞and, as pointed out by Sørensen (2016), the total pres- Wind Energ. Sci., 6, 1413–1425, 2021 https://doi.org/10.5194/wes-6-1413-2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1419 sider the tip vortex beginning at (1, 0, 0). We now determine the velocities induced at a point (x,θ,0) in cylindrical polar co-ordinates or (x cosθ,x sinθ,0) in Cartesian co-ordinates for constant p. A point on the vortex is (t(β),β,pβ) in Carte- sian co-ordinates or (t(β)cosβ,t(β)sinβ,pβ) in cylindrical polar co-ordinates, where radius t is a monotonically increas- ing function of the vortex angle β that asymptotes to the far- wake radius. Thus, 1 ≤t ≤R∞, and from here on, the depen- dence of t on β will be understood. An increment dl along the vortex is given by Ia is, clearly, dependent only on the geometry of the tip vor- tices. For an expanding wake with constant p, Eq. (4) will un- derestimate a as Ia(x) ≥Ia(0) ≥1/p when t is not constant. If p varied with β, then pβ in Eq. (36) would be replaced by R pdβ and the direct relation between ∂/∂z and (1/p)d/dβ would be lost. It is likely that an analytic expression for the integrands in Eq. (37) would not be possible. Performing the θ integration of Eq. (40) using Mathemat- ica gives iv(x) = pβ πx p p2β2 + (x + t)2 h 1 + m 2 E mp −K mp , and (42) ia(x) = −1 π p p2β2 + (x + t)2 1 + m 2 −mt 2r E mp −K mp , (43) dl = −t sinβ + dt dβ cosβ,t cosβ + dt dβ sinβ,p dβ, (34) (42) and the distance d from the point to the vortex is and the distance d from the point to the vortex is d = (x cosθ −t cosβ,x sinθ −t sinβ,−pβ), (35) (35) p −K mp , (43) (43) so that so that where m = 4xt/(p2β2+(x−t)2). E(.) and K(.) are the com- plete elliptic integrals of the second and ﬁrst kind, respec- tively, whose argument mp = m/(1 + m). 4.1 Biot–Savart analysis of expanding tip vortices Thus, v and a can be obtained by integrating Eq. (41) along the trajectory of the tip vortex, t(β) for 0 ≤β ≤∞. This must, in general, be done numerically, but several checks are possible. In de- scribing these, we continue to use the notation I = R idβ and identify the limits to the integral if they differ from (0,∞). d2 = x2 + t2 −2xt cos(β −θ) + p2β2, (36) (36) which is an even function of β and θ. A straightforward ap- plication of the Biot–Savart law yields the three velocities associated with the trailing tip vortex as (v(x,θ),w(x,θ),a(x,θ)) = 0 4π (Iv,Iw,Ia) = 0 4π ∞ Z 0 (iv(x,θ),iw(x,θ),ia(x,θ)) d3 dβ, (37) (v(x,θ),w(x,θ),a(x,θ)) = 0 4π (Iv,Iw,Ia) = If t remains constant at 1, say, and the integration is over −∞≤β ≤∞, that is, for a doubly inﬁnite vortex or vortices of constant radius and pitch, then Iv(−∞,∞) = 0 for any x, and 0 4π ∞ Z 0 (iv(x,θ),iw(x,θ),ia(x,θ)) d3 dβ, (37) (37) where 0 is the vortex strength, where 0 is the vortex strength, Ia(−∞,∞) = 2/p, for x < 1, = 1/p, for x = 1, and = 0, otherwise. (44) iv(x,θ) = −p tβ cos(β −θ) + t −β dt dβ sin(β −θ) , (38) iw(x,θ) = p x + β dt dβ −t cos(β −θ) −βt sin(β −θ) , and (39) ia(x,θ) = t2 −xt cos(β −θ) −x dt dβ sin(β −θ). (40) (44) The interior and exterior solutions in Eq. (44) are conse- quences of the KH equations, derived from the velocity po- tential. All results in Eq. (44) follow from Eq. (37). Using NIntegrate in Mathematica and MATLAB’s integral, these results were reproduced to six signiﬁcant ﬁgures for a similar range of x to that used in the main text and limits of ±1000π on the integration. For a singly inﬁnite helix, the values of Ia for z = 0 when β = 0 are half those in Eq. (44). These were reproduced numerically to the same accuracy. Iv is not avail- able from the KH equations for this case. (40) In forming the circumferential averages by integrating over 0 ≤θ ≤2π, all the sin(β −θ) terms will vanish as they are odd in θ. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics These behaviours could be miti- gated by using the well-known cut-off modiﬁcation to the limits of the Biot–Savart integrals as was done for helical vor- tices by Ricca (1994); see also Sect. 11.2 of Saffman (1992). There is, however, a simpler, heuristic alternative. The upper limit on a(x) as x →1 is taken to be a∞. A partial justiﬁca- tion for this tactic comes from the wind tunnel measurements of a model wind turbine by Krogstad and Adaramola (2012). Their Fig. 9c shows that at λ = 9.51, a ≈0 at small x but rises to the extraordinary value of around 0.8 at x = 1. Thus, Ia ≤2/p was enforced in the calculations described in the main text. Whenever this was done, Iv was assumed equal to the maximum value below the limit on Ia. Figure 2. Integrand, iv, for p = 0.1, R2∞= 1.597. ⃝, x = 0.9; □, x = 0.99,; and ⋄, x = 0.999 from Eq. (42). iv increases with x. × is the integrand in Eq. (45). For clarity, only every second data point is plotted. The solid line shows the remainders from Eq. (48). The differences with varying x are within the thickness of the line. at 1, Eq. (44) gives Ia = 1/p for any x, and the conventional momentum equation (Eq. 6) remains valid. We assume that 1/p is the minimum value of Ia, and, as explained in the Ap- pendix, we impose a ≤a∞so that 1/p ≤Ia ≤2/p. For max- imum power, the familiar derivation of the Betz–Joukowsky limit suggests R2 ∞≈2, so we investigate R∞around that value. Note, however, the use of Eq. (6) to derive this limit means that it is applicable only to a wake that expands either very slowly, as explained above, or very rapidly to t = √ 2, as Ia = 1/p for any constant t. We will show that generic wind turbine wakes at high λ expand at a rate that is intermediate between these extremes, which causes Eq. (6) to be inaccu- rate. There is no direct maximization of power output in the following analysis. Instead, the wake model is constrained as we now describe. The numerical evaluation of Ia and Iv can be improved by considering the asymptotic behaviour of ia and iv for large β, which corresponds to small m and mp. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1420 Figure 2. Integrand, iv, for p = 0.1, R2∞= 1.597. ⃝, x = 0.9; □, x = 0.99,; and ⋄, x = 0.999 from Eq. (42). iv increases with x. × is the integrand in Eq. (45). For clarity, only every second data point is plotted. The solid line shows the remainders from Eq. (48). The differences with varying x are within the thickness of the line. iv(x) ∼pβ p2β2 + x2 + t2 / q p2β2 + (x + t)2/x p2β2 + (x −t)2 , and (45) ia(x) ∼− p2β2 + x2 −t2 / q p2β2 + (x + t)2/ p2β2 + (x −t)2 , (46) (45) (46) showing that a logarithmic singularity occurs in ia despite it being the integrand for the circumferentially averaged ax- ial velocity. This is a stronger singularity than that in Chat- tot (2020) perturbation analysis of the ﬂow near the edge of the rotor, which assumes a vortex cylinder wake. There is no logarithmic singularity in iv, but the slope div/dβ increases without bound as β →0. These behaviours could be miti- gated by using the well-known cut-off modiﬁcation to the limits of the Biot–Savart integrals as was done for helical vor- tices by Ricca (1994); see also Sect. 11.2 of Saffman (1992). There is, however, a simpler, heuristic alternative. The upper limit on a(x) as x →1 is taken to be a∞. A partial justiﬁca- tion for this tactic comes from the wind tunnel measurements of a model wind turbine by Krogstad and Adaramola (2012). Their Fig. 9c shows that at λ = 9.51, a ≈0 at small x but rises to the extraordinary value of around 0.8 at x = 1. Thus, Ia ≤2/p was enforced in the calculations described in the main text. Whenever this was done, Iv was assumed equal to the maximum value below the limit on Ia. showing that a logarithmic singularity occurs in ia despite it being the integrand for the circumferentially averaged ax- ial velocity. This is a stronger singularity than that in Chat- tot (2020) perturbation analysis of the ﬂow near the edge of the rotor, which assumes a vortex cylinder wake. There is no logarithmic singularity in iv, but the slope div/dβ increases without bound as β →0. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics The leading terms are simple functions of β, allowing the inﬁnite integrals to be approximated. For Iv, we have Iv(x) ≈Iv(x, bβ) + Rv(bβ), (47) (47) where the ﬁrst term was obtained numerically over β = [0, bβ] and the remainder, Rv(bβ), is an approximation to the integral over β = [bβ,∞]. Rv(bβ) is where the ﬁrst term was obtained numerically over β = [0, bβ] and the remainder, Rv(bβ), is an approximation to the integral over β = [bβ,∞]. Rv(bβ) is Solving Eq. (37) for Ia and Iv requires p and the tip vortex trajectory. We used the very simple form: Rv(bβ) = xR2 ∞/ 2bβ3p4 . (48) (48) The remainder for Ia is independent of x: The remainder for Ia is independent of x: t = R∞−(R∞−1)exp(−kβ), (50) (50) Ra(bβ) = R2 ∞/ 2bβ2p3 . Ra(bβ) = R2 ∞/ 2bβ2p3 . (49) (49) which satisﬁes three necessary conditions: t = 1 when β = 0, t →R∞for large β, and t and its derivatives are continuous. The fourth condition is that k must satisfy the reduced ver- sion of Eq. (2): This result also follows from Eq. (41) when z ≫R∞. b This result also follows from Eq. (41) when z ≫R∞. b It was found that bβ = 200π was sufﬁcient to ensure six- ﬁgure accuracy of the integrals over the range of x considered below. Iv converged faster than Ia, reaching 99 % of the ﬁnal value by β = 2π for any x. ∞ Z 0 I 2 v −I 2 a xdx = 0. (51) Z 0 I 2 v −I 2 a xdx = 0. (51) (51) The Biot–Savart integrands in Eq. (43) are plotted in Figs. 2 and 3 for x close to the blade tip, in terms of axial distance z = pβ, where β is the vortex angle starting from zero at the rotor. The ﬁgures also show the small-β asymp- totes in Eqs. (45) and (46) and the large-β remainders de- ﬁned in Eqs. (48) and (49). If the tip vortex radius t remains This integral will be called the “expansion integral”. It uniquely ﬁxes k for any choice of R2 ∞and p. Ia and Iv were obtained using the MATLAB function integral over β = [0,200π] to an absolute tolerance of 10−6. https://doi.org/10.5194/wes-6-1413-2021 4.1 Biot–Savart analysis of expanding tip vortices The linearity of inviscid ﬂow leads to equal con- tributions to the averaged (u,w,a) from the N identical and equi-spaced trailing vortices. The simplest calculation of ia is for x = 0, for which the circumferential average a(0) = a(0,θ), and As with any Biot–Savart analysis, the behaviour of Eqs. (42) and (43) as x →t(0) = 1 must be considered. As mp →1, E(mp) ∼1, Eq. (19.6.1) of NIST DLMF (2021), and K(mp) ∼log(16/m′ p)/2, where m′ p + mp = 1, Eq. (17.3.26) of Abramowitz and Stegun (1964). The lead- ing terms in Eqs. (42) and (43) become a(0) = N0 4π ∞ Z 0 t2 t2 + p2β23/2 dβ, = N0 4πp ∞ Z 0 t2 t2 + z23/2 dz = N0 4πp ∞ Z 0 ia(0)dz, = N0 4π Ia(0). a(0) = N0 4π ∞ Z 0 t2 t2 + p2β23/2 dβ, = N0 4πp ∞ Z 0 t2 t2 + z23/2 dz = N0 4πp ∞ Z 0 ia(0)dz, = N0 4π Ia(0). (41) Wind Energ. Sci., 6, 1413–1425, 2021 https://doi.org/10.5194/wes-6-1413-2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics The remainders, This integral will be called the “expansion integral”. It uniquely ﬁxes k for any choice of R2 ∞and p. Ia and Iv were obtained using the MATLAB function integral over β = [0,200π] to an absolute tolerance of 10−6. The remainders, Wind Energ. Sci., 6, 1413–1425, 2021 https://doi.org/10.5194/wes-6-1413-2021 https://doi.org/10.5194/wes-6-1413-2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1421 Figure 3. Integrand, ia, for p = 0.1, R2∞= 1.597. ⃝, x = 0.9; □, x = 0.99,; and ⋄, x = 0.999 from Eq. (43). ia increases with x. × is the integrand in Eq. (46). For clarity, only every second data point is plotted. The solid line shows the remainders from Eq. (49). The mass ﬂux through the rotor, using Eq. (33) to re- move N0, determines a∞: 1 −a∞p 1 Z 0 Iaxdx = (1 −a∞)R2 ∞. (52) (52) Equation (32) then yields λ. A number of possible methods were considered for solving the integral in Eq. (52). ia(x,θ) can be written as ia(x,θ) = d dx x d −p2β2 d3 , (53) (53) which allows an analytic integration of ia(x,θ)x in x. The re- sulting expression is complicated and probably requires nu- merical integration in θ and β to obtain the mass ﬂux. Fur- ther, the integrand is singular at a point that varies with θ and β. The simpler alternative of numerical integration of Iax was used. Figure 3. Integrand, ia, for p = 0.1, R2∞= 1.597. ⃝, x = 0.9; □, x = 0.99,; and ⋄, x = 0.999 from Eq. (43). ia increases with x. × is the integrand in Eq. (46). For clarity, only every second data point is plotted. The solid line shows the remainders from Eq. (49). To ﬁnd the unique R2 ∞, we impose the further condition that k must match the slope of the vortex surface at the rotor. Then k in Eq. (50) equals k∗, given by dt dz(β = 0) = v(x = 1) 1 −a(x = 1) = (R∞−1) k∗ p . (54) Figure 4. Axial induction, a, and radial velocity, v, for the condi- tions in Table 1. p = 0.05: a, ⋄; v, ×. The corresponding results for p = 0.10 are the dashed lines. Note that −a is shown to make it distinct from v. D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics The solid lines are −a and v for no expansion, i.e. k = 0, R∞= 2. The x axis is logarithmic. (54) 4.2 Results The results in Table 1 were obtained using the MATLAB routine patternsearch to minimize the single objective func- tion that combined the magnitude of the expansion integral and \|k −k∗\|. This, surprisingly, occurred at a constant value of k∗/p, implying that the vortex expansion to the far-wake radius happens over a ﬁxed distance and the surface contain- ing the vortices has the same shape, independent of p or λ. g p p p CT was calculated from Eq. (1) with w2 ignored because λ is large: CT ≈N0λ/π ≈2a∞pλ ≈2a∞(1 −a∞/2), (55) (55) using Eqs. (32) and (33). We note that Eq. (1) makes the high- λ blade-element thrust constant across the rotor, whereas the familiar form involving the axial velocity equation in Eq. (6) requires a signiﬁcant variation near the tip. From conser- vation of angular momentum, and ﬁnding the power as the product of torque and angular velocity, Figure 4. Axial induction, a, and radial velocity, v, for the condi- tions in Table 1. p = 0.05: a, ⋄; v, ×. The corresponding results for p = 0.10 are the dashed lines. Note that −a is shown to make it distinct from v. The solid lines are −a and v for no expansion, i.e. k = 0, R∞= 2. The x axis is logarithmic. CP ≈CT (1 −a∞)R2 ∞, (56) (56) so the power extraction also decreases signiﬁcantly near the tip. Equation (56) and the third component of Eq. (55) also hold for the conventional analysis that leads to the Betz– Joukowsky limit. Eqs. (48) and (49), were then added. The expansion integral and the mass ﬂux integral described below were found by trapezoidal integration using the points shown in Fig. 4. The expansion integral is large for small k as v is (not obviously) maximized when there is very little vortex expansion near the rotor. Eqs. (48) and (49), were then added. The expansion integral and the mass ﬂux integral described below were found by trapezoidal integration using the points shown in Fig. 4. The expansion integral is large for small k as v is (not obviously) maximized when there is very little vortex expansion near the rotor. From Table 1, the biggest change from the familiar Betz– Joukowsky wake is the 20 % reduction in R2 ∞, which occurs because a > a∞/2 for much of the rotor (Fig. 4). In other https://doi.org/10.5194/wes-6-1413-2021 Wind Energ. https://doi.org/10.5194/wes-6-1413-2021 4.2 Results Using the data in Table 1, the integral of δP is 0.087, so the magnitude of PD is generally signiﬁcantly less than that of PU. It was shown in the previous section that the pressure integrals are equal in magnitude in the minimally expanding wake when λ = 0, but the analysis in this section shows di- vergence in the expanding Joukowsky wake at high λ. In the ﬁnal ﬁgure, Fig. 5, the ratio a/a∞documents the de- parture from the conventional relationship a∞= 2a near the rotor edge. Note that a∞is constant for a Joukowsky wake, so the ratio is non-zero for x > 1. g p g y g The integrands iv and ia Figs. 2 and 3 are large in the vicin- ity of the rotor. Their size implies that the simple assumed shape of the tip vortex trajectory, Eq. (50), is reasonable and that adding a term or terms, say, to control the approach to the far wake would not change the analysis signiﬁcantly. The low- and high-β asymptotic approximations to iv and ia are accurate at sufﬁciently large x for an appropriate range of β but are not sufﬁciently general to yield analytic approxima- tions to the integrals. The ﬁnal ﬁgure, Fig. 5, plots the ratio of a and a∞. The large differences from a∞= 2a occur near the edge of the rotor. 4.2 Results Sci., 6, 1413–1425, 2021 Table 1. Results for the expanding Joukowsky wake with constant pitch. Table 1. Results for the expanding Joukowsky wake with constant pitch. Table 1. Results for the expanding Joukowsky wake with constant pitch. p R2∞ k∗ λ a∞ CP CT C′ T 1CT 0.10 1.597 0.4947 7.13 0.574 0.557 0.819 0.866 0.047 0.05 1.592 0.2482 14.28 0.572 0.556 0.817 0.864 0.047 72 0.556 0.817 0.864 0.047 Figure 5. Ratio of axial induction at the rotor, a, to value in the far wake, a∞. p = 0.05; ⋄. p = 0.10; dashed line. Note that the x axis is logarithmic. words, more of the expansion occurs upwind of the rotor. In contrast, the maximum CP is reduced by only 6 % to 0.557 and the mass ﬂux is increased by 2 %. The second to last col- umn in Table 1 gives C′ T, the conventional thrust coefﬁcient evaluated from Eq. (6): words, more of the expansion occurs upwind of the rotor. In contrast, the maximum CP is reduced by only 6 % to 0.557 and the mass ﬂux is increased by 2 %. The second to last col- umn in Table 1 gives C′ T, the conventional thrust coefﬁcient evaluated from Eq. (6): C′ T = 8 1 Z 0 a(1 −a)xdx. (57) (57) The positive values of 1CT = C′ T −CT in Table 1 indicate the conventional equation overestimates the thrust by around 5 %. From Eqs. (29) and (30), we get the integrated asymme- try of the disc pressure distribution at high λ as 1 Z 0 PU ρ + PD ρ xdx ≈ 1 Z 0 a2 −v2 xdx. (58) (58) Figure 5. Ratio of axial induction at the rotor, a, to value in the far wake, a∞. p = 0.05; ⋄. p = 0.10; dashed line. Note that the x axis is logarithmic. Using the data in Table 1, the integral of δP is 0.087, so the magnitude of PD is generally signiﬁcantly less than that of PU. It was shown in the previous section that the pressure integrals are equal in magnitude in the minimally expanding wake when λ = 0, but the analysis in this section shows di- vergence in the expanding Joukowsky wake at high λ. Wind Energ. Sci., 6, 1413–1425, 2021 https://doi.org/10.5194/wes-6-1413-2021 5 Discussion The pressure in the expanding ﬂow ahead of a wind turbine contributes to the axial force on the rotor and a momentum deﬁcit in the ﬂow outside the rotor. Researchers have been aware of these two effects for many years, but the present analysis provides the ﬁrst quantitative determination of them in Eqs. (14) and (15) derived by approximating the rotor as an actuator disc. The effects on the disc in integral and incre- mental form depend on a2 −v2, where v is the normalized radial velocity and a is the usual axial induction factor. Fur- ther, v2 −a2 can be used to quantify the external ﬂow dis- turbed by the wind turbine and so may be useful to the study of multiple rotors in close proximity, as analyzed by, for ex- ample, Branlard and Meyer Forsting (2020). One way to do this is by deﬁning IE as Figure 4 shows that a and v at the rotor for the cases in Table 1 are independent of p. a(0) = 0.296, which is less than the Betz–Joukowsky value of 1/3, and v(0) = 0 as it must. The results for v and a = 1/3 in an unexpanding wake (k∗= 0) are plotted as solid lines. Note that a = 0 for x > 1, but v is very large near the edge of the rotor, and it is clear that the expansion integral cannot be satisﬁed. The limit a ≤ a∞was applied near the blade tip for the expanding wake, where v has increased to be nearly equal but smaller than a. Outside the wake, v > a and takes until x = 3 to fall to 0.03. Similarly shaped distributions of a and v for a Joukowsky wake are shown in Fig. 5 of van Kuik (2020), who also found that Eq. (2) was satisﬁed in his low-λ simulations. IE = ∞ Z xBS v2 −a2 xdx = − xBS Z 0 v2 −a2 xdx, (59) (59) https://doi.org/10.5194/wes-6-1413-2021 Wind Energ. Sci., 6, 1413–1425, 2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1423 locity in relating the pressure at the rear of the disc to the far wake. These errors cancel, so the main failing of the con- ventional equation is the breakdown of the relation a∞= 2a when expansion is signiﬁcant. 5 Discussion 4, where an expanding Joukowsky wake comprising tip and hub vor- tices of constant pitch was analyzed. The conventional thrust equation is altered by around 5 %–10 %, depending on the trajectory of the tip vortices because the geometrical relation in Eq. (4) is modiﬁed by the expansion. Including v in the axial momentum equation effectively adds an extra unknown to the conservation equations that may render them useless unless another equation for u, v, or w could be derived. Further, high v may cause signiﬁcant al- terations to the lift and drag of the blade elements near the tip. To our knowledge, radial velocity effects on airfoil lift and drag have not been studied in the context of blade-element theory. The ﬁrst three sections of the paper used only the standard form of control volume (CV) analysis for axial momentum to determine the thrust of the rotor and the incremental thrust of the blade elements comprising the rotor. To clarify the ef- fects of expansion, most analysis in this paper used CVs with downwind faces in the immediate vicinity of the rotor, as op- posed to their common placement in the far wake. The rotor and the ﬂow are assumed to be circumferentially uniform. We argued in the Introduction that the impulse analysis provides a simple and novel perspective on the role of the pressure. The thrust equations derived in Sect. 3 for the rotor, and in Sect. 4 for the local ﬂow at any radius, contain the pressure acting on the downwind face of the actuator disc, which must be removed to make the equations suitable for actual blade analysis. Removal can be done accurately only for very low tip speed ratios where the expansion and its effects are small. The role of the radial velocity and ﬂow expansion is prob- ably more complicated in rotors with a limited number of blades than the actuator discs considered here. Eriksen and Krogstad (2017) measured u, v, and w immediately behind the rotor of a model three-bladed turbine out to a radius 20 % larger than the blade tip radius. They used phase-locked av- eraging to obtain the ﬂow ﬁeld as seen by an observer ro- tating with the blades. 5 Discussion The previous section suggests this breakdown is due to the expanding tip vortices at high λ in the Joukowsky wake. At the rotor, the slope of the stream- surface containing the tip vortices is 53◦for maximum power extraction, Table 1, so their trajectory is intermediate be- tween very slow and very rapid expansion, either of which would require a∞= 2a. This analysis used Eq. (32) for the pitch of the tip vortex, found by moving the CV outlet to the far wake and using LW’s impulse equation for thrust. We note that van Kuik (2020) estimated the streamsurface angle at the rotor edge to be 46◦, which is close to the present value. The effect of the expansion on a was constrained so that a ≤a∞ as an alternative to using a cut-off in the Biot–Savart integral. Figure 4 shows that a increased with radius to reach a∞in the streamtube bounded by the tip vortex, suggesting a very substantial effect of expansion. Qualitatively, this large value of a is in agreement with the wind tunnel measurements of Krogstad and Adaramola (2012), who found that a increased across the rotor to reach 0.8 at the tip at high λ. where xBS is the radius of BS at any z ≤0. The last integral is a consequence of Eq. (2) being valid for SU lying anywhere in the upwind ﬂow. IE must be zero in the undisturbed up- wind ﬂow. It then increases to its maximum value at the rotor according to the present analysis. IE then decreases in the wake to be zero in the far wake. In other words, the perturba- tion to the external ﬂow is complete by the time the far wake is reached. Note that the second equality in Eq. (59) does not hold in the wake. The impulse analysis of Limacher and Wood (2020) (LW) showed that the Kutta–Joukowsky (KJ) equations for rotor thrust, Eq. (1), and for the blade-element contributions to the thrust, Eq. (3), are exact in the presence of wake ex- pansion, where “exact” means using no more assumptions or approximations than the eight listed in the Introduction. The KJ equations, containing only the circumferential veloc- ity and tip speed ratio, are not equivalent to the conventional equation involving only the axial velocity, when the ﬂow ex- pands. This is the outcome of the analysis in Sect. 5 Discussion Signiﬁcant phase variations occurred in a and v, showing that the averages a2 and v2 over a blade revolution could be large even if the mean values of a and v are small. Nevertheless, the magnitude of both a and v was largest near the angular location of the blades, suggesting that the issues with radial deﬂection will occur in real turbines. We hope that these comments, and the present analysis, will inspire further measurements to be made far enough outside the wake to help clarify the role of ﬂow expansion and the disturbances to the external ﬂow. To the rotor thrust, the pressure along the bounding streamsurface adds a term containing the integral of a2 −v2 over the rotor. This integral is equal and opposite the inte- gral for the ﬂow outside the wake, so there is no net con- tribution to the thrust determined using the CVs shown in Fig. 1. Unsurprisingly, the corresponding term in the local thrust equation at any x also contains a2 −v2. It follows that the conventional local thrust equation implies a ≈v, but a2 is generally larger than v2 over the rotor, but more precise es- timates of v do not appear to be possible. a2 > v2 implies that the pressure adds to the rotor thrust and is associated with a momentum deﬁcit in the external ﬂow. The common derivation of the axial momentum equation, which leads to the Betz–Joukowsky limit, ignores the interaction of pres- sure and external momentum and then ignores the radial ve- https://doi.org/10.5194/wes-6-1413-2021 Code and data availability. All the MATLAB codes and data used in this analysis are available from the ﬁrst author. 6 Conclusion This research was funded by a Canadian Nat- ural Sciences and Engineering Research Council Discovery Grant. – derive in Sect. 5 the following results from the model of constant pitch, expanding tip vortices: Review statement. This paper was edited by Jens Nørkær Sørensen and reviewed by Gijs van Kuik and Emmanuel Branlard. a. the angle of the tip vortex surface to the wind direc- tion was 53◦for maximum power production, inde- pendently of the tip speed ratio and vortex pitch; b. because it is neither very small nor very large, this expansion leads to an error of around 6 % in the conventional thrust equation, which would be ac- curate for both extreme expansions; 6 Conclusion This analysis started from the impulse-derived Kutta– Joukowsky equation for wind turbine thrust, which does not involve the axial velocity. The equation is valid for any amount of expansion in the upwind ﬂow and the wake and https://doi.org/10.5194/wes-6-1413-2021 Wind Energ. Sci., 6, 1413–1425, 2021 1424 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics any distribution of bound circulation on the rotor. We were able to Author contributions. This study was conceived by both authors, who shared the analytic development. DHW completed the numer- ical analysis and the ﬁrst draft of the manuscript, with both authors contributing equally to its revision thereafter. – demonstrate the conventional thrust equation containing the axial velocity can be correct only when the tip speed ratio is large; Competing interests. The contact author has declared that nei- ther they nor their co-authors have any competing interests. – derive an exact expression for the effects of ﬂow ex- pansion on the conventional momentum equation – this involves the axial induction factor and the radial veloc- ity; Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. – apply the conventional and impulse thrust equations in the far wake to give the pitch of the tip vortices in the Joukowsky wake in terms of the tip speed ratio and the far-wake induction; Acknowledgements. This paper was inspired partly by an anony- mous referee of LW who doubted the value of Eqs. (1) and (3) because they do not contain the axial velocity. We acknowledge the useful comments of Gijs van Kuik on an earlier draft of this paper. David H. Wood’s contribution to this work is part of a re- search project on wind turbine aerodynamics funded by the NSERC Discovery Program. Eric J. Limacher acknowledges receipt of an NSERC Postdoctoral Scholarship. – ﬁnd a semi-analytic solution of the Biot–Savart law for the induced velocities at the rotor by assuming the tip vortex had constant pitch – the axial velocity near the rotor tip approached the far-wake value, but was pre- vented from exceeding it as an alternative to using the familiar cut-off in the Biot–Savart integrals, and the in- crease in the rotor value contradicts the familiar relation that the axial induction factor everywhere at the rotor is half that of the far wake; and Financial support. References Abramowitz, M. and Stegun, I. A. (Eds.): Handbook of mathe- matical functions with formulas, graphs, and mathematical ta- bles (Vol. 55), US Government printing ofﬁce, Washington, DC, 1964. c. the resulting wake expands less than the familiar Betz–Joukowsky wake – for two pitch values cor- responding to tip speed ratios of 7 and 14, the far- wake area was 1.59 times the rotor area; Branlard, E. and Meyer Forsting, A. R.: Assessing the blockage ef- fect of wind turbines and wind farms using an analytical vortex model, Wind Energy, 23, 2068–2086, 2020. d. we ﬁnd the reduction in the rotor power and thrust due to expansion – the maximum power coefﬁcient and corresponding thrust coefﬁcient were 6 % less than the values given by the Betz–Joukowsky limit; and Burton, T., Jenkins, N., Sharpe, D., Bossanyi, E., and Graham, M.: Wind Energy Handbook, 3rd Edn., John Wiley & Sons, Chices- ter, UK, 2011. Chattot, J. J.: On the Edge Singularity of the Actuator Disk Model, J. Solar Energ. Eng., 143, 014502-1–014502-5, 2020. Å e. we quantify the inﬂuence of the expansion on the ﬂow outside the rotor – for example, the radial ve- locity at three rotor radii is still 3 % of the wind speed when the rotor is producing maximum power, and the axial induction factor decays to zero more rapidly than the radial velocity as radius. Eriksen, P. E. and Krogstad, P. Å.: An experimental study of the wake of a model wind turbine using phase-averaging, Int. J. Heat Fluid Flow, 67, 52–62, 2017. Glauert, H.: Airplane propellers. In Aerodynamic theory, Springer, Berlin, Heidelberg, 169–360, 1935. Goorjian, P. M.: An invalid equation in the general momentum the- ory of the actuator disc, AIAA J., 10, 543–544, 1972. Hansen, M. O.: Aerodynamics of Wind Turbines, Routledge, Lon- don, New York, 2015. Hardin, J. C.: The velocity ﬁeld induced by a helical vortex ﬁlament, Phys. Fluids, 25, 1949–1952, 1982. Wind Energ. Sci., 6, 1413–1425, 2021 https://doi.org/10.5194/wes-6-1413-2021 D. H. Wood and E. J. Limacher: Some effects of ﬂow expansion on the aerodynamics 1425 Saffman, P. G.: Vortex Dynamics, Cambridge University Press, Cambridge, 1992. Saffman, P. G.: Vortex Dynamics, Cambridge University Press, Cambridge, 1992. Kawada, S.: Induced velocity by helical vortices, J. Aeronaut. Sci., 36, 86–87, 1936. Krogstad, P. Å. and Adaramola, M. S.: Performance and near wake measurements of a model horizontal axis wind turbine, Wind En- ergy, 15, 743–756, 2012. https://doi.org/10.5194/wes-6-1413-2021 References Sørensen, J. N.: General Momentum Theory for Horizontal Axis Wind Turbines, Springer International Publishing, Heidelberg, https://doi.org/10.1007/978-3-319-22114-4, 2016. van Kuik, G. A. M.: The ﬂuid dynamic basis for actuator disc and rotor theories, IOS Press, Amsterdam, 2018. Limacher, E. J. and Wood, D. H.: An impulse-based derivation of the Kutta–Joukowsky equation for wind turbine thrust, Wind En- erg. Sci., 6, 191–201, https://doi.org/10.5194/wes-6-191-2021, 2021. van Kuik, G. A. M.: On the velocity at wind turbine and propeller actuator discs, Wind Energ. Sci., 5, 855–865, https://doi.org/10.5194/wes-5-855-2020, 2020. McCutchen, C. W.: A theorem on swirl loss in propeller wakes, J. Aircraft, 22, 344–346, 1985. Wood, D. H.: Including swirl in the actuator disk analysis of wind turbines, Wind Eng., 31, 317–323, 2007. NIST DLMF – Digital Library of Mathematical Functions, Release 1.1.1, available at: http://dlmf.nist.gov/, last access: 15 March 2021. Wood, D. H.: Maximum wind turbine performance at low tip speed ratio, J. Renew. Sustain. Energ., 7, 053126, https://doi.org/10.1063/1.4934805, 2015. Noca, F.: On the time-dependent ﬂuid-dynamic forces on bluff bod- ies, PhD thesis, California Institute of Technology, Pasadena, CA, 1997. Wood, D. H. and Okulov, V. L.: Nonlinear blade element- momentum analysis of Betz-Goldstein rotors, Renew. Energ., 107, 542–549, 2017. Okulov, V. L. and Sørensen, J. N.: Reﬁned Betz limit for rotors with a ﬁnite number of blades, Wind Energy, 11, 415–426, 2008. Wood, D. H., Okulov, V. L., and Vaz, J. R. P.: Cal- culation of the induced velocities in lifting line analy- ses of propellers and turbines, Ocean Eng., 235, 109337, https://doi.org/10.1016/j.oceaneng.2021.109337, 2021. Ricca, R. L.: The effect of torsion on the motion of a helical vortex ﬁlament, J. Fluid Mech., 273, 241–259, 1994. Wind Energ. Sci., 6, 1413–1425, 2021 https://doi.org/10.5194/wes-6-1413-2021
https://openalex.org/W3165326000	https://zenodo.org/record/3841108/files/%CE%B1-amylase%20and%20%CE%B1-glucosidase%20antidiabetic%20potential_Ojah.pdf	English	null	α-amylase and α-glucosidase antidiabetic potential of ten essential oils from Calophyllum inophyllum Linn	Iberoamerican journal of medicine	2,020	cc-by	5,708	IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 Emmanuel Onah Ojaha,* , Dorcas Olufunke Moronkolaa,b, Adeniyi-Akee Mukaram Akintundec aMedicinal Chemistry Research Group, Organic Chemistry Unit, Department of Chemistry, University of Ibadan, Ibadan, Nigeria bMarine and Biodiversity Center, Chemistry Department, Meston Building, University of Aberdeen, Scotland, United Kingdom cDepartment of Pharmaceutical Chemistry, College of Pharmacy, Igbinedion University, Okada, Edo State of Nigeria, Nigeria * Corresponding author. E-mail address: emmanuel.ojah@yahoo.com © 2020 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). http://doi.org/10.5281/zenodo.3841108 ABSTRACT Introduction: Diabetes mellitus (DM) is a multifactorial metabolic disorder which is of public health concern. Therapeutic intervention using reliable, affordable and non-toxic natural sources is crucial. Aim of the study: This research was designed to evaluate the α-amylase and α-glucosidase inhibitory activities of ten essential oils from Calophyllum inophyllum Linn. The study is part of our local sourcing for natural promising leads to ameliorating diabetes mellitus globally. g p g g g y Materials and methods: Essential oils from ten parts of C. inophyllum Linn were extracted by hydro- distillation using all-glass Clevenger-type apparatus. The percentage yields (w/v) were between 0.219 and 0.506 %. A plot of percentage inhibition versus concentration (mg/mL) of essential oils gave the IC50 values for each essential oil using non-linear regression analysis in reference to acarbose a standard anti-diabetic drug. Results: The following IC50 values (mg/mL) were obtained in the determination of α-amylase inhibition: [(Leaf, 0.043±0.05); (Leaf-stalk, 0.044±0.02); (Flower, 0.045±0.05); (Seed, 0.042±0.03); (Pod, 0.040±0.05); (Peel, 0.047±0.09); (Stem wood, 0.047±0.02); (Stem bark, 0.049±0.05); (Root wood, 0.048±0.05) and (Root bark, 0.046±0.04)] compared to acarbose (0.034±0.02). While α- glucosidase assay gave the following IC50 values (mg/mL): [(Leaf, 0.044±0.02); (Leaf-stalk, 0.043±0.03); (Flower, 0.044±0.04); (Seed, 0.048±0.02); (Pod, 0.038±0.04); (Peel, 0.048±0.03); (Stem wood, 0.048±0.04); (Stem bark, 0.048±0.02); (Root wood, 0.047±0.04) and (Root bark, 0.045±0.04)] with reference to acarbose (0.032±0.04). The high α-amylase and α-glucosidase inhibitory activity of pod essential oil in comparison with the reference drug must be due to the presence of some impact bioactive phyto-contituents in it. p y Conclusion: C. inophyllum Linn has been considered a fundamental source of potent anti-diabetic drugs which could be useful in the management of postprandial hyperglycemia. © 2020 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). occurring compounds characterized by high volatility [1]. Pharmacologically, essential oils have been used as insecticide, antimicrobial, antioxidant, pesticide, deodorants and as aroma-therapeutic agents; therefore, the 1. INTRODUCTION Once these partially digested saccharides reach the gut, they are extensively hydrolyzed by the α-amylase synthesized in the pancreas and excreted in the lumen into simpler oligosaccharides, such as maltose, maltotriose and α-limit dextrins [13]. These sugars are eventually broken down into glucose by α-glucosidases (intestinal brush border), which is in turn absorbed from the intestinal mucosa into the portal blood, by means of the glucose transporter and sodium-glucose co-transporter, leading to postprandial hyperglycaemia [14]. Impaired regulation of PPHG constitutes a common feature in type 2 diabetes mellitus (T2DM), the most prevalent form of diabetes and accounting for about 90 % of all diabetes cases [15, 16]. After a meal, α-amylase synthesized in the pancreas and released in the duodenum, catalyzes the hydrolysis of α-1,4 glycosidic linkages in partially hydrolyzed starch (amylopectin and amylose). From this reaction, intermediate unbranched, such as maltose and maltotriose, and branched (α-limit dextrins) oligosaccharides are formed. α-glucosidase present in the brush border of the intestinal epithelium (enterocytes) is responsible for the final step of carbohydrates digestion, prior to their absorption. This enzyme converts the disaccharides and oligosaccharides into glucose, which is Medicinal plants have been extensively applied in the treatment of diverse disease conditions, especially in developing economies where resources, affordability and access to modern treatment is a challenge. Volatile and non-volatile phytochemicals in medicinal plants possess several pharmacological and biological properties which have been the focus of researches targeted at prospection of reliable, affordable and potent drugs [20-22]. These constituents could be found in plant extracts or essential oils with great activity for different therapeutic applications [23, 24]. Thus, plant based natural inhibitors of α-amylase and α-glucosidase could be developed as phyto-therapeutic agents for the treatment of diabetes involving the decrease in postprandial hyperglycemia by inhibiting conversion of carbohydrate into glucose and then its absorption from the intestine. This inhibition reduces glucose absorption through delayed carbohydrate digestion and extended digestion time [25]. g The genus Calophyllum comprises of 180 to 200 species of which C. inophyllum Linn is the most abundant species. It is widespread in tropical areas, which tolerates varied kinds of soil such as coastal sand, clay or even degraded soil [26]. 1. INTRODUCTION Essential oils (EO) are complex, odoriferous and naturally IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 254 in the basolateral membrane of enterocytes, transports glucose from cytosol to blood via facilitated diffusion. The pancreatic α-amylase activity has been targeted for inhibition by means of the so-called starch blockers in order to mitigate PPHG [17]. Acarbose is the most widely prescribed α-amylase and α-glucosidase inhibitor, and in spite of its efficiency in the control of PPHG, the administration of this drug is accompanied by gastrointestinal adverse effects in diabetic patients such as; abdominal distention, flatulence and diarrhea [18]. Thus, the search and development of effective and safer therapeutic agents, able to control glucose levels without deleterious side effects is urgent for the management of type II Diabetes mellitus [19]. possibility of exploring them as anti-diabetic agent is important [2]. important [2]. Diabetes mellitus (DM) is a chronic endocrine disorder that affects the metabolism of carbohydrates. It includes a group of metabolic diseases characterized by hyperglycemia (HG), in which blood sugar levels are elevated either because the pancreas do not produce enough insulin or cells do not respond to production of insulin [4-5]. Out of about 382 million people living with Diabetes world-wide it is estimated that over 20 million people are living with the disease in sub-Saharan Africa. Nigeria has the highest number of people with diabetes with an estimated 3.9 million people of adult population [6]. An effective therapeutic approach to manage diabetes is by decreasing postprandial hyperglycemia (PPHG). It can be achieved by the regulation and/or inhibition of carbohydrate hydrolyzing enzymes like α-amylase and α- glucosidase which are utilized in the digestion of carbohydrates [7]. α-amylase is involved in the breakdown of long chain carbohydrates while α-glucosidase breaks down starch and disaccharides to glucose. These inhibitors are the potential targets utilized in the development of lead compounds for the treatment of diabetes. α-glucosidase, is a very important enzyme in carbohydrates digestion [8]. It catalyzes the 1,4- α-bonds of the unabsorbed oligo- and disaccharides, and converts them into monosaccharides, which are absorbed in the upper jejunum, resulting in PPHG [9, 10]. α-amylase catalyzes the initial hydrolysis of starch and other carbohydrate polymers into shorter oligosaccharides through cleavage of α-1,4- bonds. The salivary isozyme provides an initial partial cleavage into shorter oligomers [11, 12]. Where: Abs540 (control) is the absorbance of the control at wavelength 540 nm. Abs540 (EO) is the absorbance of essential oils at wavelength 540 nm. The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and were calculated by non-linear regression analysis from the mean inhibitory values. Acarbose was used as the reference α- amylase inhibitor. All tests were performed in triplicate [44]. 2.2. EXTRACTION OF ESSENTIAL OILS BY HYDRODISTILLATION Each separated part (leaf, stalk, flower, seed, pod, peel, stem wood, stem bark, root wood, and root bark) of C. inophyllum Linn was air-dried, pulverized and hydro- distilled for 3 hours in an all-glass Clevenger-type apparatus designed to British Pharmacopeia (BP) specifications. Essential oils were procured in 0.219 to 0.560% yields. Each of the oils had a distinct characteristic pleasant smell. The essential oils were refrigerated until the assay was carried out. 2.3. EXTRACTION OF WHEAT ALPHA-AMYLASE 1,3,5-trihydroxy-2-methoxy-xanthone, caloxanthone A, pyranojacareubin, caloxanthone B and tovopyrifolin [34]. The genus Calophyllum has been reported to be rich in coumarins [35], triterpenoids [36], and flavonoids [37]. Several coumarins isolated from two Calophyllum species were found to inhibit HIV-1 replication and cytophaticity activities. Xanthone derivative obtained from the root bark of C. inophyllum Linn has been identified as antimicrobial and cytotoxic agent [38]. Five bioactive compounds isolated from C. inophyllum Linn leaves namely; mixture of calophyllic and isocalophyllic acids, 3-oxo-friedelin-28- oic acid, canophyllic acid, amentoflavone, and shikimic acid showed dose-dependent lipid-lowering activity in in- vivo experiments [39]. Calophyllolide a complex coumarin from C. inophyllum Linn was reported as an anticoagulant and anti-inflammatory agent [40]. The plant has also been identified as a good anticancer agent [41]. Ojah et al, identified the chemical constituents of ten essential oils extracted from C. inophyllum Linn by GC-MS analysis. The study revealed that the plant is furnished with monoterpenes, sesquiterpenes and their oxygenated analogs [42]. Although some phytochemical constituents from the plant have been reported and volatile chemical constituents in the plant have been characterized in previous studies, no study has been performed on the α-amylase and α- glucosidase inhibitory potential of essential oils from different parts of the plant. 500 g of malted whole wheat flour was added slowly with mild stirring to 1 L of 0.2 % calcium acetate solution at room temperature and continuously stirred for 2 hours on a stirrer. The suspension was then centrifuged at 40 °C at 12000 g for 10 minutes. The resultant clear brown supernatant was stored at 2 °C to 3 °C prior to heat treatment. Since β-amylase interferes with the enzymatic determination of α-amylase, it was inactivated by heating the extract at 70 °C for 15 minutes. α-amylase is resistant to inactivation by this treatment at pH between 6.5 and 8.0. The pH of the extract was first adjusted to 6.6 with cold ammonium hydroxide (4%). Heat treatment was carried out at 85 °C to 90 °C and other at 72 °C to 74 °C using a water bath with continuous stirring. The extract was then cooled to 2 °C to 3 °C until use [43]. 2.4. DETERMINATION OF ALPHA-AMYLASE INHIBITION ACTIVITY The assay mixture containing 200 µL of sodium phosphate buffer (0.02 M), 20 µL of enzyme (0.25 mg/mL) and the essential oils over a concentration range 20-100 µg/mL were incubated for 10 minutes at room temperature followed by addition of 200 µL of starch (5 mg/mL) in all test tubes. The reaction was terminated with the addition of 400 µL DNS reagent and placed in boiling water bath for 5 minutes, cooled and diluted with 15 mL of distilled water and absorbance was measured at 540 nm using a Shimadzu UV-2000i double beam spectrophotometer. The control samples were prepared without the essential oils. The inhibition (%) was calculated according to the following formula (Eqn. 1) The aim of this study is to evaluate the α-amylase and α- glucosidase inhibitory activities of ten essential oils from C. inophyllum Linn. 1. INTRODUCTION The plant possesses a wide variety of uses ranging from traditional, medicinal and industrial applications; the wood has been used in general construction and boat building, as well as for flooring, furniture, musical instruments, handicrafts, and a variety of other purposes [27]. Several species of this genus are known to be used in folk medicine [28]. The extracted oil from the fruit is used as a remedy for sciatica, shingles, neuritis, rheumatism, ulcers, and skin diseases; while seed oil is reported to have medicinal and healing properties [29]. Decoction from dried leaves is widely used in curing rheumatism, skin infections, cuts and sores [30]. Extracts from leaves and stem bark expressed antihyperglycemic and antihyperlipidemic activities [31], while leaf extract was identified to inhibit oxidative stress [32]. Its fruits are effectively utilized in the treatment of dermatitis bark is locally utilized for treating vaginal disorders after childbirth, the passing of blood, gonorrhea, and internal haemorrhages [33]. The broad spectrum of biological activities expressed by C. inophyllum has been associated with the chemical composition of its different parts. The root is furnished with xanthones such as brasilixanthone, 255 IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 2.1. PLANT MATERIAL α Fresh samples of C. inophyllum Linn were collected from the Botanical garden, University of Ibadan, Ibadan, Oyo State, Nigeria. The samples were authenticated in the Herbarium, Department of Botany, University of Ibadan, Nigeria, where voucher samples were deposited with specimen voucher number UIH - 22659. The collection of the samples was done during the daytime. The plant was sorted into ten parts: leaf, stalk, flower, seed, pod, peel, stem wood, stem bark, root wood, and root bark. α 2.5. DETERMINATION OF YEAST ALPHA- GLUCOSIDASE INHIBITION ACTIVITY p-Nitrophenyl-α-D-glucopyranoside, acarbose, baker’s yeast alpha glucosidase was dissolved in 100 mM phosphate buffer (pH 6.8) and used as the enzyme extract. p-Nitrophenyl-α-D-glucopyranoside was used as the substrate. Essential oils were used in the concentration 256 IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 3.1. PERCENTAGE YIELD OF ESSENTIAL OILS FROM C.INOPHYLLUM LINN ranging from 0.02 to 0.1 mg/mL. Different concentrations of plant extracts were mixed with 320 µL of 100 mM phosphate buffer (pH = 6.8) at 30 °C for 5 minutes. 3 mL of 50 mM sodium hydroxide was added to the mixture and the absorbance was read at 410 nm using a Shimadzu UV- 2000i double beam spectrophotometer. The control samples were prepared without any essential oil. The inhibition (%) was calculated according to the formula below (Eqn. 2): Essential oils obtained from ten (10) parts of C. inophyllum Linn gave characteristic odours and colours. The oils were procured in 0.219 to 0.506 % yields (Table 1), with the highest yield from the peel, which gave 0.560 %, while the root had the lowest yield (0.219%), which may be due to its high fiber content. α α Abs410 (control) is the absorbance of the control at wavelength 410 nm. The percentage inhibition obtained for the standard anti- diabetic drug acarbose was relatively high for the concentration range used (0.1-0.02 mg/mL) as indicated on Table 2. Maximum percentage inhibition of 95.7 % was obtained for acarbose at 0.1 mg/mL and decreased slightly to 59.65 % at 0.02 mg/mL. The ten oils (Leaf, Leaf-stalk, Flower, Seed, Pod, Peel, Stem wood, Stem bark, Root wood, and Root bark) exhibited concentration-dependent inhibition similar to acarbose the standard anti-diabetic drug used. Percentage inhibition of the standard drug (95.68 %) was in close range with pod essential oil with inhibition efficiency of 80.32% at 0.1 mg/mL (Table 2). A graph of percentage inhibition versus concentration (mg/mL) of essential oils was plotted from which the IC50 values were obtained for each fraction using linear regression analysis in reference to the central standard. An inverse relationship exists between the percentage inhibition efficiency and the IC50 values. The higher the IC50 value the lower the activity of the essential oils and vice versa. The following IC50 values were obtained in the determination of α-amylase inhibition: [(Leaf, 0.043±0.05 Abs410 (EO) is the absorbance of essential oils at wavelength 410 nm. The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and were calculated by non-linear regression analysis from the mean inhibitory values. Acarbose was used as the positive control and all tests were performed in triplicate [45]. 3. RESULTS AND DISCUSSION Several medicinal plants have been reported to possess anti-diabetic activity and hence, the use of herbal drugs as complementary and alternative therapy to existing medications for the treatment of diabetes mellitus. Development of α-amylase and α-glucosidase inhibitor from natural products such as medicinal plants has been considered as a unique opportunity for a more economic management of diabetes. In recent years the popularity of alternative medicine has increased geometrically for various reasons ranging from potency to affordability [46]. Pterocarpus soyauxii a unique Nigerian medicinal plant has been identified as a potent anti-diabetic agent [47]. Aqueous extract of Salacia oblonga has been also identified as a good α-amylase and α-glucosidase inhibitor [48]. Hence the need to explore more plants with good anti-diabetic activity. mg/mL); (Leaf-stalk, 0.044±0.02 mg/mL); (Flower, 0.045±0.05 mg/mL); (Seed, 0.042±0.03 mg/mL); (Pod, 0.040±0.051 mg/mL); (Peel, 0.047±0.04 mg/mL); (Stem Table 1. Percentage (%) yield of ten essential oils from C. Inophyllum Linn Plant parts % yield (weight/volume) Color Odor Leaf 0.333 Pale Yellow Leafy Leaf-stalk 0.313 Colorless Herbal Flower 0.288 Colorless Floral Seed 0.305 Cloudy white Pleasant Pod 0.506 Pale Red Nut-like Peel 0.560 Pale Yellow Fruity Stem wood 0.341 Pale Yellow Woody Stem bark 0.307 Colorless Nut-like Root wood 0.219 Pale Red Woody Root bark 0.279 Pale Red Nut-like Table 1. Percentage (%) yield of ten essential oils from C. Inophyllum Linn Plant parts % yield (weight/volume) Color Odor Leaf 0.333 Pale Yellow Leafy Leaf-stalk 0.313 Colorless Herbal Flower 0.288 Colorless Floral Seed 0.305 Cloudy white Pleasant Pod 0.506 Pale Red Nut-like Peel 0.560 Pale Yellow Fruity Stem wood 0.341 Pale Yellow Woody Stem bark 0.307 Colorless Nut-like Root wood 0.219 Pale Red Woody Root bark 0.279 Pale Red Nut-like various reasons ranging from potency to affordability [46]. Pterocarpus soyauxii a unique Nigerian medicinal plant has been identified as a potent anti-diabetic agent [47]. Aqueous extract of Salacia oblonga has been also identified as a good α-amylase and α-glucosidase inhibitor [48]. Hence the need to explore more plants with good anti-diabetic activity. mg/mL); (Leaf-stalk, 0.044±0.02 mg/mL); (Flower, 0.045±0.05 mg/mL); (Seed, 0.042±0.03 mg/mL); (Pod, 0.040±0.051 mg/mL); (Peel, 0.047±0.04 mg/mL); (Stem Table 1. Percentage (%) yield of ten essential oils from C. 3. RESULTS AND DISCUSSION Inophyllum Linn Plant parts % yield (weight/volume) Color Odor Leaf 0.333 Pale Yellow Leafy Leaf-stalk 0.313 Colorless Herbal Flower 0.288 Colorless Floral Seed 0.305 Cloudy white Pleasant Pod 0.506 Pale Red Nut-like Peel 0.560 Pale Yellow Fruity Stem wood 0.341 Pale Yellow Woody Stem bark 0.307 Colorless Nut-like Root wood 0.219 Pale Red Woody Root bark 0.279 Pale Red Nut-like various reasons ranging from potency to affordability [46]. Pterocarpus soyauxii a unique Nigerian medicinal plant has been identified as a potent anti-diabetic agent [47]. Aqueous extract of Salacia oblonga has been also identified as a good α-amylase and α-glucosidase inhibitor [48]. Hence the need to explore more plants with good anti-diabetic activity. IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 257 d 0 047 0 02 / L) (S b k 0 049 0 05 Table 2. Percentage α-amylase inhibition of essential oils from C. Inophyllum Linn Plant parts Concentration (mg/mL) 0.1 0.08 0.06 0.04 0.02 Acarbose (standard) 95.68 83.54 72.34 65.87 59.65 Leaf 76.33 71.96 65.04 58.69 51.76 Leaf-stalk 72.80 68.74 64.78 58.57 52.34 Flower 74.98 70.81 65.90 61.96 54.69 Seed 73.86 65.98 64.80 56.97 49.87 Pod 80.32 73.33 69.40 60.73 55.22 Peel 72.16 67.74 61.77 56.63 51.94 Stem wood 70.58 66.81 63.99 58.39 50.26 Stem bark 73.46 65.11 61.31 54.54 50.21 Root wood 72.99 66.76 60.95 55.17 52.72 Root bark 71.86 67.91 62.45 59.13 53.22 Table 2. Percentage α-amylase inhibition of essential oils from C. Inophyllum Linn wood, 0.047±0.02 mg/mL); (Stem bark, 0.049±0.05 mg/mL); (Root wood, 0.048±0.05 mg/mL) and (Root bark, 0.046±0.04 mg/mL)] compared to the standard anti- diabetic drug acarbose (0.034±0.02 mg/mL). The standard anti-diabetic drug with the lowest IC50 value of 0.034±0.05 mg/mL exhibited the highest α-amylase inhibition activity followed closely by Pod essential oil (0.040±0.05 mg/mL) as indicated on the bar chart in Figure 1. The least activity was expressed by the highly fibrous stem bark essential oil 0.049±0.01 mg/mL. The high α-amylase inhibition of the pod essential oil in comparison with the standard anti- diabetic drug must be due to the presence of some important bioactive phyto-contituents. 3.3. ALPHA-GLUCOSIDASE INHIBITION OF C.INOPHYLLUM LINN The percentage inhibition obtained for the standard anti- diabetic drug acarbose was relatively high for the concentration range used (0.1-0.02 mg/mL). Optimum percentage inhibition of 96.34% was obtained for acarbose at 0.1 mg/mL and decreased slightly to 60.28 % at 0.02 mg/mL. This trend indicates that the percentage inhibition of the standard used in the study was concentration- dependent. The ten oils (Leaf, Leaf-stalk, Flower, Seed, Pod, Peel, Stem wood, Stem bark, Root wood, and Root bark) exhibited concentration-dependent inhibition similar to acarbose the standard anti-diabetic drug used. Percentage inhibition of the standard was in close range with the pod essential oil with inhibition efficiency of 80.32 % at 0.1 mg/mL as indicated in Table 3. A graph of percentage inhibition versus concentration (mg/mL) of essential oils was plotted from which the IC50 values were obtained for each essential oil using linear regression analysis in reference to the central standard. An inverse relationship exists between the percentage inhibition efficiency and the IC50 values. The higher the IC50 value the lower the activity of the essential oils and vice versa. The following IC50 values were obtained in the determination of α-amylase inhibition: [(Leaf, 0.044±0.02 mg/mL); (Leaf- stalk, 0.043±0.03 mg/mL); (Flower, 0.044±0.04 mg/mL); (Seed, 0.048±0.02 mg/mL); (pod, 0.038±0.04 mg/mL); (Peel, 0.048±0.03 mg/mL); (Stem wood, 0.048±0.04 mg/mL); (Stem bark, 0.048±0.02 mg/mL); (Root wood, 0.047±0.04 mg/mL) and (Root bark, 0.045±0.04 mg/mL)] compared to the standard anti-diabetic drug acarbose (0.032±0.04 mg/mL). The standard anti-diabetic drug with the lowest IC50 value of 0.032±0.04 mg/mL exhibited the highest α-amylase inhibition activity followed closely by the pod essential oil (0.038±0.01 mg/mL) as indicated on Figure 2. The least activity was expressed by the Seed oil (0.048±0.02 mg/mL). Figure 1: IC50 (mg/mL) values from α-amylase inhibition of Calophyllum inophyllum Linn. Data were analyzed by non-linear regression analysis on MS Excel from the mean inhibitory data. IC50 - Concentration of essential oil that scavenged 50% of acarbose. Figure 1: IC50 (mg/mL) values from α-amylase inhibition of Calophyllum inophyllum Linn. Data were analyzed by non-linear regression analysis on MS Excel from the mean inhibitory data. IC50 - Concentration of essential oil that scavenged 50% of acarbose. IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 258 Table 2. Percentage α-glucosidase inhibition of essential oils from C. 5. RECOMMENDATIONS  It is highly recommended that bio-assay guided isolation and characterization of non-volatile components from various parts of C. inophyllum Linn should be carried out to identify specific compounds responsible for the activities observed in this study. Figure 2: IC50 (mg/mL) values from α-glucosidase inhibition of Calophyllum inophyllum Linn. Data were analyzed by non-linear regression analysis on MS Excel from the mean inhibitory data. IC50 - Concentration of essential oil that scavenged 50% of acarbose. y  In vivo study should be carried out on volatile and non-volatile components of this plant prior to clinical study. 3.3. ALPHA-GLUCOSIDASE INHIBITION OF C.INOPHYLLUM LINN Inophyllum Linn Plant parts Concentration (mg/mL) 0.1 0.08 0.06 0.04 0.02 Acarbose (standard) 96.34 85.80 74.11 68.50 60.28 Leaf 74.18 72.44 65.09 59.67 52.46 Leaf-stalk 74.48 69.45 65.94 59.08 55.14 Flower 73.05 71.11 64.02 60.94 53.63 Seed 72.42 64.06 62.34 55.92 50.82 Pod 82.05 75.73 70.90 62.74 56.42 Peel 73.46 67.43 62.79 56.69 52.90 Stem wood 72.50 66.81 61.99 57.33 50.45 Stem bark 74.09 65.18 62.39 55.57 51.71 Root wood 72.16 65.56 61.45 56.66 53.04 Root bark 73.85 68.96 63.48 58.84 53.08 4. CONCLUDING REMARKS The α-amylase and α-glucosidase inhibitory activities of C. inophyllum Linn revealed that the plant could be an interesting component against postprandial hyperglycemia and other diseases associated with diabetes mellitus. Future researches are necessary to confirm this observational study. The pod essential oils showed peak α-amylase and α-alpha glucosidase activity while essential oils from other parts showed appreciable inhibition activity. The high inhibition efficiency expressed by pod essential oils in both the α- amylase and α-glucosidase inhibition studies must be triggered by the presence of bioactive constituents. Generally, the alpha amylase and alpha glucosidase inhibition of all parts of C. inophyllum Linn were concentration dependent. The high α-amylase and α- glucosidase inhibition of the pod essential oils in comparison with the standard anti-diabetic drug must be due to the presence of some impact bioactive phyto- contituents in the plant. Results obtained for α-amylase and α-glucosidase inhibition are consistent with values reported earlier in literature [49, 50]. 6. ACKNOWLEDGEMENTS We acknowledge the use of J-Organic Chemistry research laboratory facilities, Department of Chemistry, University of Ibadan, Ibadan, Nigeria in plant extractions, preparation and in-vitro antidiabetic studies. Figure 2: IC50 (mg/mL) values from α-glucosidase inhibition of Calophyllum inophyllum Linn. Data were analyzed by non-linear regression analysis on MS Excel from the mean inhibitory data. IC50 - Concentration of essential oil that scavenged 50% of acarbose. 259 IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 21. Mohammadhosseini M, Sarker SD, Akbarzadeh A. Chemical composition of the essential oils and extracts of Achillea species and their biological activities: A review. J. Ethnopharmacol. 2017;199;257-315 doi: 10.1016/j.jep.2017.02.010. 7. REFERENCES 1. Maedeh M, Hamzeh I, Hossein D, Majid A, Reza RK. Bioactivity of Essential Oil from Satureja hortensis (Laminaceae) against Three Stored- Product Insect Species. African J. Biotech. 2011;10(34):6620-7. doi: 10.5897/AJB11.469. 22. Aidi-Wannes W, Mhamdi B, Saidani-Tounsi M, Marzouk B. Lipid and volatile composition of borage (Borago officinalis L.) leaf. Trends Phytochem Res. 2017;1(3):143-8. 2. Buchbauer G. The Detailed Analysis of Essential Oils Leads to the Understanding of Their Properties. Perfum Flavor. 2000;25:64-7. doi: 10.3923/rjphyto.2011.66.69. 23. Camilo CJ, Alves Nonato CDF, Galvão-Rodrigues FF, Costa WD, Clemente GG, Sobreira Macedo MAC, et al. Acaricidal activity of essential oils: a review. Trends in Phytochem Res. 2017;1(4):183-98. 3. Ayat A, Mohamad EA, Mohamad JK, Foroogh N. In vitro α-amylase and α- glucosidases inhibitory effects of some plant extracts. Int J. Pharmacogn Phytochem Res. 2015;7(2):315-8. doi: 10.4103/0973-1296.166018. 24. Ganesan K, Xu B. Ethnobotanical studies on folkloric medicinal plants in Nainamalai, Namakkal District, Tamil Nadu, India. Trends Phytochem Res. 2017;1(3):153-68. 4. Laoufi H, Benariba N, Adjdir S, Djaziri R. In vitro α-amylase and α- glucosidase inhibitory activity of Ononis angustissima extracts. J. App. Pharm. Sci. 2007;7(2):191-8. doi: 10.7324/JAPS.2017.70227. 25. Shimabukuro M. Higa N, Chinen I, Yamakawa K, Takasu N. Effects of a single administration of acarbose on postprandial glucose excursion and endothelial dysfunction in type 2 diabetic patients: a randomized crossover study. J Clin Endocrinol Metab. 2006;91(3):837-42. doi: 10.1210/jc.2005- 1566. 5. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract. 2010;87(1):4-14. doi: 10.1016/j.diabres.2009.10.007. 26. Dweck AC, Meadowst T. Tamanu (Calophyllum inophyllum) – The African, Asian, Polynesian and Pacific Panacea. Int J Cosmetic Sci. 2002;24(6):341-8. doi: 10.1046/j.1467-2494.2002.00160.x. 6. Dahiru T, Alhaji A, Aliyu A, Shehu A. A review of Population based Studies on Diabetes Mellitus in Nigeria. Sub-saharan Afr J Med. 2016;3(2):59-61. 27. Al-Jeboury FS, Locksley HD. Xanthones in the heartwood of Calophyllum inophyllum; A geographical survey. Phytochemistry. 1971;10(3):603-6. doi: https://doi.org/10.1016/S0031-9422(00)94704-6. 7. Xiao JB, Hogger P. Dietary polyphenols and type 2 diabetes: current insights and future perspectives. Curr Med Chem. 2015;22(1):23-38. doi: 10.2174/0929867321666140706130807. 28. Dharmaratne HRW Napagoda MT, Tennakoon SBP. Xanthones from root of Calophyllum thwaitesii and their bioactivity. Nat Prod Res. 2009;23(6):539- 45. doi: 10.1080/14786410600899118. 8. Li YQ, Zhou FC, Gao F. Comparative evaluation of quercetin, isoquercetin and rutin as inhibitors of alpha-glucosidase. J Agric Food Chem. 2009;57(24):11463-8. doi: 10.1021/jf903083h. 29. Burkhil HM. The useful plants of west tropical Africa. Families E-I. 7. REFERENCES 2nd ed. XX Royal Botanic Gardens Kew. 1994;2:522. 9. Proença C, Freitas M, Ribeiro D, Oliveira EFT, Sousac JLC, Tomé SM et al. α-Glucosidase inhibition by flavonoids: an in vitro and in silico structure– activity relationship study. J Enzyme Inhib Med Chem. 2017;32(1):1216-28. doi: 10.1080/14756366.2017.1368503. 30. Uma SM., Murthy PN, Sahoo KS, Sahu KC. Formulation and evaluation of herbal tablet containing methanolic extract of Calophyllum inophyllum. Int J Pharm. 2012;2(1):181-6. 10. Zeng L, Zhang G, Liao Y, Gong D. Inhibitory mechanism of morin on α- glucosidase and its anti-glycation properties. Food Funct 2016;7: 3953-63. doi: 10.1039/c6fo00680a. 31. Silpa S, Fathima A, Shakeel I. Phytochemical screening and evaluation of anti-hyperglycemic and anti-hyperlipidimic activity of methanolic extracts of Calophyllum inophyllum on Albino Wistar Rats Int J Adv Res. 2014;2(8):743- 52. 11. Janeček Š, Svensson B, MacGregor EA. α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cell Mol Life Sci. 2014;71(7):1149-70. doi: 10.1007/s00018-013-1388-z. 32. Varsha G, Uma MB, Ramasamy M, Karunanithi M. Effect of Ethanolic Extract of Calophyllum inophyllum Leaves on Oxidative stress Complications in Mouse Model. Asian J Pharm Clin Res. 2016;9(3):250-2. 12. Sales PM, Souza PM, Simeoni LA. Α-amylase inhibitors: a review of raw material and isolated compounds from plant source. J Pharm Pharm Sci. 2012;15(1):141-83. doi: 10.18433/j35s3k. 33. Um YL Jo YW. Inhibitory effects of Calophyllum inophyllum extract on atopic dermatitis induced by DNCB in mouse. Am J Phytomed Clin Therap. 2016;4(6):165-73. 13. Brayer GD, Sidhu G, Maurus R, Rydberg EH, Braun C, Wang Y, et al. Subsite mapping of the human pancreatic α-amylase active site through structural, kinetic, and mutagenesis techniques. Biochemistry. 2000;39(16):4778-91. doi: 10.1021/bi9921182. 34. Kijjoa A, Gonzalez TG, Pinto MM, Silva AM, Anantachoke C, Herz W. Xanthones from Calophyllum teysimannii var. inophylloide. Phytochemistry. 2000;55(7):833-6. doi: 10.1016/s0031-9422(00)00289-2. 14. Patel H, Royall PG, Gaisford S, Williams GR, Edwards CH, Warren FJ, et al. Structural and enzyme kinetic studies of retrograded starch: inhibition of α- amylase and consequences for intestinal digestion of starch. Carbohydr Polym. 2017;164:154-61. doi: 10.1016/j.carbpol.2017.01.040. 35. Ee GC, Jong VYM, Sukari MA, Rahmani M, Kua ASM. Xanthones from Calophyllum inophyllum. Pertanika J Sci Technol. 2009;17(2):307-12. 36. Govindachari TR, Vishwanathan N, Pai BR, Ramadas Rao U, Srinivasan M. Triterpenes of Calophyllum inophyllum linn. Tetrahedron. 1967;23(4):1901-10. doi: https://doi.org/10.1016/S0040-4020(01)82592-8. 15. International Diabetes Federation. IDF Diabetes Atlas. 8th ed. 2017. 16. American Diabetes Association. Classification and diagnosis of diabetes: standards of medical care in diabetes. Diabetes Care. 41. Itoigawa M, Ito C, Tan HT, Kuchide M, Tokuda H, Nishino H, et al. Cancer chemo-preventive agents, 4-phenylcoumarins from Calophyllum. inophyllum. Cancer Lett. 2001;169(1):15-9. doi: 10.1016/s0304- 3835(01)00521-3. 46. Sulistiyani, Safithri M, Sari YP. Inhibition of α-glucosidase activity by ethanolic extract of Melia azedarach L. leaves. IOP Conf Ser: Earth Environ Sci. 2016;31:12-25. doi: 10.1088/1755-1315/31/1/012025. 50. Mohamed EA, Siddiqui MJ, Ang LF, Sadikun A, Chan SH, Tan SC, et al. Potent α-glucosidase and α-amylase inhibitory activities of standardized 50% ethanolic extracts and sinensetin from Orthosiphon stamineus Benth as anti- diabetic mechanism. BMC Conplement Altern Med. 2012;12:176 doi: 10.1186/1472-6882-12-176. 47. Ojah EO, Moronkola DO. In-vitro alpha-amylase and glucosidase inhibitory potential of leaf hexane, ethyl acetate and methanol fractions from Pterocarpus soyauxii Taub Trends Phytochem Res. 2020; 4(1):37-44. 48. Chelladurai GRM, Cinnachamy C. Alpha amylase and Alpha glucosidase inhibitory effects of aqueous stem extract of Salacia oblonga and its GC-MS analysis. Braz J Pharm Sci. 2018;54(1):1-10. doi: https://doi.org/10.1590/s2175-97902018000117151. 42. Ojah EO, Moronkola DO, Pettrelli R, Nzekoue FK, Cappellacci L, Giordani C, et al. Chemical Composition of ten Essential Oils from Calophyllum Inophyllum Linn And their Toxicity against Artemia Salina Eur J Pharm Med Res. 2019;6(12):185-94. 49. Kazeem MI, Dansu TV, Adeola SA. Inhibitory Effect of Azadirachta indica A. Juss Leaf Extract on the Activities of α-amylase and α-glucosidase. Pak J Biol Sci. 2013;16(21):1358-62. doi: 10.3923/pjbs.2013.1358.1362. 45. Kim YM, Jeong MH, Wang WY, Lee HI, Rhee HI. Inhibitory effect of pine extract on alpha- glucosidase activity and postprandial hyperglycemia. Nutrition. 2005;21(6):756-61. doi: 10.1016/j.nut.2004.10.014. 7. REFERENCES 2018;41(Suppl 1):S13- S27. doi: 10.2337/dc18-S002. 37. Hang NTM, Chien NQ, Van Hung N. Triterpenes from the leaves of the Vietnamese plant Calophyllum inophyllum L. Tap chi hoa hoc. 2006;44(1):115-8. 17. Butterworth PJ, Warren FJ, Ellis PR. Human α-amylase and starch digestion: an interesting marriage. Starch-Stärke. 2011;63:395-405. doi: https://doi.org/10.1002/star.201000150. 38. Patil AD, Freyer AJ, Eggleston DS, Haltiwanger RC, Bean MF, Taylor PB, et al. The inophyllum’s novel inhibitors of HIV-1 reverse transcriptase isolated from the Malaysian tree, C. inophyllum Linn. J Med Chem 1993;36(26):4131-8. doi: 10.1021/jm00078a001. 18. Etxeberria U, de la Garza AL, Campion J, Martínez JA, Milagro FI. Antidiabetic effects of natural plant extracts via inhibition of carbohydrate hydrolysis enzymes with emphasis on pancreatic alpha amylase. Expert Opin Ther Targets. 2012;16(3):269-97 doi: 10.1517/14728222.2012.664134. 39. Janki P, Atul S, Khanna AK, Bhatia G, Awasthi SK, Narender T. Antidyslipidemic and antioxidant activity of the constituents isolated from the leaves of Calophyllum inophyllum. Phytomedicine. 2012;19(14):1245-9. doi: 10.1016/j.phymed.2012.09.001. 19. Kahn SE, Cooper ME, Del-Prato S. Pathophysiology and treatment of type 2 diabetes: perspectives on the past, present, and future. Lancet. 2014;383(9922):1068-83. doi: 10.1016/S0140-6736(13)62154-6. 20. Mohammadhosseini M, Akbarzadeh A, Hashemi-Moghaddam H, Mohammadi Nafchi A, Mashayekhi HA, Aryanpour A. Chemical composition of the essential oils from the aerial parts of Artemisia sieberi by using conventional hydrodistillation and microwave assisted hydrodistillation: A comparative study. J Essent. Oil-Bear Plants 2016;19(1):32-45. doi: https://doi.org/10.1080/0972060X.2015.1119067. 19. Kahn SE, Cooper ME, Del-Prato S. Pathophysiology and treatment of type 2 diabetes: perspectives on the past, present, and future. Lancet. 2014;383(9922):1068-83. doi: 10.1016/S0140-6736(13)62154-6. 40. Arora RB, Mathur CN, Seth SD. Calophyllolide, a complex coumarin anticoagulant from C. inophyllum Linn. J Pharm Pharmacol 1962;14:534-5. doi: 10.1111/j.2042-7158.1962.tb11133.x. 20. Mohammadhosseini M, Akbarzadeh A, Hashemi-Moghaddam H, Mohammadi Nafchi A, Mashayekhi HA, Aryanpour A. Chemical composition of the essential oils from the aerial parts of Artemisia sieberi by using conventional hydrodistillation and microwave assisted hydrodistillation: A comparative study. J Essent. Oil-Bear Plants 2016;19(1):32-45. doi: https://doi.org/10.1080/0972060X.2015.1119067. 41. Itoigawa M, Ito C, Tan HT, Kuchide M, Tokuda H, Nishino H, et al. Cancer chemo-preventive agents, 4-phenylcoumarins from Calophyllum. inophyllum. Cancer Lett. 2001;169(1):15-9. doi: 10.1016/s0304- 3835(01)00521-3. 260 IBEROAMERICAN JOURNAL OF MEDICINE 04 (2020) 253-260 43. Kneen E, Sandsted RM, Hollenbeck CM. Amylase and diastatic activity. Cereal Chem. 1943;20(4):399. 44. McCue PP, Shetty K. Inhibitory effects of rosmarinic acid extracts on porcine pancreatic amylase. Asia Pac J Clin Nutr. 2004;13(1):101-6. 45. Kim YM, Jeong MH, Wang WY, Lee HI, Rhee HI. 43. Kneen E, Sandsted RM, Hollenbeck CM. Amylase and diastatic activity. Cereal Chem. 1943;20(4):399. 44. McCue PP, Shetty K. Inhibitory effects of rosmarinic acid extracts on porcine pancreatic amylase. Asia Pac J Clin Nutr. 2004;13(1):101-6. 42. Ojah EO, Moronkola DO, Pettrelli R, Nzekoue FK, Cappellacci L, Giordani C, et al. Chemical Composition of ten Essential Oils from Calophyllum Inophyllum Linn And their Toxicity against Artemia Salina Eur J Pharm Med Res. 2019;6(12):185-94. 43. Kneen E, Sandsted RM, Hollenbeck CM. Amylase and diastatic activity. Cereal Chem. 1943;20(4):399. 44. McCue PP, Shetty K. Inhibitory effects of rosmarinic acid extracts on porcine pancreatic amylase. Asia Pac J Clin Nutr. 2004;13(1):101-6. 45. Kim YM, Jeong MH, Wang WY, Lee HI, Rhee HI. Inhibitory effect of pine extract on alpha- glucosidase activity and postprandial hyperglycemia. Nutrition. 2005;21(6):756-61. doi: 10.1016/j.nut.2004.10.014. 46. Sulistiyani, Safithri M, Sari YP. Inhibition of α-glucosidase activity by ethanolic extract of Melia azedarach L. leaves. IOP Conf Ser: Earth Environ Sci. 2016;31:12-25. doi: 10.1088/1755-1315/31/1/012025. 7. REFERENCES Inhibitory effect of pine extract on alpha- glucosidase activity and postprandial hyperglycemia. Nutrition. 2005;21(6):756-61. doi: 10.1016/j.nut.2004.10.014.
https://openalex.org/W2040828254	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0040805&type=printable	English	null	A Multi-Factorial Genetic Model for Prognostic Assessment of High Risk Melanoma Patients Receiving Adjuvant Interferon	PloS one	2,012	cc-by	8,345	A Multi-Factorial Genetic Model for Prognostic Assessment of High Risk Melanoma Patients Receiving Adjuvant Interferon Ena Wang1., Yingdong Zhao2., Alessandro Monaco1, Lorenzo Uccellini1, John M. Kirkwood3, Maria Spyropoulou-Vlachou4, Monica C. Panelli3, Francesco M. Marincola1, Helen Gogas5* 1 Department of Transfusion Medicine, Clinical Center and Trans-NIH Center for Human Immunology, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America, 3 University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, Pennsylvania, United States of America, 4 Department of Immunology, National Tissue Typing Center, General Hospital of Athens, Athens, Greece, 5 First Department of Medicine, University of Athens, Medical School, Athens, Greece Abstract Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. . These authors contributed equally to this work. Despite the ability of high dose IFN-a to reduce relapse and mortality by up to 33% [6], the large majority of patients experience side effects and toxicity which outweigh the benefits. Attempts to identify a subset of patients likely to benefit from adjuvant treatment with IFN-a2b have failed to discover clinical or demographic features of true predictive value. Correlative studies undertaken over the years identified a variety of immunologic parameters that are associated with therapeutic benefit but are observable only after therapy and, therefore, have no predictive value [7,8]. Recently, mounting evidence indicates that immune cell infiltration in tumor correlates with prolonged survival suggesting a fine balance between tumor progression and immune recognition within the tumor microenvironment. This Despite the ability of high dose IFN-a to reduce relapse and mortality by up to 33% [6], the large majority of patients experience side effects and toxicity which outweigh the benefits. Abstract Purpose: IFNa was the first cytokine to demonstrate anti-tumor activity in advanced melanoma. Despite the ability of high- dose IFNa reducing relapse and mortality by up to 33%, large majority of patients experience side effects and toxicity which outweigh the benefits. The current study attempts to identify genetic markers likely to be associated with benefit from IFN- a2b treatment and predictive for survival. Experimental design: We tested the association of variants in FOXP3 microsatellites, CTLA4 SNPs and HLA genotype in 284 melanoma patients and their association with prognosis and survival of melanoma patients who received IFNa adjuvant therapy. Results: Univariate survival analysis suggested that patients bearing either the DRB115 or HLA-Cw7 allele suffered worse OS while patients bearing either HLA-Cw6 or HLA-B44 enjoyed better OS. DRB115 positive patients suffered also worse RFS and conversely HLA-Cw6 positive patients had better RFS. Multivariate analysis revealed that a five-marker genotyping signature was prognostic of OS independent of disease stage. In the multivariate Cox regression model, HLA-B38 (p = 0.021), HLA-C15 (p = 0.025), HLA-C3 (p = 0.014), DRB115 (p = 0.005) and CT60G/G (0.081) were significantly associated with OS with risk ratio of 0.097 (95% CI, 0.013–0.709), 0.387 (95% CI, 0.169–0.889), 0.449 (95% CI, 0.237–0.851), 1.948 (95% CI, 1.221– 3.109) and 1.484 (95% IC, 0.953–2.312) respectively. Conclusion: These results suggest that gene polymorphisms relevant to a biological occurrence are more likely to be informative when studied in concert to address potential redundant or conflicting functions that may limit each gene individual contribution. The five markers identified here exemplify this concept though prospective validation in independent cohorts is needed. Citation: Wang E, Zhao Y, Monaco A, Uccellini L, Kirkwood JM, et al. (2012) A Multi-Factorial Genetic Model for Prognostic Assessment of High Risk Melanoma Patients Receiving Adjuvant Interferon. PLoS ONE 7(7): e40805. doi:10.1371/journal.pone.0040805 Editor: Sophia N. Karagiannis, King’s College London, United Kingdom Received February 3, 2012; Accepted June 13, 2012; Published July 24, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or ot any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. PLoS ONE \| www.plosone.org Selection of Single Nucleotide Polymorphisms in the Human CTLA4 Gene and Microsatellite in FOXP3 Gene u a C e e a d c osate te O 3 e e Six CTLA4 SNPs, CT 60, AG 49, CT 318, JO 27, JO 30 and JO 31 were selected based on known association with autoimmune disorders [18–22]. CT 318 is located within the promoter region of the CTLA-4 gene, A/G49 is located at exon 1, while the rest of the SNPs are located at the 39 untranslated region of CTLA-4 [22]. Microsatellite (TC)n of FOXP3 located within intron 5 from +476 to +595, up to +539 (IVS5) on Xp11.23 was selected based on previous publication and tested by using the previously described PCR primer pairs for primer sequences and nomencla- ture [14]. Polymorphism Detection HLA typing was performed using previously published DNA based techniques [23–25]. Initially HLA-A,-B,-Cw,-DRB1,and - DQB1 low resolution molecular typing was performed in all subjects, with amplification of genomic DNA by polymerase chain reaction (PCR) using locus-specific primers and reverse hybrid- ization with sequence and allele-specific oligonucleotide probes (reverse PCR-SOP), using a commercially available kit (Lambda Array Beads Multi-Analyte System, LABTypeH RSS0, One Lambda, Inc). Subsequently, high resolution typing of HLA- DRB1 and -DQB1 loci was implemented by sequence specific oligonucleotide (SSO) (ELPHA HiRes, Biotest, Germany) and sequence specific primer (SSP) PCR (Olerup SSPTM, Salt- sjoebaden, Sweden) respectively. The allele assignment was made according to the HLA-visual software program. Study Subjects The present translational research protocol was approved by the Bioethics Committee of the National and Kapodistrian University of Athens School of Medicine, Ethics Committee of Athens General Hospital ‘‘G. Gennimatas’’, under the general title ‘‘Immunological studies of melanoma patients receiving adjuvant interferon’’ (A399/5-3-1999). Patients participating in this study were enrolled in Trial 13A/98, a prospective, multicenter, randomized phase III trial conducted at 13 institutions by the Hellenic Cooperative Oncology Group (HeCOG). In this trial, 364 patients with histologically documented AJCC stage IIB, IIC, or III primary cutaneous melanoma were enrolled between 1998 and 2004. For patients with clinically negative lymph nodes, stage was defined pathologically by sentinel lymph node (SLN) biopsy. Patients with positive SLN were required to undergo completion lymphadenectomy. All patients were randomized to receive treatment within 2 months of initial surgery or 1.5 months following therapeutic lymph node dissection. The regimen used was a modification of the E1684 regimen [17]. Group A patients received IFN-a2b (15 MIU/m2/day IV 5 days per week for 4 weeks) followed by observation. Group B patients received the same induction dose for 4 weeks followed by subcutaneous therapy (10 MIU/day TIW) for an additional 48 weeks. The primary endpoint for the core protocol was recurrence free survival (RFS) and overall survival (OS) and no difference in relapse free survival, distant metastasis free survival or overall survival was shown between the 2 groups [17]. FOXP3 (TC)n microsatellite analysis was performed by capillary electrophoresis using DNA isolated from 259 patients. PCR amplification was conducted using (TC)n forward primer and reverse primer (Table 1). The reaction mixture were denatured at 95uC for 15 minutes and cycled 35 times at 94uC for 30 second, 54uC for 30 second and 72uC for 30 second followed by 72uC for 30 min. After digestion with EXOSAP at 37uC for 15 min and 80uC for 15 min to remove unincorporated primers and inactivate the enzyme, the PCR product mixed with internal size standard (Gene Scan-350) and formamide were analyzed by ABI Prism 3730 XL DNA analyzer. Data were analyzed using Genemapper software (Applied Biosystems) which automatically calls fragments size. FOXP3 (TC)n microsatellite analysis was performed by capillary electrophoresis using DNA isolated from 259 patients. PCR amplification was conducted using (TC)n forward primer and reverse primer (Table 1). PLoS ONE \| www.plosone.org Introduction The incidence of melanoma in the US increased threefold in the past 3 decades [1] and an estimated 114,900 new cases were diagnosed in 2010; among them, 46,770 were noninvasive (in situ) while 68,130 were invasive resulting in 8,700 deaths [2]. The survival of patients, whose melanoma is detected early, is about 99 percent [3] and a 5 year survival of around 95% for stage I tumors [4]. However, the survival rate falls to 15 percent for those with advanced disease [5]. Attempts to identify a subset of patients likely to benefit from adjuvant treatment with IFN-a2b have failed to discover clinical or demographic features of true predictive value. Correlative studies undertaken over the years identified a variety of immunologic parameters that are associated with therapeutic benefit but are observable only after therapy and, therefore, have no predictive value [7,8]. Recently, mounting evidence indicates that immune cell infiltration in tumor correlates with prolonged survival suggesting a fine balance between tumor progression and immune recognition within the tumor microenvironment. This Interferon alpha (IFN-a) was the first cytokine to demonstrate anti-tumor activity in patients with advanced melanoma and is the only approved regimen for the adjuvant treatment for melanoma. PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40805 1 July 2012 \| Volume 7 \| Issue 7 \| e40805 Genetic Model for Prognosis of Melanoma Patients Genetic Model for Prognosis of Melanoma Patients balance relies on multiple factors important for the maintenance of normal immune function. Among them, human leukocyte antigen (HLA), Cytotoxic T lymphocyte antigen 4 (CTLA4) and FOXP3, a member of the fork head/winged-helix family of transcriptional regulators are important first and secondary signal molecules influencing T lymphocyte activation and function. HLA genotype has been associated with disease susceptibility, immune respon- siveness and prognosis [9,10]. Single nucleotide polymorphisms (SNPs) in the CTLA4 and microsatellite polymorphism in FOXP3 gene region were also reported to be associated with several autoimmune diseases including, type-1 diabetes, systemic lupus erythematosus, autoimmune thyroid diseases and celiac disease [10–16]. Based on the observation that allelic polymorphism of individual genes is generally only weakly associated with disease predisposition in multi-genic disorders like cancer, a multi-factorial contribution by distinct genes affecting a specific function (i.e. T cell function) could be hypothesized; thus, polymorphisms of CTLA4, FOXP3 and HLA genes in melanoma patients may contribute to variability in patient survival and prognosis and their combination may have stronger predictive power than that of each genes assessed as a single entity. Thus, we tested the association of variants in FOXP3 microsatellites, CTLA4 SNPs and HLA genotype in 284 melanoma patients and their association with prognosis and survival of patients with melanoma who received adjuvant therapy with IFNa. used for polymorphism testing. DNA was isolated using the BioRobotH EZ1 Genomic DNA Kit and (GenoVision, Oslo, Norway). The clinical outcome of patients was prospectively followed according to standard parameters; clinical staging consisted of medical history, physical exams, blood cell counts, blood biochemistry at 3-month intervals, and chest x-ray and liver ultrasound at 6-month intervals. Out of 364 patients, 284 were genotyped for all three genes assessed in the current study based on available DNA samples. used for polymorphism testing. DNA was isolated using the BioRobotH EZ1 Genomic DNA Kit and (GenoVision, Oslo, Norway). The clinical outcome of patients was prospectively followed according to standard parameters; clinical staging consisted of medical history, physical exams, blood cell counts, blood biochemistry at 3-month intervals, and chest x-ray and liver ultrasound at 6-month intervals. Out of 364 patients, 284 were genotyped for all three genes assessed in the current study based on available DNA samples. Data Processing and Statistic Analysis Variable coding. For HLA alleles, each patient was counted as an event and a given allele was coded as 1 if detected either in heterozygous or homozygous conditions or 0 if undetected. For each allele, an additional code classified as 1 for homozygosity and 0 for heterozygosity. For CTLA-4 and FOXP3 testing, each patient was counted as an unique event and a particular allele was coded as 1 if detected or 0 if undetected. Of all potential variant alleles only those presented at least with a frequency of 10%, i.e., a total of 71 variables were included in the analysis. Next, we tested whether the association of genotyping with survival was statistically significant. A log-rank statistic (LRd) was computed for the cross-validated Kaplan-Meier curves. We performed a statistical significance test by randomly shuffling survival data among the cases and repeating the entire cross- validation process. For each random re-shuffling, we repeated the process, created new cross-validated Kaplan-Meier curves, and computed the log-rank statistic for the random shuffling, which served as a null-distribution of the log-rank statistic created in this way. We defined the tail area of this null distribution beyond the value LRd obtained for the real data as the permutation significance level for testing the null hypothesis that there was no relation between the genotyping data and survival. y Feature selection and prediction models. OS and RFS were calculated from the date treatment was started to the date of last follow-up or the date when death from any cause or relapse first occurred. We first performed univariate analysis to identify markers (alleles) whose presence/absence correlated with survival by fitting Cox proportional hazards model [26] that computed the p value for each allele testing the hypothesis that survival was independent of the presence/absence of that allele. This backward elimination method was used for feature selection in the multivariate Cox proportional hazard model. A prognostic index for each patient was calculated as the weighted average (weighted as regression coefficient) of the variables selected for the multivariate Cox model. A high value of the prognostic index corresponded to a high hazard of death, and consequently a poor predicted survival. The patients were classified into high risk or low risk groups based on whether their prognostic index was above or below the median. Study Subjects The reaction mixture were denatured at 95uC for 15 minutes and cycled 35 times at 94uC for 30 second, 54uC for 30 second and 72uC for 30 second followed by 72uC for 30 min. After digestion with EXOSAP at 37uC for 15 min and 80uC for 15 min to remove unincorporated primers and inactivate the enzyme, the PCR product mixed with internal size standard (Gene Scan-350) and formamide were analyzed by ABI Prism 3730 XL DNA analyzer. Data were analyzed using Genemapper software (Applied Biosystems) which automatically calls fragments size. CTLA4 SNP-PCR was carried out with the primer pairs listed in Table 1. 50 ng of DNA were amplified in a 50 mL reaction containing 25 mL MasterMix (Illustra HotStart Mas- terMix, GE Healthcare, Buckinghamshire, UK) 1 mL (10 pmol) of each primer and nuclease free water. PCR were denatured at 5 minute at 95uC followed by 45 cycles of 95uC for15 seconds, 30 seconds at 56uC and 15 seconds at 72uC followed by final extension at 72uC for 5 minutes. The single strand PCR products were prepared by Streptavidin SepharoseTM High Performance beads capture (GE Healthcare, Uppsala, Sweden) after denaturation and washing (Biotage, Uppsala, Sweden) according to manual. All sequencing reactions were performed The polymorphism study reported here was conducted retro- spectively in four institutions that had participated in the core protocol. This study received separate IRB approval, and all patients had provided written informed consent for provision of biological material for such future research studies at initiation of treatment. Blood samples for CTLA-4, FOXP3 and HLA typing were drawn prior to treatment at the time of routine initial visit blood testing. The first 10 mL of blood were used for standard biochemistry and blood cell counts, and the second 3 mL were PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40805 2 Genetic Model for Prognosis of Melanoma Patients Table 1. PCR Primers for FOXP3 (TC)n amplification. SNP name Forward Reverse CT 318 59-ACCCTTGTACTCCAGGAAATTCTC 59-Biotin-GGTTTAGCTGTTACGTCGAAAAGA AG 49 59-TTTCAGCGGCACAAGGCTC 59-Biotin-GAGTGCAGGGCCAGGTCC CT 60 59-GCAAGTCATTCTTGGAAGGTATC 59-Biotin-TGCCAATTGATTTATAAAGGACTG JO 27 59-GAGCTGGTCAGCCGAGAT 59-Biotin-TGACACCACCCCTCCATAAT JO 30 59-CAAAGCAAAACGCTGCCAATAA 59-Biotin-TCCAGTGGCAATAGGAGCTTTC JO 31 59-TTGTCATGTTAGCCGTGCAGC 59-Biotin-CCACCACCACACCCAGGTAA (TC)n 59-FAM-TCCACTGTTCCCAAAGTTCTAGC 59GAGTGCTGGAGATAATGTTGGAAGT doi:10.1371/journal.pone.0040805.t001 on the PyroMarkTM ID pyrosequencer, using the PSQ 96 SNP Reagent Kit (Biotage AB), under sequence primer (Table 1) and analysis was done with PyroMarkTM ID 1.0 software. Study Subjects on the PyroMarkTM ID pyrosequencer, using the PSQ 96 SNP Reagent Kit (Biotage AB), under sequence primer (Table 1) and analysis was done with PyroMarkTM ID 1.0 software. Kaplan-Meier survival curves were plotted for the cases predicted to bear above or below average risk as for the above model. Since the risk group for each case was determined based on a predictor that did not use that case in its construction, the Kaplan-Meier curves were unbiased and the separation between the curves gave a fair representation of the predictive value of genotyping profiles on survival. Data Processing and Statistic Analysis PLoS ONE \| www.plosone.org A Five-marker Genotyping Signature may be Used as a Prognostic Index for RFS in Patients with Melanoma Receiving Adjuvant IFN-a Treatment We attempted to build the RFS model using an approach similar to the OS model. However, Kaplan-Meier plot obtained from LOOCV did not show a clear separation between the curves, indicating a poor performance of the model in predicting RFS risk. We then evaluated the five-marker OS model on RFS survival data. LOOCV was performed as predictive of OS but then was tested on RFS survival data. The LOOCV Kaplan-Meier curve demonstrated that 90 patients (44 relapses) were in the low risk group (median RFS: 36.7 months) and 194 patients (111 relapses) were in the high risk group (median RFS: 63.0 months) (log rank test p = 0.048) (Figure 4A). The permutation significance level was 0.09 (1,000 permutations), indicating the association of genotyping data to RFS data was borderline statistically significant. We attempted to build the RFS model using an approach similar to the OS model. However, Kaplan-Meier plot obtained from LOOCV did not show a clear separation between the curves, indicating a poor performance of the model in predicting RFS risk. We then evaluated the five-marker OS model on RFS survival data. LOOCV was performed as predictive of OS but then was tested on RFS survival data. The LOOCV Kaplan-Meier curve demonstrated that 90 patients (44 relapses) were in the low risk group (median RFS: 36.7 months) and 194 patients (111 relapses) were in the high risk group (median RFS: 63.0 months) (log rank test p = 0.048) (Figure 4A). The permutation significance level was 0.09 (1,000 permutations), indicating the association of genotyping data to RFS data was borderline statistically significant. LOOCV Kaplan-Meier curve (Figure 2 A) identified 90 patients (22 deaths) in the low risk group and 194 (78 deaths) in the high risk group. The median OS of the low risk group has not yet been reached, while it was 68.2 months in the high risk group (log rank test p = 0.0026). The permutation p-value was 0.04 (500 permu- tations) indicating that the association of genotyping with OS was statistically significant. Time-dependent ROC curves showed that the AUCs at 5 and 7 year were 0.645 and 0.72, respectively (Figure 2B, 7 year survival ROC curve). We finally evaluated if the combined survival risk model, i.e., genotyping + stage model built on OS data, can also be used to predict RFS. A Five-marker Genotyping Signature is an Independent Predictor of OS Independent of Disease Stage To assess whether the prediction of OS by the five marker signature was affected by stage of disease, multivariate Cox regression analysis was performed by adding stage as a factor. Hazard ratios (HR), 95% CI of HR, and p values are shown for the five markers and disease stage (Table 5). LOOCV Kaplan-Meier curve (Figure 3A) showed that 131 patients (31 deaths) were in the low risk group and 153 patients (69 deaths) were in the high risk group. The median OS of the low risk group had not yet been reached, while it was 64.3 months in the high risk group (log rank test p = 0.00009). The permutation significance level was 0.05 (500 permutations), indicating that the association of genotyping data with OS data was statistically significant. Time-dependent ROC curve showed that the AUC for 7 year survival was 0.735. This, the five-marker genotyping signature was an independent predictor of OS after controlling for disease stage (Figure 3B) while stage of disease was an independent risk factor (Table 3). A Five-marker Genotyping Signature has Prognostic Significance on OS of Patients with Melanoma Receiving Adjuvant Therapy with IFN-a Multivariate analysis revealed that a five-marker genotyping signature was prognostic of OS. In the final multivariate Cox regression model, HLA-B38 (p = 0.021), HLA-C15 (p = 0.025), HLA-C3 (p = 0.014), DRB115 (p = 0.005) and CT60G/G (0.081) (Table 3) were significantly associated with OS with risk ratio of 0.097 (95% CI, 0.013 to 0.709), 0.387 (95% CI, 0.169 to 0.889), 0.449 (95% CI, 0.237 to 0.851), 1.948 (95% CI, 1.221 to 3.109) and 1.484 (95% IC, 0.953 to 2.312) respectively (Table 4). A prognostic index for a patient with a specific genotyping profile could be calculated as the weighted average (weighted as regression coefficient) of the above five variables. Data Processing and Statistic Analysis PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40805 PLoS ONE \| www.plosone.org 3 Genetic Model for Prognosis of Melanoma Patients Table 3. Univariate relapse free survival (RFS) analysis. Variables P value HR 95% CI of HR Cw6 0.021 0.593 [0.380, 0.924] DRB115 0.026 1.543 [1.054, 2.260] doi:10.1371/journal.pone.0040805.t003 Table 3. Univariate relapse free survival (RFS) analysis. A Five-marker Genotyping Signature may be Used as a Prognostic Index for RFS in Patients with Melanoma Receiving Adjuvant IFN-a Treatment The Leave-One-Out Cross Validated Kaplan-Meier curve is shown in Figure 4B, with 131 patients (60 relapses) in the low risk group (median RFS 29.9 months) and 153 patients (95 relapses) in the high risk group (median RFS 65.3 months) (log rank test p = 0.001). The permutation significance level was 0.06 (500 permutations), indicating the association of genotyping data and stage to RFS data was borderline statistically significant. Table 2. Univariate overall survival (OS) analysis. Variables P value HR 95% CI of HR DRB115 0.028 1.676 [1.056, 2.659] Cw6 0.029 0.510 [0.279, 0.933] Cw7 0.030 1.547 [1.044, 2.291] B44 0.040 0.421 [0.184, 0.961] doi:10.1371/journal.pone.0040805.t002 Table 2. Univariate overall survival (OS) analysis. Results Individual Genotyping Markers Predict OS and RFS of Patients with Melanoma Receiving Adjuvant IFN-a Therapy py Univariate survival analysis suggested that patients bearing either the DRB115 or HLA-Cw7 allele suffered worse OS while patients bearing either HLA-Cw6 or HLA-B44 enjoyed better OS. DRB115 positive patients suffered also worse RFS and conversely HLA-Cw6 positive patients had better RFS. The p values, Hazard Ratio (HR), and 95% Confidence Interval (CI) of HR for the significant markers (Wald test p value ,0.05) are listed in Table 2 and Table 3, for OS and RFS, respectively. Kaplan-Meier curves for DRB115 are shown in Figure 1A and 1B for OS (log rank p value = 0.027) and RFS (log rank p value = 0.025), respectively. Comparison of FOXP3 microsatellites did not demonstrate significant associations (Univariate survival analysis for FoxP3 microsatellites, Table S1) and, therefore, the final data presented here using a model excluded FOXP3 from the analysis. We would however emphasize that this results should not exclude completely lack of association between FOXP3 genetic variants and outcome of IFN-a therapy as other SNPs not analyzed in this study could have provided different information. Such analysis is the subject of future studies. A Five-marker Genotyping Signature is an Independent Predictor of OS Independent of Disease Stage Data Processing and Statistic Analysis We then compared the combined survival risk model (i.e., genotyping + covariate) to the model based only on the covariate (i.e., stage) using as a test statistic the difference between the cross- validated log-rank statistic for the combined model minus the log- rank statistic for cross-validated Kaplan–Meier curves for the covariate model. The null distribution of the test statistic was generated based on permuting the genotyping vectors among cases. In these permutations, the correspondence between survival times, censoring indicators and the covariate were not disrupted. The null hypothesis tested was that the genotyping data were independent of survival and the covariate. This approach is described more fully in R Simon et al. [27]. Leave-One-Out-Cross-Validation (LOOCV) was applied to evaluate the predictive accuracy of survival risk classifiers based on high-dimensional data [27]. A single event (patient) was omitted and the entire procedure described above was performed to create a prognostic index. This function was created from scratch on the training set with the one case removed, including the feature selection step in the multivariate Cox model. Having determined a prognostic index function for that training set, this was used to compute a prognostic index for the omitted event. That observed value was compared to the prediction of the prognostic index for the n-1 cases included in that training set. The omitted patient was placed into either a high risk group or a low risk group based on his/her prognostic index . = or , the median of the prognostic index in the training set. This analysis was repeated from scratch n times, leaving out a different patient each time. We finally evaluated if the survival risk model built on OS data can be used to predict RFS. LOOCV was performed by building the survival risk model on the training set OS data but validating the model on the test set RFS data. Considering the arbitrary cut-off percentiles specified for defining the risk groups (50th percentile cut-off was used in our case), we created a time-dependent ROC curve from censored survival data [28], indicating how well the marker predicted the survival time for the subjects in the dataset by TP (True Positive), FP (False Positive), AUC (Area Under (ROC) Curve) at the time point of interest (e.g., 7 year survival). Discussion In the past decade, great efforts have been dedicated to identify and validate prognostic markers in patients with advanced melanoma. Such markers could assist the selection by physicians of the most suitable treatments on a patient-specific basis. In the case of IFN-a therapy, although robust predictors of response to therapy have not yet been identified weak prognostic associations July 2012 \| Volume 7 \| Issue 7 \| e40805 July 2012 \| Volume 7 \| Issue 7 \| e40805 4 Genetic Model for Prognosis of Melanoma Patients Figure 1. Kaplan-Meier curves for DRB115 for OS and RFS. The Leave-One-Out Cross Validated (LOOCV) Kaplan-Meier curves to compare DRB115 positive (red line) and negative patients (green line) for OS (log rank p value = 0.027) (Figure 1A) and RFS (log rank p value = 0.025) (Figure 1B), respectively. doi:10.1371/journal.pone.0040805.g001 Figure 1. Kaplan-Meier curves for DRB115 for OS and RFS. The Leave-One-Out Cross Validated (LOOCV) Kaplan-Meier curves to compare DRB115 positive (red line) and negative patients (green line) for OS (log rank p value = 0.027) (Figure 1A) and RFS (log rank p value = 0.025) (Figure 1B), respectively. doi:10.1371/journal.pone.0040805.g001 have previously been described [29]. Recently, increased infiltra- tion of lymphocytes within tumors was recognized to bear positive predictive value and correlate with good prognosis in patients with various cancer types independent of treatment suggesting an important role of the adaptive immune response in controlling disease progression [30–33]. This may in turn bear predictive and prognostic significance in patients with melanoma treated with IFN-a as this cytokine is believed to exerts its effects at least in part through activation of T cells within the tumor microenvironment [34]. Therefore, polymorphism of genes involved in the modula- tion of T cell function could play important role in promoting or inhibiting cancer progression. It has been reported that IFN-c (+874A–T) polymorphism is significantly associated with RFS and OS of patients with melanoma. Combined with two weakly or marginally associated polymorphisms of IL10 and ERCC1, patients could be stratified into distinct groups with different clinical outcomes [35]. Furthermore, polymorphism of CCR5, a chemokine receptor preferentially expressed by Th1 T cells and responsible for homing to the site of inflammation via interaction with its ligands has been reported to have clinical relevance; the D32 deletion of CCR5 was reported to be associated with decreased survival in patients with melanoma receiving immuno- therapy [36]. Discussion A combination of three polymorphism of CDKN2A spanning the coding exon 1a (rs2811710), the first intron (rs2518720), and the 59 regulatory region (rs2811708) was also reported significantly associated with decreased OS in melanoma [37]. In the current study, CTLA4, FOXP3 and HLA were selected because T lymphocytes play a major role both in tumor immunity and autoimmunity and these genes are well known to directly or indirectly affect T cell function. CD28, CTLA4 and inducible co- stimulator (ICOS) molecules are key secondary signal molecules in T lymphocyte activation. SNPs in the CD28/CTLA4/ICOS gene region were reported to be associated with several autoimmune diseases including, type-1 diabetes, systemic lupus erythematosus, autoimmune thyroid diseases and celiac disease [11,20,38]. High expression of FOXP3 in CD4+ and CD25+ T helper cells is indicative of immune suppressive function [39,40]. However, recent publications indicate that increased FOXP3 expression in CD8+ T cell serves as a good prognosis marker in melanoma patients undergoing high does IL-2 combined with peptide vaccination therapy [41]. We observed that FOXP3 gene up regulation in tumor infiltrating lymphocytes expanded in vitro for adopted transfer therapy is associated with likelihood of response to therapy (unpublished observation). Correlation of HLA phenotypes with clinical outcome have been studied in infectious disease, transplantation rejection, disease susceptibility and prog- nosis of cancer [42]. Table 4. Multivariate overall survival (OS) analysis. Alleles HR 95% CI of HR P value HLA-B38 0.097 [0.013, 0.709] 0.021 HLA-C15 0.387 [0.169, 0.889] 0.025 HLA-C3 0.449 [0.237, 0.851] 0.014 DRB115 1.948 [1.221, 3.109] 0.005 CT60G/G 1.484 [0.953, 2.312] 0.081 doi:10.1371/journal.pone.0040805.t004 Table 4. Multivariate overall survival (OS) analysis. Previously, we reported that RFS and OS did not differ significantly between patients with distinct CTLA4 polymorphisms when assessed alone [12]. Similarly, individual HLA class I and II alleles were not informative in predicting recurrence in patients receiving adjuvant IFN-a with the exception of HLA-Cw06 which was associated with a better RFS and OS [43]. In the current analysis, we found that patients carrying either DRB115 or HLA-Cw7 suffered worse OS while patients with either HLA- July 2012 \| Volume 7 \| Issue 7 \| e40805 5 Genetic Model for Prognosis of Melanoma Patients Figure 2. LOOCV Kaplan-Meier and Time-dependent ROC curves. A prognostic index for a patient with a specific genotyping profile were calculated as the weighted average (weighted as regression coefficient) of the five-marker genotypes. Discussion LOOCV Kaplan-Meier curve (Figure 2 A) identified 90 patients (22 deaths) in the low risk group (green line) and 194 (78 deaths) in the high risk group (red line). Time-dependent ROC curves showed that the AUCs at 5 and 7 year were 0.645 and 0.720, respectively (Figure 2B, 7 year survival ROC curve). doi:10.1371/journal.pone.0040805.g002 Figure 2. LOOCV Kaplan-Meier and Time-dependent ROC curves. A prognostic index for a patient with a specific genotyping profile were calculated as the weighted average (weighted as regression coefficient) of the five-marker genotypes. LOOCV Kaplan-Meier curve (Figure 2 A) identified 90 patients (22 deaths) in the low risk group (green line) and 194 (78 deaths) in the high risk group (red line). Time-dependent ROC curves showed that the AUCs at 5 and 7 year were 0.645 and 0.720, respectively (Figure 2B, 7 year survival ROC curve). doi:10.1371/journal.pone.0040805.g002 Cw6 or HLA-B44 had better OS. However, only Cw6 and DRB115 could inversely predict RFS (p = 0.021, HR 0.593 and p = 0.026, 1.543 respectively). and HLA genotype taken as single factors has marginal bearing on survival risk. However, the analytic strategy based on Cox’s proportional hazards model identified a combination that had predictive value on clinical outcome: HLA-B38, HLA-C15, HLA- Cw3, DRB115 and CTLA4 CT60G/G gene polymorphisms. As high throughput and high resolution technologies are applied in human study, increasing evidence suggests that a given immune physiological and pathological condition is regulated and governed by coordinated gene networks and overlapping pathways [44–46]. Million years of evolution and selection acquired redundant machineries to compensate for single allele variants such as synonymous polymorphism or silent mutation that alone would impact of critical functions. This may have resulted in not one single gene playing a high weight in determining the fate of complex diseases, but rather a combination of modifiers affecting a giving phenotype. Thus, multigenic correlations are more likely to be effective in predicting patient outcomes [47,48]. In this study, the association of SNPs in the CTLA4, microsatellite in FOXP3 Among the five identified markers, HLA-B38, HLA-C15 and HLA-C3 were associated with low risk of RFS. HLA-B38 previously had been positively associated with several autoimmune disorders [49] and negatively with childhood acute leukemia [50]. HLA-Cw3 has been reported as a favorable prognostic biomarker. Discussion In a prospective randomized, observation-controlled, phase III trial of adjuvant Melacine as allogeneic stage IV melanoma vaccine study, patients expressing $2 specific class I antigens (HLA-A2, HLA-A28, HLAB44, HLA-B45 and HLA-C3) and carrying HLA-Cw3 and/or HLA-A2 genotype enjoyed significant benefit from adjuvant therapy (5-year RFS for vaccinated patients was 77%, compared with 64% in the observation group, P = 0.004) [51]. Table 5. Multivariate overall survival (OS) analysis with stage correction. Alleles HR 95% CI of HR P value HLA-B38 0.089 [0.012, 0.648] 0.017 HLA-C15 0.443 [0.192, 1.020] 0.056 HLA-C3 0.446 [0.235, 0.847] 0.014 DRB115 1.729 [1.078, 2.773] 0.023 CT60G/G 1.715 [1.093, 2.691] 0.019 Stage 2.062 [1.314, 3.234] 0.002 doi:10.1371/journal.pone.0040805.t005 Table 5. Multivariate overall survival (OS) analysis with stage correction. Conversely, the DRB115 allele was found to be a high risk marker in our prediction model. DRB115 is one of the susceptibility factors for multiple sclerosis [52] and DRB115 alleles is associated with significantly increased risk to develop hepatocellular carcinoma in Asians (OR = 2.88, 95%CI: 1.77– 4.69, P,0.001) [53]. The DRB115-DQB106 haplotype is also associated with predisposition to suffer HPV infection (p(c) ,0.05) and develop cervical cancer (p(c) ,0.05) [54] suggesting that this genotype is associated with malignance predisposition. g yp g p p In addition to HLA, CT 60 G/G allele, one of the CTLA4 polymorphisms was identified as a high risk markers in this multivariate analysis associated with shorter OS. CTLA4 polymorphism has been reported in association with degree of responsiveness in melanoma patient treated by CTL-4 blockade July 2012 \| Volume 7 \| Issue 7 \| e40805 6 Genetic Model for Prognosis of Melanoma Patients Figure 3. Multivariate Cox regression analysis with stage as a factor. LOOCV Kaplan-Meier curve (Figure 3A) showed that 131 patients (31 deaths) were in the low risk group (green line) and 153 patients (69 deaths) were in the high risk group (red line). The Leave-One-Out Cross Validated time dependent (7 year) ROC curves (Figure 3B) to compare different prognostic models: Stage only (dashed line); Stage and five-marker genotyping signature (red line); Five-marker genotyping signature only (green line). doi:10.1371/journal.pone.0040805.g003 Figure 3. Multivariate Cox regression analysis with stage as a factor. LOOCV Kaplan-Meier curve (Figure 3A) showed that 131 patients (31 deaths) were in the low risk group (green line) and 153 patients (69 deaths) were in the high risk group (red line). References of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis. Curr Opin Rheumatol; 15(4): 430–435. of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis. Curr Opin Rheumatol; 15(4): 430–435. 1. Garbe C, Eigentler TK, Keilholz U, Hauschild A, Kirkwood JM (2011) Systematic review of medical treatment in melanoma: current status and future prospects. Oncologist; 16(1): 5–24. 1. Garbe C, Eigentler TK, Keilholz U, Hauschild A, Kirkwood JM (2011) Systematic review of medical treatment in melanoma: current status and future prospects. Oncologist; 16(1): 5–24. p ( ) 17. Pectasides D, Dafni U, Bafaloukos D, Skarlos D, Polyzos A, et al. (2009) Randomized phase III study of 1 month versus 1 year of adjuvant high-dose interferon alfa-2b in patients with resected high-risk melanoma. J Clin Oncol; 27(6): 939–944. p p g ( ) 2. American Academy of Dermatology. Melanoma Fact Sheet Available: http:// www aad org/media-resources/stats-and-facts/conditions/skin-cancer 2011. 3. Huang CL, Halpern AC (2005) Management of the patient with melanoma In Rigel DS, Friedman RJ, Dzubow LM, Reintgen DS, Bystryn JC, Marks R eds. Cancer of the skin. New York, NY: Elsevier Saunders 2005: 265–75. 18. Kristiansen OP, Larsen ZM, Pociot F (2000) CTLA-4 in autoimmune diseases–a general susceptibility gene to autoimmunity? Genes Immun; 1(3): 170–184. 19. Thompson CB, Allison JP (1997) The emerging role of CTLA-4 as an immune attenuator. Immunity; 7(4): 445–450. 4. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, et al. (2009) Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol; 27(36): 6199–6206. 20. Ueda H, Howson JM, Esposito L, Heward J, Snook H, et al. (2003) Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature; 423(6939): 506–511. 5. American Cancer Society. Cancer Facts & Figures 2010 Available: http:// www cancer org/acs/groups/content/@epidemiologysurveilance/documents/ document/acspc-026238 pdf 2011. 21. Gough SC, Walker LS, Sansom DM (2005) CTLA4 gene polymorphism and autoimmunity. Immunol Rev; 204: 102–115. 6. Kirkwood JM, Ibrahim JG, Sosman JA, Sondak VK, Agarwala SS, et al.(2001) High-dose interferon alfa-2b significantly prolongs relapse-free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with resected stage IIB-III melanoma: results of intergroup trial E1694/S9512/C509801. J Clin Oncol; 19(9): 2370–2380. 22. Sanderson K, Scotland R, Lee P, Liu D, Groshen S, et al. References (2005) Autoimmunity in a phase I trial of a fully human anti-cytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma. J Clin Oncol; 23(4): 741– 750. 7. Kirkwood JM, Richards T, Zarour HM, Sosman J, Ernstoff M, et al. (2002) Immunomodulatory effects of high-dose and low-dose interferon alpha2b in patients with high-risk resected melanoma: the E2690 laboratory corollary of intergroup adjuvant trial E1690. Cancer; 95(5): 1101–1112. 23. Saiki RK, Walsh PS, Levenson CH, Erlich HA (1989) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A; 86: 6230–6234. 24. Olerup O, Zetterquist H (1992) HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens; 39(5): 225–235. 8. Yurkovetsky ZR, Kirkwood JM, Edington HD, Marrangoni AM, Velikokhat- naya L, et al. (2007) Multiplex analysis of serum cytokines in melanoma patients treated with interferon-alpha2b. Clin Cancer Res; 13(8): 2422–2428. 9. Nestle FO, Kaplan DH, Barker J Psoriasis (2009) N Engl J Med; 361(5): 496– 509. 25. Marsh SG, Albert ED, Bodmer WF, Bontrop RE, Dupont B, et al. (2002) Nomenclature for factors of the HLA system, 2002. Eur J Immunogen; 29(6): 463–515. 10. Muto M, Deguchi H, Tanaka A, Inoue T, Ichimiya M (2011) Association between T-lymphocyte regulatory gene CTLA4 single nucleotide polymorphism at position 49 in exon 1 and HLA-DRB108 in Japanese patients with psoriasis vulgaris. J Dermatol Sci; 62(1): 70–71. 26. Cox DR (1972) Regression models and life-tables. J Roy Stat Soc Ser; 34(2): 187–220. 27. Simon RM, Subramanian J, LI MC, Menezes S (2011) Using cross-validation to evaluate predictive accuracy of survival risk classifiers based on high-dimensional data. Brief Bioinform; 12(3): 203–214. 11. Bouwhuis MG, Gast A, Figl A, Eggermont AM, Hemminki K, et al. (2010) Polymorphisms in the CD28/CTLA4/ICOS genes: role in malignant melanoma susceptibility and prognosis? Cancer Immunol Immunother; 59(2): 303–312. 28. Heagerty PJ, Lumley T, Pepe MS (2000) Time-dependent ROC curves for censored survival data and a diagnostic marker. Biometrics; 56(2): 337–344. 12. Gogas H, Dafni U, Koon H, Spyropoulou-Vlachou M, Metaxas Y, et al.(2010) Evaluation of six CTLA-4 polymorphisms in high-risk melanoma patients receiving adjuvant interferon therapy in the He13A/98 multicenter trial. J Transl Med; 8: 108. 29. Genetic Model for Prognosis of Melanoma Patients [55]. CTLA4 haplotypes has also been documented in association with susceptibility to develop esophageal squamous cell carcinoma and osteosarcoma [56,57]. However, no significant association between CT 60 G/G allele and with risk of developing cancer has been previously reported (CT60: OR, 1.02; 95% CI, 0.80–1.29 for AA + AG vs GG) based on 48 case-control meta analysis studies from 27 articles were analyzed [58]. excision of the melanoma but had different survival outcomes; thus, the study was aimed at testing the genetic bearing within the cancer population on clinical outcome. A population not undergoing this treatment is currently not available to these investigators and, therefore, this study cannot conclusively address the issue of whether the combination of genetic markers is predictive or response to therapy rather than reflecting an overall genetic predisposition to better prognosis. Author Contributions Conceived and designed the experiments: EW JMK FMM HG MCP. Performed the experiments: EW AM LU. Analyzed the data: EW AM LU. Contributed reagents/materials/analysis tools: EW AM LU JMK MSV MCP FMM HG. Wrote the paper: EW YZ. Performed the experiments: EW AM LU. Analyzed the data: EW AM LU. Contributed reagents/materials/analysis tools: EW AM LU JMK MSV MCP FMM HG. Wrote the paper: EW YZ. Contributed reagents/materials/analysis tools: EW AM LU JMK MSV MCP FMM HG. Wrote the paper: EW YZ. Supporting Information Our study suggests that single nucleotide variants and their association with a given condition should be explored together with polymorphisms genes with relevant and compensatory function that are thought to contribute to biological changes. Genome wide association analysis would be an ideal way to explore the complexity of genetic variants associated with disease predisposition, prognosis and treatment response, if patient population and funding issue is not challenged. The five markers identified in the study need to be validated prospectively in an independent cohort of patients receiving the same therapy. Moreover, all patients underwent IFNa treatment following Table S1 Univariate survival analysis for FoxP3 micro- satellites. (DOCX) Discussion The Leave-One-Out Cross Validated time dependent (7 year) ROC curves (Figure 3B) to compare different prognostic models: Stage only (dashed line); Stage and five-marker genotyping signature (red line); Five-marker genotyping signature only (green line). doi:10.1371/journal.pone.0040805.g003 ined survival risk model. The LOOCV Kaplan-Meier curves showed that Figure 4. The LOOV Validated Kaplan-Meier curve based on combined survival risk model. The LOOCV Kaplan-Meier curves showed that the five-marker OS model (Figure 4A) and the combined model (five-marker + stage model) (Figure 4B) may be used to predict RFS survival data. In each round of LOOCV, the model was built in the training set using the OS information and tested on the left out sample using the RFS information. doi:10.1371/journal.pone.0040805.g004 Figure 4. The LOOV Validated Kaplan-Meier curve based on combined survival risk model. The LOOCV Kaplan-Meier curves showed that the five-marker OS model (Figure 4A) and the combined model (five-marker + stage model) (Figure 4B) may be used to predict RFS survival data. In each round of LOOCV, the model was built in the training set using the OS information and tested on the left out sample using the RFS information. doi:10.1371/journal.pone.0040805.g004 July 2012 \| Volume 7 \| Issue 7 \| e40805 PLoS ONE \| www.plosone.org 7 7 Genetic Model for Prognosis of Melanoma Patients Genetic Model for Prognosis of Melanoma Patients Genetic Model for Prognosis of Melanoma Patients 35. Liu D, O’Day SJ, Yang D, Boasberg P, Milford R, et al. (2005) Impact of gene polymorphisms on clinical outcome for stage IV melanoma patients treated with biochemotherapy: an exploratory study. Clin Cancer Res; 11(3): 1237–1246. 48. Li Y, Sun Z, Cunningham JM, Aubry MC, Wampfler JA, et al. (2011) Genetic variations in multiple drug action pathways and survival in advanced stage non- small cell lung cancer treated with chemotherapy. Clin Cancer Res; 17(11): 3830–3840. 36. Ugurel S, Schrama D, Keller G, Schadendorf D, Brocker EB, et al. (2007) Impact of the CCR5 gene polymorphism on the survival of metastatic melanoma patients receiving immunotherapy. Cancer Immunol Immunother; 57(5): 685–691. 49. Colovai AI, Suciu-Foca N, Baiulescu GE, Harris PE (1994) HLA class I self peptides isolated from a T-cell leukemia reveal the allele-specific motif of HLA- B38. Tissue Antigens; 44(2): 65–72. 37. Casula M, Budroni M, Cossu A, Ascierto PA, Mozzillo N, et al. (2010) The susceptibility CDKN2 locus may have a role on prognosis of melanoma patients. Ann Oncol; 21(6): 1379–1380. 50. Ozdilli K, Oguz FS, Anak S, Kekik C, Carin M, et al. (2010) The frequency of HLA class I and II alleles in Turkish childhood acute leukaemia patients. J Int Med Res; 38(5): 1835–1844. 38. Amundsen SS, Naluai AT, Ascher H, Ek J, Gudjonsdottir AH, et al. (2004) Genetic analysis of the CD28/CTLA4/ICOS (CELIAC3) region in coeliac disease. Tissue Antigens; 64(5): 593–599. 51. Sosman JA, Unger JM, Liu PY, Flaherty LE, Park MS, et al. (2002) Adjuvant immunotherapy of resected, intermediate-thickness, node-negative melanoma with an allogeneic tumor vaccine: impact of HLA class I antigen expression on outcome. J Clin Oncol; 20(8): 2067–2075. 39. Niu J, Jiang C, Li C, Liu L, Li K, et al. (2011) Foxp3 expression in melanoma cells as a possible mechanism of resistance to immune destruction. Cancer Immunol Immunother; 60(8): 1109–1118. outcome. J Clin Oncol; 20(8): 2067–2075. 52. Banwell B, Bar-Or A, Arnold DL, Sadovnick D, Narayanan S, et al. (2011) Clinical, environmental, and genetic determinants of multiple sclerosis in children with acute demyelination: a prospective national cohort study. Lancet Neurol; 10(5): 436–445. 40. Sakaguchi S (2011) Regulatory T cells: history and perspective. Methods Mol Biol; 707: 3–17. 41. Schwartzentruber DJ, Lawson DH, Richards JM, Conry RM, Miller DM, et al. (2011) gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma. References Ascierto PA, Kirkwood JM (2008) Adjuvant therapy of melanoma with interferon: lessons of the past decade. J Transl Med; 6(1): 62. 30. Clemente CG, Mihm MCJ, Bufalino R, Zurrida S, Collini P, et al. (1996) Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer; 77(7): 1303–1310. 13. Benhatchi K, Jochmanova I, Habalova V, Wagnerova H, Lazurova I (2011) CTLA4 exon1 A49G polymorphism in Slovak patients with rheumatoid arthritis and Hashimoto thyroiditis-results and the review of the literature. Clin Rheumatol; 30(10): 1319–24. 31. Deschoolmeester V, Baay M, Van ME, Weyler J, Vermeulen P, et al. (2010) Tumor infiltrating lymphocytes: an intriguing player in the survival of colorectal cancer patients. BMC Immunol; 11: 19. 14. Bassuny WM, Ihara K, Sasaki Y, Kuromaru R, Kohno H, et al. (2003) A functional polymorphism in the promoter/enhancer region of the FOXP3/ Scurfin gene associated with type 1 diabetes. Immunogenetics; 55(3): 149–156. 32. Gooden MJ, de Bock GH, Leffers N, Daemen T, Nijman HW (2011) The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis. Br J Cancer; 105(1): 93–103. 15. Ban Y, Tozaki T, Tobe T, Ban Y, Jacobson EM, et al. (2007) The regulatory T cell gene FOXP3 and genetic susceptibility to thyroid autoimmunity: an association analysis in Caucasian and Japanese cohorts. J Autoimmun; 28(4): 201–207. y 33. Mahmoud SM, Paish EC, Powe DG, Macmillan RD, Grainge MJ, et al. (2011) Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer. J Clin Oncol; 29(15): 1949–1955. 34. Rizza P, Capone I, Moretti F, Proietti E, Belardelli F (2011) IFN-alpha as a vaccine adjuvant: recent insights into the mechanisms and perspectives for its clinical use. Expert Rev Vaccines; 10(4): 487–498. 16. Gambineri E, Torgerson TR, Ochs HD (2003) Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40805 July 2012 \| Volume 7 \| Issue 7 \| e40805 8 PLoS ONE \| www.plosone.org Genetic Model for Prognosis of Melanoma Patients N Engl J Med; 364(22): 2119–2127. 53. Lin ZH, Xin YN, Dong QJ, Wang Q, Jiang XJ, et al. (2010) Association between HLA-DRB1 alleles polymorphism and hepatocellular carcinoma: a meta- analysis. BMC Gastroenterol; 10: 145. g J ( ) 42. Hoon DS, Okamoto T, Wang HJ, Elashoff R, Nizze AJ, et al. (1998) Is the survival of melanoma patients receiving polyvalent melanoma cell vaccine linked to the human leukocyte antigen phenotype of patients? J Clin Oncol; 16(4): 1430–1437. 54. Kohaar I, Hussain S, Thakur N, Tiwari P, Nasare V, et al. (2009) Association between human leukocyte antigen class II alleles and human papillomavirus- mediated cervical cancer in Indian women. Hum Immunol; 70(4): 222–229. 55. Breunis WB, Tarazona-Santos E, Chen R, Kiley M, Rosenberg SA, et al. (2008) Influence of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) common polymorphisms on outcome in treatment of melanoma patients with CTLA-4 blockade. J Immunother; 31(6): 586–590. 43. Gogas H, Kirkwood JM, Falk CS, Dafni U, Sondak VK, et al. (2010) Correlation of molecular human leukocyte antigen typing and outcome in high- risk melanoma patients receiving adjuvant interferon. Cancer; 116(18): 4326– 4333. 56. Cheng XL, Ning T, Xu CQ, Li Y, Zhu BS, et al. (2011) Haplotype analysis of CTLA4 gene and risk of esophageal squamous cell carcinoma in Anyang area of China. Hepatogastroenterology; 58(106): 432–437. 44. Wang E, Worschech A, Marincola FM. (2008) The immunologic constant of rejection. Trends Immunol; 29(6): 256–262. 45. Ascierto ML, De Giorgi V, Liu Q, Bedognetti D, Murtas D, et al. (2011) An immunologic portrait of cancer. J Transl Med; 9: 146. 57. Liu Y, He Z, Feng D, Shi G, Gao R, et al. (2011) Cytotoxic T-lymphocyte Antigen-4 Polymorphisms and Susceptibility to Osteosarcoma. DNA Cell Biol; 30(12): 1051–1055. 46. Spivey TL, Uccellini L, Ascierto ML, Zoppoli G, De G, V, et al. 2011 Gene expression profiling in acute allograft rejection: challenging the immunologic constant of rejection hypothesis. J Transl Med; 9: 174. 58. Zhang Y, Zhang J, Deng Y, Tian C, Li X, et al. (2011) Polymorphisms in the cytotoxic T-lymphocyte antigen 4 gene and cancer risk: A meta-analysis. Cancer; 15;117(18): 4312–24. j yp 47. Briggs FB, Shao X, Goldstein BA, Oksenberg JR, Barcellos LF, et al. (2011) Genome-wide association study of severity in multiple sclerosis. Genes Immun; 12(8): 615–625. PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40805 9
https://openalex.org/W2753680582	https://breast-cancer-research.biomedcentral.com/track/pdf/10.1186/s13058-017-0894-6	English	null	Breast Tissue Organisation and its Association with Breast Cancer Risk	Breast cancer research	2,017	cc-by	10,452	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Mammographic percentage density is an established and important risk factor for breast cancer. In this paper, we investigate the role of the spatial organisation of (dense vs. fatty) regions of the breast defined from mammographic images in terms of breast cancer risk. Methods: We present a novel approach that provides a thorough description of the spatial organisation of different types of tissue in the breast. Each mammogram is first segmented into four regions (fatty, semi-fatty, semi-dense and dense tissue). The spatial relations between each pair of regions is described using so-called forces histograms (FHs) and summarised using functional principal component analysis. In our main analysis, association with case–control status is assessed using a Swedish population-based case–control study (1,170 cases and 1283 controls), for which digitised mammograms were available. We also carried out a small validation study based on digital images. Results: For our main analysis, we obtained a global p value of 2 × 10−7 indicating a significant association between the spatial relations of the four segmented regions and breast cancer status after adjustment for percentage density and other important breast cancer risk factors. Our (spatial relations) score had a per standard deviation odds ratio 1.29, after accounting for overfitting (percentage density had a per standard deviation odds ratio of 1.34). The spatial relations between the fatty and semi-fatty tissue and the spatial relations between the fatty and dense tissue were the most significant. The spatial relations between the fatty and semi-fatty tissue were associated with parity and age at first birth (p = 6×10−4). Using digital images, we were able to verify that the same characteristics of tissue organisation can be identified and we validated the association for the spatial relations between the fatty and semi-fatty tissue. Conclusions: Our findings are consistent with the notion that fibroglandular and adipose tissue plays a role in breast cancer risk and, more specifically, they suggest that fatty tissue in the lower quadrants and the absence of density in the retromammary space, as shown in mediolateral oblique images, are protective against breast cancer. Keywords: Mammography, Spatial organisation, Breast cancer risk, Adipose distribution Keywords: Mammography, Spatial organisation, Breast cancer risk, Adipose distribution PD). Women exhibiting a high PD, e.g. over 75%, have an approximately sixfold increased risk of breast cancer compared to women with a low PD (< 5%) [1]. However, the underlying mechanisms of this association are still unclear. Alsheh Ali et al. Breast Cancer Research (2017) 19:103 DOI 10.1186/s13058-017-0894-6 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 DOI 10.1186/s13058-017-0894-6 Correspondence: Keith.Humphreys@ki.se 1Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 2Swedish eScience Research Centre (SeRC), Karolinska Institutet, Stockholm, Sweden Full list of author information is available at the end of the article Abstract Although PD aims at measuring the amount of dense tissue in the breast, it indirectly reflects the quantity of fat. The role of non-dense tissue in cancer develop- ment has been investigated by several researchers with contrasting outcomes [2, 3]. Maya Alsheh Ali1,2, Kamila Czene1, Louise Eriksson1,3, Per Hall1 and Keith Humphreys1,2 Maya Alsheh Ali1,2, Kamila Czene1, Louise Eriksson1,3, Per Hall1 and Keith Humphreys1,2* Background Recent years have seen intensive efforts put into searching for relevant information from mammograms to assist the prediction of breast cancer risk. Mammographic breast density, which represents the amount of fibroglandular tissue in the breast, is the only strongly established image- based risk factor for breast cancer [1]. It is measured quan- titatively either as the total dense area or the percentage of dense area on the mammogram (percentage density or Mammograms present a two-dimensional projection of the breast, superimposing several layers of tissue into a single image. Hence, some researchers argue that mea- sures of dense mammographic volume should be more accurate for classifying risk than measures of dense mam- mographic area [4]. These measures have still not been Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Page 2 of 13 Page 2 of 13 studiedasextensively as area-basedmethodsandKelleret al. [5] suggest that volumetric and areal density measures may be complementary for breast cancer risk assessment. medical image analysis [12, 13] and we recently described how they can be applied to analyse mammograms [14]. In [14], we also carried out a pilot case–control study on 500 mammographic images. Aside from mammographic breast density, additional relevant information can be extracted from mammo- grams. Numerous studies have investigated the relations between cancer risk and the heterogeneity of the mammo- graphic parenchymal pattern using quantitative texture descriptors. These particular features have been extracted on different scales, from the entire breast region [6, 7] to specific regions of interest such as the retroareolar area [8] and the central area of the breast [9]. In our main analysis based on digitised mammograms, we show, using 1170 cases and 1283 controls, that spatial relation FHs hold important information for discriminat- ing between breast cancer cases and controls, after taking into account PD and other important breast cancer risk factors. We present additional analyses that shed light on the biological information captured by the FHs that are most strongly associated with breast cancer risk. We also present a small validation study based on 300 digital mammograms (69 cases and 231 controls). Another type of information present in mammograms, though seldom measured, is the spatial organisation (rela- tions) of the different types of tissue in the breast. Background Whilst mammographic breast density summarises the relative amounts of dense and fatty tissue in the breast and different texture features measure the local interactions between pixel intensities, spatial relations quantitatively capture the global layout of dense and fatty tissue. It has been previously suspected that the relative distribution of adipose and fibroglandular tissue is involved in breast cancer development [10]. Figure 1 shows three different mammograms to illustrate different types of distributions of fatty and dense tissue inside the breast. Such differ- ences could be identified by, for instance, applying basic shape descriptors on the segmented dense tissue and by measuring the distance from its centroid to the skin line [5]. However, measuring only a specific spatial relation is rarely sufficient to describe fully the possibly complex relationships between two objects. In this article, we use a novel approach that provides a more complete descrip- tion of the spatial organisation of different types of tissue in the breast. We use the so-called forces histograms (FHs), which represent quantitative fuzzy spatial relation descriptors of the pairwise relations of different regions of interest. A single FH takes into account both the direc- tional and distance relationships as well as the shapes of two objects [11]. FHs have been used in other fields of Methods Materials Our main analysis is based on CAHRES (Cancer and Hormone Replacement Study), a population-based post- menopausal breast cancer case–control study of Swedish residents born in Sweden and aged 50 to 74 years, between 1 October 1993 and 31 March 1995. The study includes approximately 6000 women (3000 cases and 3000 age group matched controls) from which area-based PD mea- surements of digitised mammograms are available [15]. Body mass index (BMI) was recorded at entrance to the study, whereas age, in this study, was assessed according to date of mammography. All cases had primary invasive breast cancer. We used the mediolateral oblique (MLO) view since it offers the best opportunity to visualise the maximum amount of breast tissue in a single image. Mam- mograms were digitised with an Array 2905HD Laser Film Digitizer.1 Density resolution was set at 12 bits, spatial res- olution at 5.0 mm and optical density at 0–4.7. The size of the images was 4770 × 3580 pixels with 0.05 mm per pixel. For the present study, we used 2453 mammograms (1170 cases and 1283 controls) for which data on PD, age, BMI, hormone replacement therapy (HRT) status, parity and age at first birth (AFB) were available for all women Fig. 1 Examples of mammograms exhibiting different distributions of fatty and dense tissue. a The dense tissue is mainly located on the lower part of the breast. b The bulk of the dense tissue is concentrated in the retroareolar area. c The dense tissue is scattered but falls into two clusters, one next to the nipple and the other in the upper part of the breast Fig. 1 Examples of mammograms exhibiting different distributions of fatty and dense tissue. a The dense tissue is mainly located on the lower part of the breast. b The bulk of the dense tissue is concentrated in the retroareolar area. c The dense tissue is scattered but falls into two clusters, one next to the nipple and the other in the upper part of the breast Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Page 3 of 13 included. Of these, 500 (250 cases and 250 controls) were previously included in our pilot study [14]. used an automated measure of area PD, which has been shown to perform in line with other established density measures in terms of breast cancer risk association [20]. Methods Materials We carried out a small validation study using digi- tal mammograms from the Karolinska Mammography cohort (KARMA) study (http://karmastudy.org/), which is a prospective cohort study that was initiated in January 2011. Recruitment ended in March 2013. It comprises women attending mammography screening or clinical mammography at four hospitals in Sweden [16]. Participants answered a comprehensive web-based questionnaire, allowed storage of mammograms and accepted linkage to national breast cancer registers. Iden- tification of KARMA participants as cases or controls for the present study was based on linkage with the Swedish Cancer Registry (last updated 31 December 2013). Here, we included 69 incident cases (59 with primary invasive cancer and 10 with ductal carcinoma in situ) with full field digital mammography images (raw MLO images) from GE Medical Systems, model Senographe Essential ver- sion ADS 53.40. We selected an additional 231 healthy controls with images taken with the same machine (so that, in total, our validation study was based on 300 post- menopausal women). The size of the images was 3062 × 2394 pixels with 0.1 mm per pixel. Information on age, BMI, HRT status and reproductive history was collected via a web-based questionnaire at study entry. Spatial relations measurements Image preprocessing The main aim of the preprocessing step is to separate the breast from other objects in the mammogram (i.e. labels, tags and screening artefacts) with a minimum loss of breast tissue. In general, two independent steps are per- formed. The first aims to segment the breast region, while the second separates the pectoral muscle from the rest of the breast area. The pectoral muscles were removed from both digital and digitised images using the texture gradient-based approach proposed by Bora et al. [21]. Digitised mammograms were rescaled to have pixel val- ues between 0 and 1 and denoised using pixelwise adap- tive Wiener filtering. The strong signal-to-noise ratio of the digital images allowed us to segment the breast region by applying a simple thresholding to the image. To seg- ment the breast profile, the contrast of the image first needed to be enhanced to brighten the low-intensity pixels close to the skin line. This was achieved using a loga- rithmic transformation of the image. The image was then segmented into three classes according to Otsu’s multi- thresholding method. The two brightest classes were kept since they correspond to the breast region and the other objects in the mammogram, while the darkest corre- sponds to the background. The breast mask was finally extracted as the largest group of connected components and smoothed using morphological filtering. In both studies, for cases, all mammograms were taken less than 3 years before diagnosis (and at latest, at date of diagnosis). For cases, we used the mammogram contralat- eral to the tumour to ensure that image measurements were not affected by the tumours, whereas for controls an image of a single side was selected at random. This is common practice in case–control studies using mammo- graphic images [17, 18]. For KARMA controls, all mam- mograms were taken at questionnaire date and, therefore, had a confirmed negative follow-up of at least 9 months. For CAHRES, mammograms of the controls were taken within 3 years of questionnaire date (the majority of mam- mograms were from before questionnaire date, but some were from several months after) and were all from before the diagnosis date of the last incident case, so that they were free from a breast cancer diagnosis. Partition of breast images Af b i g After breast region segmentation, pectoral muscle removal and contrast enhancement for raw digital images, breast regions representing different types of tissue need to be defined. In the literature, there is no standard for the number of regions that can be defined from an X-ray image of the breast, and the number used has varied from two to 13 [22], often depending on the purpose of the study. In our work, we defined four different regions in the breast. The choice of four regions is common in the literature and corresponds to the number of original parenchymal patterns defined by Wolfe [23], who cate- gorised images according to both the extent of densities and their characteristics (prominence of ducts and dys- plasia). Also, four regions representing very dense tissue (both fibrotic stromal and glandular tissue), the fatty background of the mammogram and the fatty breast edge have been used for extracting textural descriptions [24]. Here, we used a fully automated segmentation method, the fuzzy C-Means clustering, to divide the breast into four regions loosely representing the fatty, semi-fatty, semi-dense and dense tissue. Statistical methods Then, for each line, a weight is calculated as the sum of the inverted squared Euclidean distances between all pairs of pixels, such that the first pixel belongs to object A and the other to B. Finally, the weights of all the lines are summed to generate the value of the FH along this particular direction, which corre- sponds to a single bin of the histogram. This procedure is repeated for a series of angles evenly distributed between 0° and 360°. The number of angles considered defines the length of the histogram and its angular precision, hence shorter descriptions are less accurate. Each image of the breast, segmented into four regions, is then described by a set of six FHs (4 choose 2) measur- ing the relative positions of the different pairs of regions. We use FHij to denote the FH that measures the rela- tive positions of regions i and j. FH descriptions for an example mammographic image are shown as part of Fig. 3 (which is an overview of the complete strategy for our main analysis, which we continue to describe in ‘Statistical methods’). The computation of the FH follows a complex- ity of O(an√n), where n is the number of pixels in the image and a is the number of angles considered. To reduce the computation time while retaining the overall spatial organisation of the tissue, each image was rescaled to 0.25 mm per pixel and we set the number of angles to 180 (a step of 2°). For more details, see [14]. We evaluated the association between the fPCs and case–control status in CAHRES by fitting logistic regres- sion models treating case–control status as a dependent variable and the fPCs as independent variables. All fPCs were standardised to zero mean and unit variation prior to being included in logistic regression models. We included age, BMI, PD, HRT, parity and AFB (the variables parity and AFB were combined into a single categorical variable with five categories; see Table 1) in all logistic regression models as adjustment variables. In our main analysis, we chose to transform PD by taking its square root prior to including it as an adjustment variable, although we also Fig. 2 Illustration of the computation of FHs between two objects A (green) and B (orange). a Both objects and the parallel lines sweeping across the image oriented by a specific angle θ°. Statistical methods Prior to testing for association with breast cancer status, for each study, we compressed the key information gath- ered in our six FHs (described by, in total, 6 × 180 FH variables) into a small number of variables. Each FH can be viewed as a function of the angle θ. To summarise the information contained in each FH, we apply approaches that are suitable for analysing data. These provide infor- mation about phenomena varying over a continuum (i.e. curves). Specifically, we chose to extract the dominant modes of variations included in each FH by carrying out a functional principal component (fPC) analysis [25]. We used the method described in [26], which is imple- mented in PACE,2 a MATLAB package for functional data analysis. This approach transforms the FH data to K-dimensional multivariate data consisting of the first K(fPC) scores, which account for a cumulative variance of 85%. In CAHRES, for all FHs, we retained two fPCs per FH except for the one relating regions 2 and 4, for which we retained three fPCs (13 fPCs, in total). Exactly the same number of fPCs (for all FHs) was retained when analysing the KARMA images (again 13 fPCs, in total). We will denote the kth fPC for the FH that measures the rel- ative positions of regions i and j by fPCk ij. For CAHRES, we carried out our main association analysis by describing breast tissue organisation using all 13 fPC variables. This step of the analysis is represented by the box ‘functional PCA’ in Fig. 3. The FHmethod is applied to describe comprehensively the relative positions of the different regions of tissue. Since the FH encapsulates in a single histogram the directional and the distance relationships between the regions con- sidered as well as their shapes, we construct an exhaustive description of the spatial organisation of tissue in a mam- mogram by computing the FH between all pairs of regions. Figure 2 illustrates the principle of the computation of the FH between two sets of pixels, in this example compar- ing pixels within a region A (in green) with pixels within a region B (in orange). The value of the FH for a spe- cific angle θ is obtained using the following algorithm. First, a series of parallel lines sweeping across the image in direction θ is defined. Percentage density measurement used in this study Percentage density measurement used in this study For CAHRES, an area-based PD for each image was mea- sured using the user-assisted approach of Cumulus [19]. Cumulus is the most widely used software for measure- ment of mammographic density in analogue images. The user first needs to trace and remove the pectoral mus- cle manually. Then, they use sliders to perform global (breast region) and then local (dense region) interactive thresholding. A trained user (LE) carried out the Cumulus measurements blinded to case–control status. KARMA images were not read by Cumulus. For these images, we Page 4 of 13 Page 4 of 13 Page 4 of 13 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Statistical methods b The final FH (with angles between 0° and 360°) describing the spatial relationships between A and B. In this example, the maximum value of the FH is obtained for an angle of approximately 18° and is empty for values between 50° and 350°, since no line meets both objects A and B along these orientations. FH forces histogram Fig. 2 Illustration of the computation of FHs between two objects A (green) and B (orange). a Both objects and the parallel lines sweeping across the image oriented by a specific angle θ°. b The final FH (with angles between 0° and 360°) describing the spatial relationships between A and B. In this example, the maximum value of the FH is obtained for an angle of approximately 18° and is empty for values between 50° and 350°, since no line meets both objects A and B along these orientations. FH forces histogram Page 5 of 13 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Fig. 3 Overview of main analyses. A given breast image is first segmented into four regions: dense (red), semi-dense (yellow), semi-fatty (light blue) and fatty (dark blue) tissue. The spatial relations between each pair of regions is described by a forces histogram. The information captured by each forces histogram is compressed into a small number of variables using a functional principal component analysis and then the association between these variables (representing the spatial organisation of the regions) and breast cancer status is evaluated through a statistical test. PCA principal component analysis Fig. 3 Overview of main analyses. A given breast image is first segmented into four regions: dense (red), semi-dense (yellow), semi-fatty (light blue) and fatty (dark blue) tissue. The spatial relations between each pair of regions is described by a forces histogram. The information captured by each forces histogram is compressed into a small number of variables using a functional principal component analysis and then the association between these variables (representing the spatial organisation of the regions) and breast cancer status is evaluated through a statistical test. PCA principal component analysis (which we will refer to as an apparent estimate). We, therefore, used a bootstrapping procedure that provides a nearly unbiased (honest) estimate of the effect size (the procedure is, in fact, slightly biased in the direction of underestimating the effect); see Harrell et al. [27]. Statistical methods We also estimated area under the receiver operating characteristic curve (AUC) values (honest values were calculated using the bootstrap procedure) and used Delong’s test [28] for comparing apparent AUC values. repeated our analysis with PD on its original scale as well as categorised into five groups ([ 0, 5[, [ 5, 10[, [ 10, 20[, [ 20, 40[ and [ 40, 100]). In our data, the overall model fit was best using square root PD. To evaluate whether our selected features (fPCs) were, overall, associated with breast cancer status (after adjustment for potential con- founding variables), we carried out a likelihood ratio test. This step of the analysis is represented by the box ‘Associ- ation with breast cancer risk’ in Fig. 3. After carrying out our global test of association and estimating AUC values and an effect size for a spatial relation score, to interpret our results, we identified the most important fPCs using a stepwise selection procedure (using the step function in R). Two fPCs were selected. For these two fPCs, to identify the angle(s) (in their cor- responding FHs) for which they predominantly capture variability, we visualised plots of modes of variations [29] and eigenfunctions (see Figure 6 in the Appendix). To understand the fPCs better, we studied their association with risk factors considered in our case–control analysis. Since (both) selected fPCs were approximately normally distributed, we did this by fitting linear regression models with normal error distributions. We estimated an effect size for a risk score constructed from the fPC variables of the spatial relations. Statistical methods If a single dataset is used naively both to train a score and to eval- uate its effect size, the effect size will be overestimated Table 1 Key characteristics of individuals (CAHRES) Characteristic Cases Controls P value Number 1,170 1,283 HRT use 2 × 10−9 Never 791 (68%) 998 (78%) Past 98 (8%) 40 (3%) Current 281 (24%) 245 (19%) Parity and AFB 7 × 10−7 Nulliparous 157 (13%) 129 (10%) Parity ≤2 and AFB ≤25 349 (30%) 354 (28%) Parity ≤2 and AFB > 25 372 (32%) 351 (27%) Parity > 2 and AFB ≤25 214 (18%) 351 (27%) Parity > 2 and AFB > 25 78 (7%) 98 (8%) Age 62.6 (±6.5) 63.6 (±6.4) 8 × 10−5 BMI 25.2 (±3.6) 25.0 (±3.8) 0.26 PD 18.7 (±14.6) 14.8 (±13.2) 4 × 10−12 √ PD 3.9 (±1.7) 3.5 (±1.7) 6 × 10−14 Means (with standard deviations in parentheses) are given for continuous variables and counts (with percentages in parentheses) are given for categorical variables. P values are obtained using likelihood ratio tests based on fitting logistic regression models without adjustment for additional covariates AFB age at first birth, BMI body mass index, HRT hormone replacement therapy, PD percentage density For our validation study (KARMA), we retained only the two fPCs that were selected in our analysis based on CAHRES data. We verified that the fPCs extracted from CAHRES and KARMA carried corresponding information (using plots of modes of variations and eigenfunctions; see Figure 7 in the Appendix). Associ- ational analysis in KARMA was also based on logistic regression analysis. Main analysis For CAHRES, key characteristics of cases and controls included in our analyses are described in Table 1. We refer to the four density regions segmented inside the breast as 1, 2, 3 and 4, corresponding to the fatty, semi-fatty, semi- dense and dense tissue, respectively. The 13 fPCs, which Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Page 6 of 13 Page 6 of 13 Table 2 Logistic regression results with age, BMI, √ PD, parity and AFB, HRT and fPCs as covariates (CAHRES) Covariate Estimated Standard P value coefficient error Intercept −1.828 0.667 0.006 Age −0.008 0.007 0.259 BMI 0.066 0.015 2 × 10−5 √ PD 0.199 0.040 6 × 10−7 Parity and AFB Nulliparous Parity ≤2 and AFB ≤25 −0.186 0.149 0.213 Parity ≤2 and AFB > 25 −0.139 0.147 0.346 Parity > 2 and AFB ≤25 −0.666 0.156 2 × 10−5 Parity > 2 and AFB > 25 −0.393 0.201 0.051 HRT use Never Past 0.737 0.201 2 × 10−4 Current 0.270 0.108 0.013 Spatial relations fPCsa fPC1 12 −0.150 0.130 0.250 fPC2 12 −0.395 0.100 8 × 10−5 fPC1 13 0.004 0.098 0.967 fPC2 13 0.144 0.092 0.118 fPC1 14 −0.208 0.091 0.023 fPC2 14 0.039 0.066 0.554 fPC1 23 0.148 0.121 0.219 fPC2 23 0.097 0.091 0.283 fPC1 24 −0.022 0.093 0.812 fPC2 24 0.158 0.078 0.042 fPC3 24 0.006 0.066 0.926 fPC1 34 0.118 0.065 0.069 fPC2 34 0.081 0.066 0.217 AFB age at first birth, BMI body mass index, HRT hormone replacement therapy, PD percentage density ap = 2 × 10−7 Table 2 Logistic regression results with age, BMI, √ PD, parity and AFB, HRT and fPCs as covariates (CAHRES) describe breast tissue organisation (see ‘Statistical meth- ods’), were used as covariates in the logistic regression model to investigate the association of tissue organisation with breast cancer status. Parameter estimates of the logistic regression with all covariates (fPCs) and adjustment variables are shown in Table 2. We obtained a global p value of 2 × 10−7 from a likelihood ratio test on 13 degrees of freedom, indicat- ing significant association between the spatial relations of the four segmented regions and breast cancer status. To ensure that this association is independent of breast size (it is possible that the FHs, to some extent, capture breast size), we also adjusted for the breast mammographic area; the p value was then 1 × 10−7. Main analysis We also noted that the global p value was largely unchanged when PD was on its original scale (p = 5 × 10−8), or when it was coded as a categorical variable (p = 7 × 10−7). For an independent validation of our pilot study result, we removed the 500 images that had been included in [14]. When doing so, we obtained a p value of 8 × 10−5. p We next constructed a spatial relations score, as a sum of the fPC values weighted by the estimated coefficients (for the fPCs), as shown in Table 2. From fitting a logis- tic regression model with this score (instead of the 13 fPC variables), along with PD and the other breast can- cer risk factors, and multiplying the standard deviation of the score by its regression coefficient, we obtained a naive (biased) per standard deviation effect size estimate of 0.33 (the per standard deviation odds ratio was 1.39) and an apparent AUC value of 0.654. Delong’s test gave a p value of 6.46 × 10−4 when comparing this model to one that excluded our score (which had an apparent AUC of 0.630). Using a bootstrapping procedure (based on 1000 boot- strap samples), we obtained an honest estimate of 1.29 for the per standard deviation odds ratio for the fPC-based score, and an honest estimate of 0.637 for the AUC for the full model (the honest estimate of AUC for the model excluding our score was 0.621). We note that from fitting a model that excluded all spatial relations variables, we esti- mated the per standard deviation odds ratio for PD to be 1.34 (the per standard deviation odds ratio for PD actually increased, to 1.40, when the fPCs of the spatial relations were included as covariates in the model). corresponding estimated coefficients for fPC2 12 and fPC1 14 were −0.124 with a p value of 0.006, and −0.201 with a p value of 3 × 10−5, respectively. The combined p value for these two fPCs was 7 × 10−8. These two fPCs were mod- erately correlated (r = 0.3) and both were approximately normally distributed. The main features of the fPCs have to be identified to gain insight into the biological reasons for the associa- tion of the fPCs with breast cancer status. Due to the organisation of the segmented regions, it is expected that different fPCs carry overlapping information. Main analysis Based on the full model (Table 2), it appears that the fPCs describ- ing the relative position of the different regions to the fatty breast edge (region 1) are most important. After using stepwise selection, we retained fPC2 12 and fPC1 14. When fit- ting a logistic regression model with these two variables as covariates and with all the considered confounders, the To visualise the variation in FHs between regions 1 and 2, captured by fPC2 12, we plotted the second mode of variation, along with its FH data and eigenfunction; see Figure 6 (a to c) in the Appendix. From the mode of variation and the cor- responding eigenfunction it can be seen that fPC2 12 captures variability at angles of 54°, 152° and 268°. We note that we Page 7 of 13 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 tissue in the lower part of the breast (fatty and semi- fatty tissue are collected in the lower quadrants of the images). obtained a multiple R2 value of 0.99 based on fitting a lin- ear regression model to fPC2 12 with FH12(54°), FH12(152°) and FH12(268°) included as covariates. FH12(54°) and FH12(268°) are positively associated with fPC2 12 whereas FH12(152°) is negatively associated with fPC2 12. From the first mode of variation and its corresponding eigenfunction for the FHs between regions 1 and 4, it can be seen that fPC1 14 captures variability at an angle of 192°; see Figure 6 (d to f) in the Appendix. FH14(192°) is posi- tively associated with fPC1 14 and we obtained an R2 value of 0.93 based on fitting a linear regression model to fPC1 14 with FH4(192°) as a covariate. Four images selected for having high or low values of fPC1 14 are shown in Fig. 5. These images (a to d) have PD values of 39, 3, 19 and 3, and standardised fPC1 14 values of 4.4, 3.3, −1, 9 and −1.6, respectively. The value of FH14(192°) for each image is marked with a vertical red line on the histogram of the values of FH14(192°) for all images (shown above the corresponding image). fPC1 14 provides information about We next selected images with high and low values of fPC2 12; four images are shown in Fig. 4. Main analysis These images (a to d) have PD values of 53, 9, 35 and 9, and standardised fPC2 12 values of 2.5, 3.0, −1.9 and −1.9, respectively. His- tograms for FH12(54°), FH12(152°) and FH12(268°) are included above each image, with the specific FH value of an image marked as a vertical red line. From scrutinising the images and their FH values, it seems that low fPC2 12 values are linked to regular distributions of both fatty and semi-fatty tissue along the skin line of the breast, whereas high fPC2 12 values are related to thinning regions near the retroareolar area and irregular spreading of semi-fatty a c b d Fig. 4 Examples of mammograms with high ((a) and (b)) and low ((c) and (d)) values of fPC2 12. Original mammograms are shown next to their segmented regions 1 (dark blue) and 2 (light blue). Histograms of FH12 values at angles of 54°, 152° and 268° are included above each image with the value for the specific image marked as a vertical red line. A low fPC2 12 value, after adjustment for PD and other covariates, is associated with increased risk of breast cancer d b Fig. 4 Examples of mammograms with high ((a) and (b)) and low ((c) and (d)) values of fPC2 12. Original mammograms are shown next to their segmented regions 1 (dark blue) and 2 (light blue). Histograms of FH12 values at angles of 54°, 152° and 268° are included above each image with the value for the specific image marked as a vertical red line. A low fPC2 12 value, after adjustment for PD and other covariates, is associated with increased risk of breast cancer Page 8 of 13 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 more heterogeneous distribution of dense tissue (i.e. it is more scattered). the location of dense tissue in relation to the fatty region. From viewing images (a) and (c), two images with mod- erately high PD, it appears that low values of this fPC can be synonymous with the dense region being located in the retromammary space of the image (high values capture an absence of dense tissue in the retromammary space). From viewing the images (b) and (d), it may be plausible that, in low PD images, a low value of fPC1 14 is synonymous with a the location of dense tissue in relation to the fatty region. Main analysis Breast Cancer Research (2017) 19:103 Page 9 of 13 Page 9 of 13 Table 4 Linear regression model for fPC1 14 with breast cancer risk factor covariates (CAHRES) PD, parity and AFB, and BMI are all positively associated with fPC2 12. Parameter estimates for the model for fPC2 12 are shown in Table 3. For fPC1 14, only BMI and PD have statistically significant coefficients; see Table 4. Table 4 Linear regression model for fPC14 with breast cancer risk factor covariates (CAHRES) Covariate Estimated Standard P value coefficient error Intercept −2.24 0.260 < 2 × 10−16 Age −0.003 0.003 0.224 BMI 0.078 0.005 < 2 × 10−16 PD 0.028 0.001 < 2 × 10−16 Parity and AFB Nulliparous Parity ≤2 and AFB ≤25 0.052 0.066 0.420 Parity ≤2 and AFB > 25 0.086 0.065 0.188 Parity > 2 and AFB ≤25 0.015 0.068 0.826 Parity > 2 and AFB > 25 0.004 0.089 0.959 HRT use Never Past −0.005 0.085 0.953 Current 0.016 0.048 0.732 Pearson product–moment correlation coefficients between fPC1 14 and variables age, BMI and PD are −0.10 (p = 8 × 10−7), 0.15 (p = 7 × 10−14) and 0.29 (p < 2 × 10−16) respectively AFB age at first birth, BMI body mass index, HRT hormone replacement therapy, PD percentage density In the main association analysis (Table 2), we concen- trated on looking for the main effects of fPCs. It is, of course, possible that particular features are important within narrow ranges of PD. However, when dividing the samples into three groups defined by PD ([ 0, 7[, [ 7, 25[ and [ 25, 100]; the number of images per group being respectively 717, 1,176 and 560), fPC2 12 and fPC1 14 were jointly significantly associated with breast cancer status in all groups (with p values of 0.009, 8 × 10−4 and 0.006) and the signs of the coefficients were consistent across all groups. We note that, with the exception of breastfeeding, we believe that we adjusted for the potentially most impor- tant confounders in our main analysis (Table 2). Because information on breastfeeding was missing for a substantial number of individuals (451), we did not include breast- feeding as a covariate in the main analysis. We did, how- ever, carry out a sub-analysis. Main analysis When we re-performed our analysis on a reduced data set (2002 individuals and images) with complete information on breastfeeding (yes or no), and including breastfeeding, along with the other covariates, the p value for association between the 13 fPCs and breast cancer risk was 2×10−6, which was unchanged when breastfeeding was excluded from the model. Based on fitting linear regression models with fPCs as outcome variables, breastfeeding was not significantly associated with either fPC2 12 or fPC1 14 after adjustment for other covariates (e.g. parity and AFB). Table 3 Linear regression model for fPC2 12 with breast cancer risk factor covariates (CAHRES) Covariate Estimated Standard P value coefficient error Intercept −1.332 0.272 1 × 10−6 Age 0.003 0.003 0.373 BMI 0.025 0.006 8 × 10−6 PD 0.022 0.002 < 2 × 10−16 Parity and AFBa Nulliparous Parity ≤2 and AFB ≤25 0.252 0.069 2 × 10−4 Parity ≤2 and AFB > 25 0.100 0.068 0.142 Parity > 2 and AFB ≤25 0.229 0.071 0.001 Parity > 2 and AFB > 25 0.108 0.093 0.243 HRT use Never Past −0.121 0.089 0.175 Current −0.074 0.050 0.140 Pearson product–moment correlation coefficients between fPC2 12 and variables age, BMI and PD are −0.04 (p = 0.05), −0.01 (p = 0.82) and 0.25 (p < 2 × 10−16), respectively AFB age at first birth, BMI body mass index, HRT hormone replacement therapy, PD percentage density ap = 6 × 10−4 Table 3 Linear regression model for fPC2 12 with breast cancer risk factor covariates (CAHRES) Main analysis From viewing images (a) and (c), two images with mod- erately high PD, it appears that low values of this fPC can be synonymous with the dense region being located in the retromammary space of the image (high values capture an absence of dense tissue in the retromammary space). From viewing the images (b) and (d), it may be plausible that, in low PD images, a low value of fPC1 14 is synonymous with a To trace any potential determinants of fPC2 12 (key char- acteristics of the spatial distribution of adipose tissue) and fPC1 14 (position of dense tissue relative to fatty tissue), we fitted linear regression models with, in turn, fPC2 12 and fPC1 14 as dependent variables, and the risk factors con- sidered in the earlier models as independent variables. Fig. 5 Examples of mammograms with high ((a) and (b)) and low ((c) and (d)) values of fPC1 14. Original mammograms are shown next to their segmented regions 1 (dark blue) and 4 (red). The histograms of FH14 at an angle of 192° are included above each image with the value for the specific image marked as a vertical red line. A low fPC1 14 value, after adjustment for PD and other covariates, is associated with increased risk of breast cancer. PD percentage density Fig. 5 Examples of mammograms with high ((a) and (b)) and low ((c) and (d)) values of fPC1 14. Original mammograms are shown regions 1 (dark blue) and 4 (red). The histograms of FH14 at an angle of 192° are included above each image with the value for the s vertical red line. A low fPC1 14 value, after adjustment for PD and other covariates, is associated with increased risk of breast canc Fig. 5 Examples of mammograms with high ((a) and (b)) and low ((c) and (d)) values of fPC1 14. Original mammograms are shown next to their segmented regions 1 (dark blue) and 4 (red). The histograms of FH14 at an angle of 192° are included above each image with the value for the specific image marked as a vertical red line. A low fPC1 14 value, after adjustment for PD and other covariates, is associated with increased risk of breast cancer. PD percentage density Alsheh Ali et al. Validation A major purpose of our validation study was to check that we can identify the same characteristics of breast tissue organisation from digital images as we can from digitised analogue images. That we have a relatively small number of cases with digital images means that we have relatively low power to validate case–control associations. Key char- acteristics of cases and controls selected from KARMA are displayed in Table 5 in Appendix. For KARMA, as in CAHRES, 13 fPCs were retained that describe breast tissue organisation. For further validation, we considered the two fPCs that were retained using stepwise selection in CAHRES (fPC2 12 and fPC1 14). After constructing and examining mode of variations and eigenfunction plots (see Figure 7 in Appendix), we could confirm that these fPCs identified the same features in the KARMA digital images as they did in CAHRES ana- logue images. The angles capturing maximum variability in fPC2 12 in KARMA were 56°, 156° and 270°, which were very close to the angles captured by fPC2 12 in CAHRES. Similarly, the angle of maximum variability was almost the same over the two studies for fPC1 14. When fitting a logistic regression model to case–control status, with fPC2 12 and fPC1 14 as covariates along with the considered Page 10 of 13 Page 10 of 13 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 confounders, the corresponding estimated coefficients for fPC2 12 and fPC1 14 were −0.344 with a p value of 0.031, and 0.243 with a p value of 0.128, respectively (Table 6 in Appendix). That is, despite the small number of cases, we were able to validate the association with fPC2 12. This association had a p value that was lower than that for the association between case–control status and PD in this dataset. The apparent and honest AUCs for the model with no fPCs were 0.687 and 0.634, respectively, whilst for the model with the selected fPCs, the apparent and hon- est AUCs were 0.703 and 0.643, respectively. Delong’s test (based on the apparent AUCs) gave a p value of 0.386. approaches could be employed. Moreover, the segmen- tation method used in this study will always divide the breast into four regions, even for a very low-density mam- mogram. Conclusions Several studies have already explored the role of breast fatty tissue, but have reported conflicting results [2, 3]. However, it has been noted that studies reporting a neg- ative association between adipose tissue area and breast cancer risk based their measurements on the craniocau- dal view, whereas those reporting a positive association used the mediolateral view. Our results show that the location of adipose tissue (both our fatty and semi-fatty regions) has an impact on breast cancer risk and provides additional information to explain these contrasting stud- ies and may help in understanding the role of the fatty tissue. To define better estimates for breast cancer risk, it will likely be important to clarify the biological mecha- nisms regulating the spatial distribution of adipose tissue inside the breast. Much attention has been paid to the dense and non-dense areas of the breast and the role of their sizes as breast can- cer risk factors. In this study, we go further and find an association between the spatial organisation of breast tis- sue and breast cancer risk that is independent of (overall) mammographic density and a number of other established risk factors for breast cancer. The concentration of adi- pose tissue in the lower quadrants (which is associated with high parity and young age at first birth) and the absence of dense tissue in the retromammary space can be protective against breast cancer. These findings are com- pletely novel and may provide a basis for more detailed biological hypotheses concerning the role of breast tissue in breast cancer. In our validation study, we were able to verify that it is possible to identify the same characteristics of breast tissue organisation in digital images as in analogue images. Moreover, despite the small sample size of our validation study, we were able to validate the association between risk and our measure of the spatial relations between the fatty and semi-fatty tissue. It would, though, be valuable to study the association between the two specific FH vari- ables identified here (or related measures developed to capture directly the important spatial features identified here) with breast cancer status using larger digital external datasets and to investigate their use in breast cancer risk prediction. Validation Since the proposed features are unavoidably influenced by the choice of the segmentation method, an adaptive approach taking into consideration the quantity of dense tissue in the breast might yield more precise descriptors, which could be easier to interpret. Our approach could also be used on other types of images. In this study, we included only MLO mammo- grams. It would be interesting to investigate the spatial organisation by including craniocaudal views to give a more precise and more exhaustive description of the spa- tial relations of regions of density inside the breast. It would also be interesting to extend the method described here to digital breast tomosynthesis images, which pro- vide a detailed three-dimensional view of the breast where patterns of fibroglandular tissue are not subject to over- lapping tissue. Finally, we point out that the approach we described here, for capturing relevant patterns in the organisation of breast tissue, may be useful in numerous contexts, for example, in studying in detail the role of tissue density in screening sensitivity. Discussion In this paper, we investigated the role of the spatial organi- sation of different regions comprising the breast, on mam- mograms, in terms of the risk of developing breast cancer. The results of our main analysis showed that the spa- tial relations between the fatty and semi-fatty tissue along with the spatial relations between the fatty and dense tis- sue are associated with breast cancer risk after adjustment for PD and other possible confounders. These findings are consistent with the idea that fibroglandular and adipose tissue play a role in breast cancer risk. Endnotes 1 Array Corporation, Hampton, NH, USA 2 http://www.stat.ucdavis.edu/PACE/ Modes of variation plots For a particular fPC, of a particular FH, the set of functions (defined across a range of values of α) viewed on the modes of variations plots were calculated as V = μ(θ) ± α √ λφ(θ), where μ(θ) is the mean function of the considered FH, λ is the corresponding eigenvalue and φ(θ) is the correspond- ing eigenfunction. where μ(θ) is the mean function of the considered FH, λ is the corresponding eigenvalue and φ(θ) is the correspond- ing eigenfunction. We have adopted one approach for segmenting the images prior to carrying out statistical analyses. Other Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Page 11 of 13 Fig. 6 Visualisation of variability in fPC2 12 and fPC1 14 in CAHRES (analogue images). a and d show FH12 and FH14 for all images. The second mode of variation of FH12 and its eigenfunction are displayed in b and c, respectively. The red lines at angles 54°, 152° and 268° indicate the locations of the maxima of variations in the set of all the FH12. The first mode of variation of FH14 and its eigenfunction are displayed in e and f, respectively. The red line at angle 192° indicates the maximum of variation in the set of all the FH14 Fig. 6 Visualisation of variability in fPC2 12 and fPC1 14 in CAHRES (analogue images). a and d show FH12 and FH14 for all images. The second mode of variation of FH12 and its eigenfunction are displayed in b and c, respectively. The red lines at angles 54°, 152° and 268° indicate the locations of the maxima of variations in the set of all the FH12. The first mode of variation of FH14 and its eigenfunction are displayed in e and f, respectively. The red line at angle 192° indicates the maximum of variation in the set of all the FH14 a b c d e f Fig. 7 Visualisation of variability in fPC2 12 and fPC1 14 in KARMA (digital images). a and d show FH12 and FH14 for all images. The second mode of variation of FH12 and its eigenfunction are displayed in b and c, respectively. The red lines at angles 56°, 156° and 270° indicate the locations of the maxima of variations in the set of all the FH12. Availability of data and materials Subject to participants’ consent and legal requirements, data can be made available upon request to the primary department of the corresponding author. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Table 6 Logistic regression results with age, BMI, √ PD, Parity and AFB, HRT and fPCs as covariates (KARMA) Table 6 Logistic regression results with age, BMI, √ PD, Parity and AFB, HRT and fPCs as covariates (KARMA) Covariate Estimated Standard P value coefficient error Intercept −8.680 2.300 2 × 10−04 Age 0.072 0.024 0.002 BMI 0.070 0.046 0.128 √ PD 0.388 0.186 0.037 Parity and AFB Nulliparous Parity ≤2 and AFB ≤25 −0.490 0.502 0.330 Parity ≤2 and AFB > 25 −0.022 0.485 0.963 Parity > 2 and AFB ≤25 −0.125 0.528 0.816 Parity > 2 and AFB > 25 −1.331 0.857 0.121 HRT use Never Past −0.772 0.347 0.026 Current 0.246 0.476 0.605 Spatial relations fPCs fPC2 12 −0.344 0.160 0.031 fPC1 14 0.243 0.160 0.128 AFB age at first birth, BMI body mass index, fPC Functional principal component, HRT hormone replacement therapy, PD percentage density Authors’ contributions MAA and KH were involved in the development and application of the methodology, were responsible for interpreting the results, and drafting and revising the manuscript. LE helped in interpreting the results, and in revising the manuscript. KC and PH helped in collection and assembly of data and in revising the manuscript. All authors read and approved the final manuscript. Ethical approval and consent to participate All participants in the studies had provided written informed consent, and the studies had the approval of the ethics review board at Karolinska Institutet, Stockholm, Sweden. Competing interests The authors declare that they have no competing interests. Validation study (KARMA) Validation study (KARMA) Abbreviations AFB: Age at first birth; AUC: Area under the receiver operating characteristic curve; BMI: Body mass index; FH: Forces histogram; FHxy(θ): Forces histogram between regions x and y at angle θ; fPC: Functional principal component; fPCz xy: Functional principal component number z from the forces histogram between regions x and y; HRT: Hormone replacement therapy; MLO: Mediolateral oblique; PCA: Principal component analysis; PD: Percentage density Table 5 Key characteristics of individuals (KARMA) Modes of variation plots The first mode of variation of FH14 and its eigenfunction are displayed in e and f, respectively. The red line at angle 196° indicates the maximum of variation in the set of all the FH14 a c b a b b e c a b c e f a d d f e Fig. 7 Visualisation of variability in fPC2 12 and fPC1 14 in KARMA (digital images). a and d show FH12 and FH14 for all images. The second mode of variation of FH12 and its eigenfunction are displayed in b and c, respectively. The red lines at angles 56°, 156° and 270° indicate the locations of the maxima of variations in the set of all the FH12. The first mode of variation of FH14 and its eigenfunction are displayed in e and f, respectively. The red line at angle 196° indicates the maximum of variation in the set of all the FH14 Page 12 of 13 Page 12 of 13 Page 12 of 13 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Validation study (KARMA) Table 5 Key characteristics of individuals (KARMA) Characteristic Cases Controls P value Number 69 231 HRT use 0.179 Never 42 (61%) 125 (54%) Past 18 (26%) 86 (37%) Current 9 13%) 20 (9%) Parity and AFB 0.192 Nulliparous 9 (13%) 26 (11%) Parity ≤2 and AFB ≤25 19 (27%) 75 (33%) Parity ≤2 and AFB > 25 24 (35%) 66 (29%) Parity > 2 and AFB ≤25 15 (22%) 40 (17%) Parity > 2 and AFB > 25 2 (3%) 24 (10%) Age 63.06 (±6.05) 60.95 (±6.71) 0.021 BMI 25.93 (±4.48) 25.37 (±3.65) 0.283 PD 20.156 (±7.268) 18.908 (±8.252) 0.258 √ PD 4.410 (±0.8481) 4.238 (±.976) 0.187 Means (with standard deviations in parentheses) are given for continuous variables and counts (with percentages in parentheses) are given for categorical variables. P values are obtained using likelihood ratio tests based on fitting logistic regression models without adjustment for additional covariates AFB age at first birth, BMI body mass index, HRT hormone replacement therapy, PD percentage density Author details 1Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. 2Swedish eScience Research Centre (SeRC), Karolinska Institutet, Stockholm, Sweden. 3Department of Oncology and Pathology, Cancer Centre Karolinska, Karolinska Institutet and University Hospital, Stockholm, Sweden. Intercept 8.680 2.300 2 × 10 Age 0.072 0.024 0.002 BMI 0.070 0.046 0.128 √ PD 0.388 0.186 0.037 Parity and AFB Nulliparous Parity ≤2 and AFB ≤25 −0.490 0.502 0.330 Parity ≤2 and AFB > 25 −0.022 0.485 0.963 Parity > 2 and AFB ≤25 −0.125 0.528 0.816 Parity > 2 and AFB > 25 −1.331 0.857 0.121 HRT use Never Past −0.772 0.347 0.026 Current 0.246 0.476 0.605 Spatial relations fPCs fPC2 12 −0.344 0.160 0.031 fPC1 14 0.243 0.160 0.128 AFB age at first birth, BMI body mass index, fPC Functional principal component, HRT hormone replacement therapy, PD percentage density y p Stockholm, Sweden. Received: 3 April 2017 Accepted: 7 August 2017 References 1. Boyd NF, Guo H, Martin LJ, Sun L, Stone J, Fishell E, et al. Mammographic density and the risk and detection of breast cancer. N Engl J Med. 2007;356(3):227–36. 2. Lokate M, Peeters PH, Peelen LM, Haars G, Veldhuis WrB, van Gils CH. Mammographic density and breast cancer risk: the role of the fat surrounding the fibroglandular tissue. Breast Cancer Res. 2011;13(5):1–8. 3. Pettersson A, Hankinson SE, Willett WC, Lagiou P, Trichopoulos D, Tamimi RM. Nondense mammographic area and risk of breast cancer. Breast Cancer Res. 2011;13(5):1–10. 4. Shepherd JA, Kerlikowske K, Ma L, Duewer F, Fan B, Wang J, et al. Volume of mammographic density and risk of breast cancer. Cancer Epidemiol Biomarkers Prev. 2011;20(7):1473–82. 5. Keller BM, Conant EF, Oh H, Kontos D. Breast cancer risk prediction via area and volumetric estimates of breast density. In: Breast imaging: 11th International Workshop. Berlin Heidelberg: Springer; 2012. p. 236–43. 6. Nielsen M, Vachon CM, Scott CG, Chernoff K, Karemore G, Karssemeijer N, et al. Mammographic texture resemblance generalizes as an independent risk factor for breast cancer. Breast Cancer Res. 2014;16:R37. 7. Haberle L, Wagner F, Fasching PA, Jud SM, Heusinger K, Loehberg CR, et al. Characterizing mammographic images by using generic texture features. Breast Cancer Res. 2012;14(2):R59. Received: 3 April 2017 Accepted: 7 August 2017 Received: 3 April 2017 Accepted: 7 August 2017 References 1. Boyd NF, Guo H, Martin LJ, Sun L, Stone J, Fishell E, et al. Mammographic density and the risk and detection of breast cancer. Funding Funding This research was supported by the Swedish Cancer Society (grant CAN 2014/472), the Swedish Research Council (2016-01245), the Cancer Health Risk Prediction Centre (CRISP; www.crispcenter.org) and a Linneus Centre (contract ID 70867902) financed by the Swedish Research Council. Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 8. Wei J, Chan HP, Wu YT, Zhou C, Helvie MA, Tsodikov A, et al. Association of computerized mammographic parenchymal pattern measure with breast cancer risk: a pilot case–control study. Radiology. 2011;260(1):42–9. 9. Manduca A, Carston MJ, Heine JJ, Scott CG, Pankratz VS, Brandt KR, et al. Texture features from mammographic images and risk of breast cancer. Cancer Epidemiol Biomarkers Prev. 2009;18(3):837–45. 10. Pereira SMP, McCormack VA, Moss SM, dos Santos Silva I. The spatial distribution of radiodense breast tissue: a longitudinal study. Breast Cancer Res. 2009;11(3):1–12. 11. Matsakis P. Understanding the spatial organization of image regions by means of force histograms: a guided tour In: Matsakis S, editor. Applying soft computing in defining spatial relations. Heidelberg: Springer. Physica-Verlag; 2002. p. 99–122. 12. Shyu CR, Matsakis P. Spatial lesion indexing for medical image databases using force histograms. In: Computer vision and pattern recognition IEEE, vol. 2. IEEE; 2001. p. 603–608. 13. Garnier M, Alsheh Ali M, Seguin J, Mignet N, Hurtut T, Wendling L. Grading cancer from liver histology images using inter and intra region spatial relations. In: International conference image analysis and recognition. Portugal: Springer; 2014. p. 247–254. 14. Alsheh Ali M, Garnier M, Humphreys K. Spatial relations of mammographic density regions and their association with breast cancer risk. Procedia Comput Sci. 2016;90:169–74. 14. Alsheh Ali M, Garnier M, Humphreys K. Spatial relations of mammographic density regions and their association with breast cancer risk. Procedia Comput Sci. 2016;90:169–74. 15. Eriksson L, Czene K, Rosenberg L, Humphreys K, Hall P. The influence of mammographic density on breast tumor characteristics. Breast Cancer Res Treat. 2012;134(2):859–66. 15. Eriksson L, Czene K, Rosenberg L, Humphreys K, Hall P. The influence of mammographic density on breast tumor characteristics. Breast Cancer Res Treat. 2012;134(2):859–66. 16. Gabrielson M, Eriksson M, Hammarström M, Borgquist S, Leifland K, Czene K, et al. Cohort profile: The Karolinska Mammography Project for Risk Prediction of Breast Cancer (KARMA). Int J Epidemiol. 2017. doi:10.1093/ije/dyw357. 17. Bertrand KA, Tamimi RM, Scott CG, Jensen MR, Pankratz VS, Visscher D, et al. Mammographic density and risk of breast cancer by age and tumor characteristics. Breast Cancer Res. 2013;15(6):R104. 18. Aitken Z, McCormack VA, Highnam RP, Martin L, Gunasekara A, Melnichouk O, et al. Screen-film mammographic density and breast cancer risk: a comparison of the volumetric standard mammogram form and the interactive threshold measurement methods. Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Cancer Epidemiol Biomarkers Prev. 2010;19(2):418–28. 19. Byng JW, Boyd N, Fishell E, Jong R, Yaffe MJ. The quantitative analysis of mammographic densities. Phys Med Biol. 1994;39(10):1629. 20. Cheddad A, Czene K, Eriksson Ml, Li J, Easton D, Hall P, et al. Area and volumetric density estimation in processed full-field digital mammograms for risk assessment of breast cancer. PloS ONE. 2014;9(10):e110690. 21. Bora VB, Kothari AG, Keskar AG. Robust automatic pectoral muscle segmentation from mammograms using texture gradient and Euclidean distance regression. J Digit Imaging. 2016;29(1):115–25. 22. He W, Juette A, Denton ERE, Oliver A, Martí R, Zwiggelaar R. A review on automatic mammographic density and parenchymal segmentation. Int J Breast cancer. 2015;2015. http://dx.doi.org/10.1155/2015/276217. 23. Wolfe JN. Breast patterns as an index of risk for developing breast cancer. Am J Roentgenol. 1976;126(6):1130–7. 23. Wolfe JN. Breast patterns as an index of risk for developing breast cancer. Am J Roentgenol. 1976;126(6):1130–7. 23. Wolfe JN. Breast patterns as an index of Am J Roentgenol. 1976;126(6):1130–7. 24. Gong YC, Brady M, Petroudi S. Texture based mammogram classification and segmentation. In: Digital mammography: 8th International Workshop. Berlin Heidelberg: Springer; 2006. p. 616–25. 24. Gong YC, Brady M, Petroudi S. Texture based mammogram classification and segmentation. In: Digital mammography: 8th International Workshop. Berlin Heidelberg: Springer; 2006. p. 616–25. 25. Shang HL. A survey of functional principal component analysis. AStA Adv Stat Anal. 2014;98(2):121–42. 25. Shang HL. A survey of functional principal component analysis. AStA Adv Stat Anal. 2014;98(2):121–42. Author details N Engl J Med. 2007;356(3):227–36. 2. Lokate M, Peeters PH, Peelen LM, Haars G, Veldhuis WrB, van Gils CH. Mammographic density and breast cancer risk: the role of the fat surrounding the fibroglandular tissue. Breast Cancer Res. 2011;13(5):1–8. 3. Pettersson A, Hankinson SE, Willett WC, Lagiou P, Trichopoulos D, Tamimi RM. Nondense mammographic area and risk of breast cancer. Breast Cancer Res. 2011;13(5):1–10. 4. Shepherd JA, Kerlikowske K, Ma L, Duewer F, Fan B, Wang J, et al. Volume of mammographic density and risk of breast cancer. Cancer Epidemiol Biomarkers Prev. 2011;20(7):1473–82. 5. Keller BM, Conant EF, Oh H, Kontos D. Breast cancer risk prediction via area and volumetric estimates of breast density. In: Breast imaging: 11th International Workshop. Berlin Heidelberg: Springer; 2012. p. 236–43. 6. Nielsen M, Vachon CM, Scott CG, Chernoff K, Karemore G, Karssemeijer N et al. Mammographic texture resemblance generalizes as an independent risk factor for breast cancer. Breast Cancer Res. 2014;16:R37. 7. Haberle L, Wagner F, Fasching PA, Jud SM, Heusinger K, Loehberg CR, et al. Characterizing mammographic images by using generic texture features. Breast Cancer Res. 2012;14(2):R59. Page 13 of 13 Alsheh Ali et al. Breast Cancer Research (2017) 19:103 Submit your next manuscript to BioMed Central and we will help you at every step: 26. Yao F, Müller HG, Wang JL. Functional data analysis for sparse longitudinal data. J Am Stat Assoc. 2005;100(470):577–90. 26. Yao F, Müller HG, Wang JL. Functional data analysis for sparse longitudinal data. J Am Stat Assoc. 2005;100(470):577–90. 27. Harrell FE, Lee KL, Mark DB. Tutorial in biostatistics multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy,andmeasuring and reducing errors. Stat Med. 1996;15:361–87. 27. Harrell FE, Lee KL, Mark DB. Tutorial in biostatistics multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy,andmeasuring and reducing errors. Stat Med. 1996;15:361–87. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit p y y p • We accept pre-submission inquiries 28. DeLong ER, DeLong DM. Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics. 1988;44:837–45. 28. DeLong ER, DeLong DM. Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics. 1988;44:837–45. 29. Jones MC, Rice JA. Displaying the important features of large collections of similar curves. Am Stat. 1992;46(2):140–5. 29. Jones MC, Rice JA. Displaying the important features of large collections of similar curves. Am Stat. 1992;46(2):140–5.
https://openalex.org/W3199464486	https://www.mdpi.com/1996-1073/14/18/5786/pdf?version=1631615769	English	null	Energy Monitoring in the Wild: Platform Development and Lessons Learned from a Real-World Demonstrator	Energies	2,021	cc-by	8,830	Citation: Quintal, F.; Garigalli, D.; Vasconcelos, D.; Cavaleiro, J.; Santos, W.; Pereira, L. Energy Monitoring in the Wild: Platform Development and Lessons Learned from a Real-World Demonstrator. Energies 2021, 14, 5786. https://doi.org/10.3390/en14185786 Keywords: electricity monitoring; case study; long-term; prototyping; retroﬁtting; platform energies energies Energy Monitoring in the Wild: Platform Development and Lessons Learned from a Real-World Demonstrator 1 ITI, LARSyS, Universidade da Madeira, 9000-072 Funchal, Portugal; daniel.pestana@m-iti.org (D.G.); dino.vasconcelos@iti.larsys.pt (D.V.); jonathan.cavaleiro@iti.larsys.pt (J.C.) 2 prsma.com, 9020-105 Funchal, Portugal; wilson.santos@prsma.com 3 ITI, LARSyS, Técnico Lisboa, 1049-001 Lisboa, Portugal * Correspondence: ﬁlipe.quintal@staff.uma.pt (F.Q.); lucas.pereira@tecnico.ulisboa.pt (L.P.) † These authors contributed equally to this work. 1 ITI, LARSyS, Universidade da Madeira, 9000-072 Funchal, Portugal; daniel.pestana@m-iti.org (D.G.); dino.vasconcelos@iti.larsys.pt (D.V.); jonathan.cavaleiro@iti.larsys.pt (J.C.) 2 prsma.com, 9020-105 Funchal, Portugal; wilson.santos@prsma.com 3 ITI, LARSyS, Técnico Lisboa, 1049-001 Lisboa, Portugal * Correspondence: ﬁlipe.quintal@staff.uma.pt (F.Q.); lucas.pereira@tecnico.ulisboa.pt (L.P.) † These authors contributed equally to this work. p , , g ; p 3 ITI, LARSyS, Técnico Lisboa, 1049-001 Lisboa, Portugal y g * Correspondence: ﬁlipe.quintal@staff.uma.pt (F.Q.); lucas.pereira@tecnico.ulisboa.pt (L.P.) † These authors contributed equally to this work. Abstract: This paper presents the development and evaluation of EnnerSpectrum, a platform for electricity monitoring. The development was motivated by a gap between academic, fully custom- made monitoring solutions and commercial proprietary monitoring approaches. EnnerSpectrum is composed of two main entities, the back end, and the Gateway. The back end is a server comprised of ﬂexible entities that can be conﬁgured to different monitoring scenarios. The Gateway interacts with equipment at a site that cannot interact directly with the back end. The paper presents the architecture and conﬁguration of EnnerSpectrum for a long-term case study with 13 prosumers of electricity for approximately 36 months. During this period, the proposed system was able to adapt to several building and monitoring conditions while acquiring 95% of all the available consumption data. To ﬁnalize, the paper presents a set of lessons learned from running such a long-term study in the real world. 1. Introduction Energy monitoring is not a new topic, ever since the ﬁrst electric grid was created, some kind of energy monitoring technology started to be installed for billing or control [1]. As the electric grid evolved, so did the monitoring technologies, returning more frequent, ﬁne-grained, and precise data. Promoted by the advent of the smart grid, these advance- ments eventually reached the edge of the grid into the houses of electricity end-consumers, with the deployment of Advanced Metering Infrastructure and smart-meters [2]. A multi- tude of services that rely on smart-meter data are currently under extensive research, and demonstration [3,4]. Services, such as Demand Side Management (DSM), Electric Vehi- cle Charging (EVC), Distributed Storage and Generation, rely on precise and continuous data [5]. However, a new set of services developed for the household are emerging, which cannot all be met by the smart-metering infrastructure alone [6–9]. For example, services in which the consumption/production information is not the primary purpose, for example, household occupation detection, automation or comfort [10,11]. It is common for these services to deploy some kind of energy monitoring solution that meets their requirement, for example, the SMATM portal needs the installation of a gateway in the mains, LGTM batteries need a separated inverter, which in their turn could need dedicated monitoring equipment. These solutions are often proprietary and rely upon unique protocols which cannot be shared between services or vendors or were simply built without considering integration [12,13]. This leads to households with duplicated or competing monitoring solutions, increasing installation, update, or maintenance costs. Academic Editor: Adel Merabet Received: 10 August 2021 Accepted: 6 September 2021 Published: 14 September 2021 Received: 10 August 2021 Accepted: 6 September 2021 Published: 14 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). g p Energy monitoring is still a very proliﬁc ﬁeld of research with contributions often focusing on the evaluation of particular aspects of monitoring solutions (e.g., [9,14,15]). Or https://www.mdpi.com/journal/energies Energies 2021, 14, 5786. 1. Introduction https://doi.org/10.3390/en14185786 Energies 2021, 14, 5786 2 of 14 the development of monitoring solutions for particular scenarios in which the monitoring is not the main outcome of the research (for example Abdulsalam Alghamdi employed a monitoring solution to collect data as the input for an viability study of PV installations in Saudi Arabia [16]). the development of monitoring solutions for particular scenarios in which the monitoring is not the main outcome of the research (for example Abdulsalam Alghamdi employed a monitoring solution to collect data as the input for an viability study of PV installations in Saudi Arabia [16]). In both cases, most of the systems proposed by academia are often one-off prototypes developed with a speciﬁc goal, with limited applicability in the real world. Furthermore, buildings go through several improvements and renovations during their long lifespan [17]. Consequently, monitoring solutions in the real world are expected to be retroﬁtted in the existing infrastructure, hindering the viability of certain academic contributions in the real world. These limitations mean that researchers often have to build monitoring from scratch for particular needs. This phenomenon delays the knowledge transfer from academia to industry, and it also increases the development time of new academic contributions. y p Against this background, this paper presents the EnnerSpectrum, an energy monitoring platform that emerged from academic research and was later expanded and demonstrated in a real-world demonstrator in the scope of the Horizon 2020 SMILE project. This system proposes an energy monitoring infrastructure composed of the monitoring hardware, a server to aggregate the consumption data, and a set of custom-made hardware and software components needed for integration. EnnerSpectrum provides a completely open platform for energy monitoring, allowing the undemanding integration of monitoring hardware, new data sources, visualizations for end consumers, and making all the collected information available to interested parties. This approach pursuits a centralized energy monitoring service for the household, addressing the shortcomings of having multiple closed solutions for different systems. The remainder of this article ﬁrst presents the state- of-the-art in energy monitoring, highlighting the contributions that motivated the proposed approach. The following section presents the EnnerSpectrum system, all its components, and integration. The paper then presents a case study of the EnnerSpectrum System in 13 sites. To ﬁnalize, a discussion of the ﬁndings and conclusions are presented. 2. The State of the Art of Electricity Monitoring Electricity monitoring is an essential aspect of a broad scope of studies. Ranging from work that looked purely at the technology to accurately measure electricity to studies that focused on the human side of energy consumption and research which conceptualizes how different technologies could consolidate into new services. The work presented bellow, represents a diverse sample from the state of the art which leveraged electricity monitoring, either as the main contribution of the work, or as a tool to a goal. Therefore, this literature review aims to focus on the implemented monitoring technologies that motivated the proposed approach. p p pp Several authors opted to purposely custom-made hardware and software to measure electricity consumption. Anbya et al. [15] propose an entirely custom-made system based on IC ADE7753 energy meter, which uses a Zigbee mesh to communicate with a base station. Depending on the conditions, the proposed system measured electricity consumption with a precision error between 0.03% and 1.23%. In [18], the authors propose an energy monitoring solution using a notebook. The built-in audio Analog to Digital Converter (ADC) was used to sample current and voltage, whereas the mini display and the speakers provided feedback to the consumers. The Wi-Fi card enabled communication over the Internet. Another approach followed by the same authors [19] employs an energy monitor- ing solution based on a multi-channel data acquisition system (DAQ) that was developed to be installed in the main entry point of apartment blocks, hence enabling the collection of consumption data without having to install equipment in each apartment. Pereira et al. disclosed a set of lessons learned and a comparison of the two approaches presented before [20]. Another custom-made system was proposed by Yang et al. [21]. The authors propose an infrastructure for real-time electricity monitoring, which uses a non-invasive sensor and an energy harvester to power the data capture module. These modules are to be installed Energies 2021, 14, 5786 3 of 14 in the internal grid of a building and operate in a completely autonomous manner and periodically push the data to a central unit connected to a desktop computer. Frei et al. [22] developed a series of wireless sensors for estimating building performance. The sensors are organized in a ZigbeeTM mesh and measure different aspects of the building. The proposed system measures electricity consumption through a light sensor to measure the pulse ﬂashes from the household electricity meters. 2. The State of the Art of Electricity Monitoring The sensor kit contains a gateway based on an Arduino TM(https://www.arduino.com, accessed on 10 August 2021) microprocessor that pushes the data to a web server containing a database and graphical user interface. Nevertheless, the most common approach is to develop monitoring infrastructures that combine commercial equipment (e.g., sensors or meters) with custom-made hardware or software (web servers, gateways). This approach often allows the implementation of more ﬂexible solutions (compared with using only commercial systems), with a lower development time (compared to the custom-made equipment presented before). Abdulsalam Alghamdi [16] presented a viability study for the potential of rooftop- mounted PV Power Generation to meet electrical demand in Saudi Arabia. The author used a Raspberry Pi, connected to a series of sensors, to measure electricity consumption, but also other metrics such as CO2. This custom-made solution also allowed the data to be easily pushed to an online server. y p Chen et al. [23] used an electricity monitoring infrastructure that combined off-the-shelf equipment with custom-made integration and communication. Intending to measure dis- aggregated per-plug consumption, the authors modiﬁed a Kill-a-WattTM meter, which communicated with a central gateway pushed the data to a web server. A user study with the system disclosed that 78% of participants were encouraged to reduce their consumption, and 20% reduced their consumption once it was made public. Weiss et al. [24] also pre- sented a combined approach, this contribution focuses on the human-computer-interaction aspect of behavior change to reduce electricity consumption. To measure electricity the authors used an common smart meter (from Landis + GyrTM, https://www.landisgyr.com/, accessed on 10 August 2021). A custom-made gateway connected to the meter incorporated a parser, database, and local web-server, which participants could connect and query their electricity consumption. Another study focused on the human side of the energy ﬁeld was performed by Sundramoorthy et al. [25]. In this study, the author proposes a system that uses commercial sensors, RF clamps, and commercial smart-plugs that push data to a gateway. The gateway then publishes the data in a server that contains a series of graphical representations intended for the end-user. Modern electric installations are often deployed with monitoring solutions, although mostly proprietary with limited interface options. Researchers from the energy ﬁeld presented studies that used exclusively commercial systems. 3. EnnerSpectrum System Motivated by the work presented above, the proposed system aims at collecting precise and accurate electricity data while providing a ﬂexible solution that allows the integration of new data sources and services. The EnnerSpectrum System is a software-based solution for monitoring energy at a selected site. On top of the monitoring, EnnerSpectrum can also provide services such as, for example, visualizations for the end consumer and administrator. In most scenarios, EnnerSpectrum requires employing one hardware device to assure smooth integration with other local grid components. In the remainder of this article, the site device will be referred to as the gateway (GT) and the cloud component as the EnnerSpectrum Back End (ESB). 2. The State of the Art of Electricity Monitoring One of the most popular is the SMATM SunnyBoy (https://www.sma.de/en/products/solarinverters/sunny-boy- 30-36-40-50-60.html, accessed on 10 August 2021), which can be installed with a two-way energy meter, and provides a Web Portal for their customers. Conceptually this system is very similar to the approaches discussed above, which employ metering equipment, a gateway, and a web server. Cieslik et al. [26] leveraged the data collected from a PV installation through the SMA web portal to perform an experimental analysis of the installation’s output to meet the demand of an electric vehicle. The broad deployment of smart meters also allowed researchers to obtain consumption data for their studies. Geelen et al. [27] used electricity and gas data from 519 smart meters in the Netherlands. Participants were divided into two groups. One of the groups used an application that allowed the monitoring of household consumption. Among other results, the authors disclose that participants using the moni- toring application invested in changing their behavior to conserve energy. Schultz et al. [28] carried out a similar study focused on measuring the energy conservation behaviors from a sample of 431 single-family homes. For this study, the smart meter data was paired with a custom-made in-home display. Energies 2021, 14, 5786 4 of 14 From the body of work presented above its clear that all the reviewed approaches have their own strengths, weaknesses and challenges. A common trend is that when researchers need more ﬁne-grained and frequent data, they will opt for a fully custom-built solution or combine capable commercial equipment with custom-built hardware/software. For research focused on ofﬂine studies such as viability analysis, algorithm development, or studies focused on the human aspect of sustainability, the data from commercial systems is often adequate. Withal, the most common approaches found in the literature are a combination of custom-built and commercial equipment. In most of the reviewed work, the primary purpose of this integration is to retrieve that data from the sensing equipment, and push it to a centralized location, usually a web server. The proposed approach, which will be discussed in detail in the following sections, aims at combining the strengths of both approaches presented in this literature review, employing a ﬂexible infrastructure that can be adapted to different scenarios while allowing the integration of industry-grade equipment for precise measurements. 3.2. Gateway The main hardware component is the GT, this device is composed of a Raspberry Pi 3B+ microcomputer, together with a Juice4Halt (https://juice4halt.com/, accessed on 10 August 2021) UPS to protect the equipment from sudden drops in the supply of electricity. The UPS shield also bundles a Real-Time-Clock and RS485 modules, which are used for communication using the MODBUS protocol. This protocol is commonly used by energy meters and similar equipment. This interface guarantees that the GT can communicate with a plethora of monitoring solutions. All these components are enclosed in a plastic box with DIN ﬁxing points. Each GT was ﬁtted with a 32GB SD card. g p The decision of using Raspberry Pi running RasbianOS without a GUI, allowed to take advantage of signiﬁcant performance when considering the small size of the device. It also allowed custom code development to interact with monitoring equipment and run third-party applications for services such as performance monitoring or remote access. The Raspberry Pi also provides effective I/O options, which were especially useful when connecting the device to the internet, either by WiFi, Ethernet or with a 3G USB dongle. 3.1. Architecture The ﬂexibility goal discussed before is mainly achieved by a set of architectural deci- sions in the development of the ESB. The back end is organized into ﬁve main concepts: sources schemas, sources, producers, devices and devices groups The architecture was developed to facilitate solubility and integration with different parties, for example moni- toring equipment or visualizations. g q p A source schema describes a particular kind of data that the system is capable of capturing. For instance, “webScraper” is a source schema corresponding to sources that are capable of capturing information by parsing HTML pages. Currently, there are two types of schemas, “webScraper“ and “jsonListener”, both structured in JSON format. A source is an implementation of a source schema. Each source handles converting its samples to the appropriate type and writing them to the data store. A producer is an entity that produces data for a particular source. In many cases, each installation A producer may have multiple devices which access the system. The distinction is made between producers and devices so that access tokens can be generated per device instead of storing the user’s credentials at each location. Devices can be grouped to show their data aggregated in the same view. Every device has its group in order to see its data individually, and another group has to be created containing all the devices of the producer to have a global view. Other groups can be created combining only devices that share the same type of installation, for example, having one device measuring grid and many devices measuring production or one device measuring consumption and many devices measuring production. The device groups are created through the administrator interface. Figure 1 presents the conﬁguration and relation between the Producer, Source, and Schema entities for monitoring at a particular site. 5 of 14 Energies 2021, 14, 5786 Figure 1. Relationship between the Producers, Sources and Schemas in the ESB, exempliﬁed by the conﬁguration used for S11. Figure 1. Relationship between the Producers, Sources and Schemas in the ESB, exempliﬁed by the conﬁguration used for S11. Energy Monitoring The EnnerSpectrum system is smart-meter agnostic. i.e., the goal is to support any smart meters that support the MODBUS communication protocol. However, in the case study presented in this article, the GT was mainly installed together with two commercial Carlo GavazziTM (CG) energy monitors the EM111 (https://gavazzi.se/produkter/em111 dinav81xs1pfb/), accessed on 10 August 2021 for measuring PV production and EM112 (https://gavazzi.se/produkter/em112dinav01xs1pfb/, accessed on 10 August 2021) for consumption (3 phase installations used the EM340 (https://gavazzi.se/produkter/em340 dinav23xs1pfb/, accessed on 10 August 2021)) see Figure 2. The GT communicates with the CG meter, and communication requests are forwarded through the RS485 module using the MODBUS protocol. The consumption and production data are saved locally (in the GT) in a database. Each data point is composed of a: times- tamp, active power, reactive power, apparent power, frequency, power factor, current, and voltage. The measurements are collected each second and aggregated in memory. Every 60 s, the database is updated with the data collected in the past minute. If the readings fail for more than 60 consecutive attempts, the system notiﬁes the research team about a possible issue with the GT or CG monitors. Every minute the GT also calculates the average of all the collected ﬁelds and sends it to the ESB. There is also an inspection for any previously unsent data, which is also pushed to the ESB. Furthermore, every day at 01:00 the GT creates a backup with all raw data in a Energies 2021, 14, 5786 6 of 14 GoogleDrive (https://drive.google.com/, accessed on 10 August 2021) account, the data is sent as two CVS ﬁles, one for the production and another for consumption, with a unique name that matches the day that data belongs too. After a successful backup, the data is removed from the local database in the GT to keep any database transactions as fast as possible. GoogleDrive (https://drive.google.com/, accessed on 10 August 2021) account, the data is sent as two CVS ﬁles, one for the production and another for consumption, with a unique name that matches the day that data belongs too. After a successful backup, the data is removed from the local database in the GT to keep any database transactions as fast as possible. p Certain sites already had monitoring equipment installed. In those cases, the GT is used to interact with the metering equipment at the site. Energy Monitoring Figure 3 depicts the components, communication, and architecture of all the elements that comprise the gateway. In some situations, a site could already have monitoring equipment installed. In those cases, the GT can directly interact with the equipment at the site. When this approach was not possible EnnerSpectrum used an HTML-Scrapper to retrieve consumption information from the equipment vendors’ website. In these situations, there was no need to install the GT. Figure 2. Example of two installation from the case study. On the site at left all the equipment was installed in the site main breaker box. The picture at right depicts another site in which all the installed equipment had to be installed in a adjunct box. Figure 2. Example of two installation from the case study. On the site at left all the equipment was installed in the site main breaker box. The picture at right depicts another site in which all the installed equipment had to be installed in a adjunct box. Figure 3. Left: Gateway used in the EnnerSpectrum System, the DIN box used for the installation, and the components that make up the GT; Right: Summary of the procedures carried out by the GT. Figure 3. Left: Gateway used in the EnnerSpectrum System, the DIN box used for the installation, and the components that make up the GT; Right: Summary of the procedures carried out by the GT. 3.3. Services As mentioned before, one of the goals of the EnnerSpectrum system is to provide services on top of the aggregated consumption data. The ESB contains an API with more than sixty endpoints for all the services provided by the system. These services range from traditional CRUD operations like reading the consumption from a site within a set time Energies 2021, 14, 5786 7 of 14 7 of 14 frame or averaged by the hour, day, week, or month, to other functions that provide all the operations needed to set up new producers and sources or devices at a site. There is also the opportunity to develop new services that combine different endpoints to provide value to the end electricity consumer, or to, the TSO and DSO. Two examples of services enabled by EnnerSpectrum are an end-consumer visualization and administration tool described in the following sub-sections. 3.3.1. Graphical User Interface for the End Consumer The ESB interface directed at end consumers has three main areas, see Figure 4. Current consumption, historical consumption, and comparisons. The energy information is displayed using metrics such as kWh, Euros, or grams of CO2. In these areas, the end-user can query graphs that display consumption and production information. More concretely, the current consumption area displays the real-time production, consumption, and waste information. It can also group the data by 1 h, 24 h, one week, and one month, the energy saved by the PV installation is also displayed here (again in kWh, CO2 and Euros). The history page allows the user to visualize the consumed, produced, and wasted energy for a selected date. This page also allows the user to group the data by day, week, and month for a more comprehensive overview of the consumption. The day visualization presents 60 data points per hour, while the week and month graphs use 2 and 1 sample per hour, respectively. Finally the comparison provides a tool in which users can select 2 different dates, which could 2 days, weeks or months (see Figure 4 right). The result of the selection is then presented in three different graphs that show the energy metrics for the selected dates. Figure 4. User interface of the ESB. Left: main screen of the web application showing the real-time electricity production, consumption, and a graph with the 24 h; Right: Comparison screen from the web application, comparing the consumption by week. Figure 4. User interface of the ESB. Left: main screen of the web application showing the real-time electricity production, consumption, and a graph with the 24 h; Right: Comparison screen from the web application, comparing the consumption by week. 3.4. Cyber-Security and Privacy Considerations Several privacy and security measures were adopted during the development of EnnerSpectrum. Currently, the ESB is conﬁgured to use HTTPS, which guarantees the privacy and integrity of the exchanged data while in transit to the server. It also protects against man-in-the-middle attacks. HTTP requests are automatically redirected to HTTPS. Currently, authentication is required for producers and devices. This is intended to prevent spurious data from being posted to an account and disallow the querying of private information. HTTP Basic authentication is the strategy used for the API. Concerning web interfaces (admin and client), a login is used. To avoid access to sensitive data stored at the ESB, this kind of information is hashed in the database. This includes passwords, device auth tokens, and producer tokens, which means that they’re no longer recoverable once in the database. 4. Case Study: Prosumer Monitoring This section reports the ﬁndings during the deployment of the EnnerSpectrum system. Installation sites were recruited with an advertisement campaign in the local newspapers and with the help of the local, and only, electricity company. More than forty sites were considered, and eventually, twenty-seven sites were recruited. This paper presents the ﬁndings for a subset of thirteen sites, which entered the case study in similar periods. The recruitment process was aligned with the goals of the H2020 SMILE project since one of the project’s goals was to study the viability of installing battery storage systems to optimize PV installations. All the selected sites had a PV installation directed for self-consumption, no feed-in tariff was in place, and the feed-in was restricted in certain installations. EnnerSpectrum system was responsible for monitoring both the electricity consumption and production in all the installation sites. The monitoring equipment was installed between May and August of 2018, and its been continuously ruining until the present day. This section describes the general results from running the pilot until the 31 March 2021. 3.3.2. Administration Tool The EBS Administration tool is where the research team managed the deployed equipment and monitored the status of the pilot, see Figure 5. This service allows its users to create, remove and edit producers, and view its data. It also allows the creation and removal of devices associated with a producer and the management of groups. The Administration tool permits creating an access token for each device and cleaning all the device’s data. The administrator can also create, edit and remove sources and set up which devices push data to it. Other features were implemented, which are helpful during a real-world pilot’s progress, such as cleaning the ESB cashes, managing data backups, exporting data for a CSV ﬁle, and a Map visualization with an overview of all the deployed equipment (see Figure 5, right). Energies 2021, 14, 5786 8 of 14 Figure 5. Admin screen of the ESB. Left: Main screen of the administration interface; Right: Map view showing a sub sample of monitored sites. Figure 5. Admin screen of the ESB. Left: Main screen of the administration interface; Right: Map view showing a sub sample of monitored sites. 4.1. Installation and Integration Installation Installation S1 Firstly: 2 CG Meters and 1 Gateway; Secondly: Integration with the voltronic-power meter through the GT S2 2 CG Meters and 1 GT S3 Integration with the EmonCMS system S4 2 CG Meters and 1 GT S5 2 CG Meters and 1 GT S6 2 CG Meters and 1 GT S7 2 CG Meters and 1 GT S8 Firstly: Integration with the SMA Portal; Secondly: Integration with the voltronic-power meter through the GT S9 Integration with the EmonCMS system S10 Integration with the Effekta system S11 Firstly: 2 CG Meters and 1 GT; Secondly: Firstly: 3 CG Meters and 1 GT S12 2 CG Meters and 1 GT S13 2 CG Meters and 1 GT Table 1. Installation and metering approach for each site. Installation Installation S1 Firstly: 2 CG Meters and 1 Gateway; Secondly: Integration with the voltronic-power meter through the GT S2 2 CG Meters and 1 GT S3 Integration with the EmonCMS system S4 2 CG Meters and 1 GT S5 2 CG Meters and 1 GT S6 2 CG Meters and 1 GT S7 2 CG Meters and 1 GT S8 Firstly: Integration with the SMA Portal; Secondly: Integration with the voltronic-power meter through the GT S9 Integration with the EmonCMS system S10 Integration with the Effekta system S11 Firstly: 2 CG Meters and 1 GT; Secondly: Firstly: 3 CG Meters and 1 GT S12 2 CG Meters and 1 GT S13 2 CG Meters and 1 GT Table 1. Installation and metering approach for each site. Figure 6. Typologies of the self-consumption facilities encountered during the installations. Figure 6. Typologies of the self-consumption facilities encountered during the installations. 4.1. Installation and Integration Nine of the 13 sites had no metering equipment installed. In those situations, all the monitoring had to be retroﬁtted. Hence the GT and the CG energy monitors were installed in the mains. The objective was to measure electricity consumption and production accurately. Therefore, the CG meters were positioned between the grid and local loads to measure the grid consumption and between the inverted and the local loads to measure PV production. Two major typologies were found, see Figure 6, CG Meter 1 is responsible for measuring the PV production, and CG meter 2 measures the consumption. The installation was different for every site, in some instances all the equipment was enclosed in the main fuse-box, in other cases a box had to be installed adjacent to the rest of the equipment. Figure 2 presents an example of two of the installations. There was also a situation (S11), in Energies 2021, 14, 5786 9 of 14 which the consumption and production had to be measured apart, in that situation another gateway had to be installed. In the remaining four installations, a hybrid approach was followed. In those sites, there was already equipment responsible for monitoring electricity consumption and(or) production. Such cases were addressed by querying the already installed device for the needed consumption/production data. This procedure could be accomplished by the GT or the ESB, connecting directly to the monitoring equipment, or parsing content from a vendor website. Table 1 presents the installation approach followed at each site. Table 1. Installation and metering approach for each site. Installation Installation S1 Firstly: 2 CG Meters and 1 Gateway; Secondly: Integration with the voltronic-power meter through the GT S2 2 CG Meters and 1 GT S3 Integration with the EmonCMS system S4 2 CG Meters and 1 GT S5 2 CG Meters and 1 GT S6 2 CG Meters and 1 GT S7 2 CG Meters and 1 GT S8 Firstly: Integration with the SMA Portal; Secondly: Integration with the voltronic-power meter through the GT S9 Integration with the EmonCMS system S10 Integration with the Effekta system S11 Firstly: 2 CG Meters and 1 GT; Secondly: Firstly: 3 CG Meters and 1 GT S12 2 CG Meters and 1 GT S13 2 CG Meters and 1 GT Figure 6. Typologies of the self-consumption facilities encountered during the installations. 4.2. Running the Case Study Table 1. Installation and metering approach for each site. 4.2. Running the Case Study In general, it was observed that even though the proposed infrastructure is still a prototype, it behave robustly during the pilot. All the sites reported in this article are using the system continuously for approximately three years. Nonetheless, speciﬁc issues resulted in data missing at the ESB. These issues affected the GT and made it impossible to push the site’s data to the server. The leading cause of problems was the SD card used by the Raspberry Pi at the GT. In speciﬁc usage scenarios, the SD card got corrupted. These issues were more common during blackouts (which turned the system off without notice) and in periods where there was an overload of writing and reading commands. Since the Raspberry Pi’s SD cards holds the operating system, Energies 2021, 14, 5786 10 of 14 10 of 14 programs, and all the user data, an event such as described above effectively disabled the GT. The only solution for this issue was to write a copy of the operating system and programs to a new SD card and replace the damaged one at the affected sites. During the case study, those issues were solved by adding a UPS to the GT, which assured a smoother electricity delivery and shut down during a blackout. An higher rate SD cards were also installed at the GT, and ﬁnally, the writing procedures were re-designed so that all the writes happen in bulk every minute, reducing the stress in the SD card. In tandem, these procedures eliminated the issues mentioned above. p Other obstacles encountered during the case study were mainly related to the GT communication to the ESB. Several conditions could cause this issue, for example, the site owner changed the WiFi password, or the location of the router (which could reduce the WiFi signal strength), or even the 3G USB dongle got loose. In all these situations, the research team ﬁrst attempted to solve the issue remotely. If no solution was found, a visit to the site had to be scheduled to solve the problem. A common solution was to changed the communication method, for example, from WiFi to 3G if the WiFi coverage was compromised. Finally, it is also important to consider that the monitoring does not happen in a vacuum. Each site represents a household or small commercial area. 4.2.1. Data Collection Campaign The monitoring approach returned the expected results. When using the GT meters the system was able to acquire detailed information about the site’s consumption and production. The GT managed to push the consumption data at 1 minute intervals whenever it was possible to establish a connection with the ESB. The GT also managed to recover from periods without an internet connection, pushing all the data accumulated in that period. This situation happened in several sites, in which systems remained ofﬂine for more than two weeks but were able to recover once the internet connection was re-established. This behavior was veriﬁable both for the low and high-frequency data. This strategy is, of course, limited by the memory of the GT. If the systems remain ofﬂine for a longer period (>1 month), their memory could ﬁll up, rendering the system unusable. There were no instances of this phenomenon during the presented case study. During the case study, EnnerSpectrum was capable of capturing 95% of all the production and consumption samples measured by the system. Table 2 presents a summary of all the captured electricity consumption and production data. 4.2. Running the Case Study During this long-term case study, site owners often needed to introduce changes to their installation, such as increasing the PV installation capacity, improving the reliability or safety of the site grid, or even more broad renovations at the site. This fact meant that the sensing also had to evolve, Table 1 presents the sensing approaches employed. It is noticeable that for sites S1,S8 and S11 the approach changed during this period, and new strategies had to be implemented. The ﬂexibility of the proposed system allowed it to cope with all the introduced changes at the sites. 5. Lessons Learned and Recommendations This section discusses the results presented above and provides several recommenda- tions that emerged from the long-term case study. 5.1. Buildings Characteristics When running the pilot, one of the biggest challenges was adapting the monitoring equipment to the building characteristics(see Table 1). As discussed before, EnnerSpectrum had a standard conﬁguration that ﬁtted most sites but had to be adapted for particular installations. However, the most signiﬁcant challenge was not adapting the metering but understanding the installation at each site. In some of the sites, the installations were new and well documented. In other sites, the electric installations were old, complex, and were subject to more than one renovation intervention throughout the years. The screening visits organized before the deployment were crucial to learn the installation characteristics and prepare the EnnerSpectrum system before the deployment. The presence of a qualiﬁed technician who accompanied the team during these visits was essential since he provided hints about necessary equipment, a ﬁtting location, installation duration, and the overall feasibility of the site. 4.2.2. Interfacing One of the goals of retroﬁtting monitoring equipment in existing buildings is to make the consumption information available for other services. In this case study, after the consumption data was pushed to ESB, it was made available for services such as the administration and end user services already mentioned in this article. The ESB also made the information available to other services, such as a battery energy storage system control algorithm. Furthermore the high frequency data was also used ofﬂine, to study each site consumption patterns. Energies 2021, 14, 5786 11 of 14 Table 2. Summary of the captured data for the 13 sites part of the case study. Installation First Measurement % of Consumption Captured % of PV Captured S1 18 June 2018 99.37% 99.39% S2 4 June 2018 86.5% 84.76% S3 16 July 2018 99.38% 99.51% S4 5 June 2018 93.01% 93% S5 30 April 2018 94.59% 94.41% S6 24 May 2018 99.05% 99.92% S7 1 June 2018 95.29% 95.25% S8 1 March 2018 94.45% 94.45% S9 1 March 2018 94.97% 94.97% S10 29 August 2018 97.49% 97.49% S11 29 Mary 2018 86.24% 99.94% S12 08 June 2018 98.55% 98.51% S13 28 March 2018 98.79% 98.79% Table 2. Summary of the captured data for the 13 sites part of the case study. 6. Limitations It is crucial to scrutinize the results as an exercise to uncover limitations and opportu- nities for future research. Firstly, the cloud nature of EnnerSpectrum requires an internet connection at the site. Even though this requirement seems uncomplicated since most households have Internet access, this observation is not accurate in all countries, and, as it was experienced during the case study, even modern sites with a good internet connection can have dead spots, especially in the more concealed places. This issue was mitigated using a 3G USB dongle for communication, yet this solution was not effective 100% of the time. As it was broadly discussed in the introductory sections, some monitoring equipment vendors do not provide easy cloud access to the data. To tackle this issue, the proposed system employed the use of HTML parsers from ESB. This approach was practical during the case study. However, it relies on the vendors not changing the organization of those HTML documents. Lastly, it is relevant to note that retroﬁtting the sensing approach can be cumbersome for particular sites since the GC are pass-through meters. The installation needs to cut the electricity from the site. However, the research team remains conﬁdent that this was the best decision given the quality of the data returned from GC, which was required for other studies during the case study. 5.3. Running the Pilot 5.3. Running the Pilot Running a pilot of electricity monitoring for more than 2 years presents its challenges and lessons learned. The research team encountered some issues during the ﬁrst months of the case study, primarily related to the internet connection and the SD-Card. After those initial issues, the (non-planed) visits to each site were rare. The installation of a GT with a constant internet connection allowed the team to update the sensing software several times during the study without visiting the site. One of the updates was installing a third-party service to monitor the state of each installation and, if necessary, alert the research team through a Slack bot. Withal, perhaps the main challenge running this study in the real-world is related to the “real world” aspect. The equipment was installed in households, which sometimes introduced changes in routines that had to be assimilated by the occupants. Participants had questions and were seldom apprehensive regarding the equipment and the impact on their household. Questions regarding the interface directed and the participants were also typical. After approximately 15 months, it was decided to hire a new member to the research team whose primary job was to handle the human side of the study, answering participants queries, scheduling routine visits, and actively engaging with participants regarding updates in the pilot (for example informing about a new visualization added to the front end). This decision signiﬁcantly improved the pilot’s handling for both the participants and the rest of the research team. 5.2. Hardware Deployment As discussed before, the different typologies and building characteristics (see Figure 6 and Table 1) can signiﬁcantly hinder installing new monitoring equipment. It is clear that for most buildings monitoring services such as presented here need to be retroﬁtted since the cost of signiﬁcantly changing the installation to accommodate monitoring equipment is prohibitive. This requires a platform that can adapt to different equipment, data models, locations, or acquisition frequency. In the presented case study, the infrastructure interacted with the standard CG meters. Nonetheless, it also collected data from DC meters, web services, web parsers, totaling six different metering approaches and two different typolo- gies. Since the start of the case study, the EnnerSpectrum system was also used to control battery storage systems, electric vehicle charging, monitor humidity and temperature from the sites, aggregate sub-station level data, and even monitoring electricity and water usage in industrial kitchens, further validating its ﬂexibility. This was possible since the EnnerSpectrum architecture employs two main computational elements. The GT and the ESB. The GT interacted directly with the hardware on the site, organizing the data in a consistent data model and working as a local database for periods without an internet connection. The ESB aggregated all the data and provided services that combined different data sources, such as the visualizations presented here. This way, one can argue that the proposed architecture allows interaction with almost all monitoring equipment that uses open protocols for communication. 12 of 14 12 of 14 Energies 2021, 14, 5786 7. Conclusions This paper presented the EnnerSpectrum system, a sensing agnostic infrastructure to monitor electricity consumption in buildings. The system was deployed in 13 sites for approximately 36 months. In this case study, the infrastructure was adapted to different sce- narios to measure electricity consumption and production. On average, the infrastructure was capable of capturing 95% of all the possible data points. Considering that three sites had interventions that left the site ofﬂine for several days, it is conceivable to argue that the proposed approach successfully reach its purpose in a heterogeneous sample of prosumers. The presented work also uncovered other results regarding running a long-term pilot in the real world. The EnnerSpectrum system managed to cope with several changes in the site’s infrastructure. Furthermore, the research team also had to adapt for such long-term deployment, incorporating human resources to meet participants’ expectations regarding the communication of the pilot progress and to assess the state of each site infrastructure before and during the study. This paper focused on the method used to measure electricity consumption and production. However, since the start of the deployment, EnnerSpectrum integrated other Energies 2021, 14, 5786 13 of 14 13 of 14 data sources, such as vehicle charging or battery consumption. The centralized nature of the proposed architecture also allowed to develop services that consider data from different sources, such as vehicle or battery charging optimization and control. It is also essential to consider that, while this paper focused on the technical description of the sensing approach used by EnnerSpectrum, further studies should consider how households adopted the technology and if it produced any changes in behavior. To ﬁnalize, it is suitable to lay out future research paths for the proposed approach. Apart from the user and data-centric possibilities mentioned in the previous paragraph, further research should also consider the technological aspects of the solution. The system already evolved to integrate the control of battery storage systems and to acquire plug- level data. However, the energy ﬁeld is in constant evolution, and new services will emerge that can challenge the ﬂexibility of EnnerSpectrum for example, the integration with speciﬁc loads like cooling and heating or other sensors, furthermore the centralized nature of EnnerSpectrum can enable the development of new services which take into account data from different sources. 7. Conclusions On a more practical level, other improvements should consider an ofﬂine mode that could rely on the data present on the GT if no internet connection is available. Furthermore, a real-time connection to the GT would be an interesting improvement. Currently, new data is available every minute. With real-time communication, services could push some of the processing to the GT, to, for example, directly alert consumers once an unusual event happens. Another important improvement to the solution would be implementing a cashing system for the most common queries to improve the system’s scalability. Author Contributions: Conceptualization, F.Q. and L.P.; Data curation, J.C. and W.S.; Funding acquisition, F.Q. and L.P.; Investigation, D.G. and D.V.; Methodology, F.Q. and L.P.; Software, J.C. and W.S.; Supervision, F.Q. and L.P.; Validation, D.G.; Visualization, F.Q. and L.P.; Writing—original draft, F.Q.; Writing—review & editing, F.Q. and L.P. All authors have read and agreed to the published version of the manuscript. Funding: This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 731249. Lucas Pereira has received funding from the Portuguese Foundation for Science and Technology (FCT) under grant CEECIND/01179/2017. Filipe Quintal and Lucas Pereira received funding from FCT under grant UIDB/50009/2020. This work reﬂects only the author’s view. Data Availability Statement: Data sharing not applicable. The consumption and site information collected during the case study was authorized by an informed consent signed by each household. Data sharing is only authorized for selected partners within the SMILE project H2020 consortium. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Emziane, M.; Al Ali, M. Performance assessment of rooftop PV systems in Abu Dhabi. Energy Build. 2015, 108, 101–105. [CrossRef] 9. Ali, A.S.; Zanzinger, Z.; Debose, D.; Stephens, B. Open Source Building Science Sensors (OSBSS): A low-cost Arduino-based platform for long-term indoor environmental data collection. Build. Environ. 2016, 100, 114–126. [CrossRef] P.; Stankovic, V.; Stankovic, L. Can non-intrusive load monitoring be used for identifying an appliance’s ur? Appl. Energy 2019, 238, 796–805. [CrossRef] 10. Rashid, H.; Singh, P.; Stankovic, V.; Stankovic, L. Can non-intrusive load monitoring be used for id anomalous behaviour? Appl. Energy 2019, 238, 796–805. [CrossRef] 11. Ferreira, P.M.; Ruano, A.E.; Silva, S.; Conceição, E.Z.E. Neural networks based predictive control for thermal comfort and energy savings in public buildings. Energy Build. 2012, 55, 238–251. [CrossRef] 12. LG-Chem. LG Chem Catalog Global; LG: Wonjoon Suh, Korea, 2018. 12. LG-Chem. LG Chem Catalog Global; LG: Wonjoon Suh, Korea, 2018. 13. WEBCONNECT, SMA Solar Technology AG, S.S.T. Available online: https://www.sma.de/en/produ webconnect.html (accessed on 10 August 2021) . 14. Frei, M.; Deb, C.; Nagy, Z.; Hischier, I.; Schlueter, A. Building Energy Performance Assessment Using an Easily Deployable Sensor Kit: Process, Risks, and Lessons Learned. Front. Built Environ. 2021, 6. [CrossRef] 15. Anbya, M.F.B.; Salehuddin, M.; Hadisupadmo, S.; Leksono, E. Wireless sensor network for single phase electricity monitoring system via Zigbee protocol. In Proceedings of the 2012 IEEE Conference on Control, Systems Industrial Informatics, Bandung, Indonesia, 23–26 September 2012; pp. 261–266. [CrossRef] p pp 16. Alghamdi, A.S. Potential for Rooftop-Mounted PV Power Generation to Meet Domestic Electrical Demand in Saudi Arabia: Case Study of a Villa in Jeddah. Energies 2019, 12, 4411. [CrossRef] 17. Ma, Z.; Cooper, P.; Daly, D.; Ledo, L. Existing building retroﬁts: Methodology and state-of-the-art. Energy Build. 2012, 55, 889–902. [CrossRef] 18. Pereira, L.; Quintal, F.; Nunes, N.; Bergés, M. The Design of a Hardware-Software Platform for Long-Term Energy Eco-Feedback Research. In ACM SIGCHI Symposium on Engineering Interactive Computing Systems (EICS ’12); ACM: Copenhagen, Denmark, 2012; pp. 221–230. [CrossRef] pp 19. Quintal, F.; Pereira, L.; Nunes, N.; Nisi, V.; Barreto, M. WATTSBurning: Design and Evaluation of an Innovative Eco-Feedback System; In Lecture Notes in Computer Science; Springer: Berlin/Heidelberg, Germany, 2013; pp. 453–470. [CrossRef] y g g y 20. Pereira, L.; Nunes, N. Understanding the Practical Issues of Deploying Energy Monitoring and Eco-Feedback Technology in the Wild: Lesson Learned from Three Long-Term Deployments. Energy Rep. 2019, 6, 94–106. 21. Abbreviations The following abbreviations are used in this manuscript: g DSM Demand Side Management EVC Electric Vehicle Charging PV Photovoltaics ADC Analog to Digital Converter DAQ Data Acquisition GT Gateway ESB EnnerSpectrum Back End GC Carlo Gavazzi energy meter DC Direct Current API Aplication Programming Interface UPS Uninterruptible Power Supply 14 of 14 Energies 2021, 14, 5786 References 1. MediaWiki. Electric Meter ETHW. 2020. Available online: http://ethw.org/Electric_Meter (accessed on 10 August 2021). 2. Hassan, R.; Radman, G. Survey on Smart Grid. In Proceedings of the IEEE SoutheastCon 2010 (SoutheastCon), Concord, NC, p g ( g ) 2. Hassan, R.; Radman, G. Survey on Smart Grid. In Proceedings of the IEEE SoutheastCon 2010 (SoutheastCon), Concord, NC, USA 18 21 March 2010; pp 210 213 [CrossRef] 2. Hassan, R.; Radman, G. Survey on Smart Grid. In Proceedings of the IEEE SoutheastCon 2010 (SoutheastCon), Concord, NC, USA, 18–21 March 2010; pp. 210–213. [CrossRef] USA, 18–21 March 2010; pp. 210–213. [CrossRef] pp 3. Wang, Y.; Chen, Q.; Hong, T.; Kang, C. Review of Smart Meter Data Analytics: Applications, Methodologies, and Challenges. IEEE Trans. Smart Grid 2019, 10, 3125–3148. [CrossRef] 4. Völker, B.; Reinhardt, A.; Faustine, A.; Pereira, L. Watt’s up at Home? Smart Meter Data Analytics from a Consumer-Centric Perspective. Energies 2021, 14, 719. [CrossRef] 5. Ahmad, S.; Ahmad, A.; Naeem, M.; Ejaz, W.; Kim, H.S. A Compendium of Performance Metrics, Pricing Schemes, Optimization Objectives, and Solution Methodologies of Demand Side Management for the Smart Grid. Energies 2018, 11, 2801. [CrossRef] 6. Zhou, B.; Li, W.; Chan, K.W.; Cao, Y.; Kuang, Y.; Liu, X.; Wang, X. Smart home energy manage conﬁgurations, and scheduling strategies. Renew. Sustain. Energy Rev. 2016, 61, 30–40. [CrossRef] han, K.W.; Cao, Y.; Kuang, Y.; Liu, X.; Wang, X. Smart home energy management systems: Concept scheduling strategies. Renew. Sustain. Energy Rev. 2016, 61, 30–40. [CrossRef] 6. Zhou, B.; Li, W.; Chan, K.W.; Cao, Y.; Kuang, Y.; Liu, X.; Wang, X. Smart home energy management systems: Concept, conﬁgurations, and scheduling strategies. Renew. Sustain. Energy Rev. 2016, 61, 30–40. [CrossRef] 7 Z i M J S G F L S l S Ch i f El i V hi l i P i E i 2020 13 298 [C R f] g , g g gy , , [ ] 7. Zweistra, M.; Janssen, S.; Geerts, F. Large Scale Smart Charging of Electric Vehicles in Practice. Energies 2020, 13, 298. [CrossRef] 7. Zweistra, M.; Janssen, S.; Geerts, F. Large Scale Smart Charging of Electric Vehicles in Practice. Energies 7. Zweistra, M.; Janssen, S.; Geerts, F. Large Scale Smart Charging of Electric Vehicles in Practice. Energies 2020, 13, 298. [CrossRef] 8. Emziane, M.; Al Ali, M. Performance assessment of rooftop PV systems in Abu Dhabi. Energy Build. 2015, 108, 101–105. [CrossRef] 8. References Yang, Z.; Zarabi, S.; Fernandes, E.; Rua-Taborda, M.I.; Debéda, H.; Salehian, A.; Nairn, D.; Wei, L. A Simple Wireless Sensor Node System for Electricity Monitoring Applications: Design, Integration, and Testing with Different Piezoelectric Energy Harvesters. Sensors 2018, 18, 3733. [CrossRef] [PubMed] 22. Frei, M.; Deb, C.; Stadler, R.; Nagy, Z.; Schlueter, A. Wireless sensor network for estimating building performance. Autom. Constr. 2020, 111, 103043. [CrossRef] 23. Chen, V.L.; Delmas, M.A.; Kaiser, W.J. Real-time, appliance-level electricity use feedback system: How to engage users? Energy Build. 2014, 70, 455–462. [CrossRef] 24. Weiss, M.; Mattern, F.; Graml, T.; Staake, T.; Fleisch, E. Handy feedback: Connecting smart meters with mobile phones. In Proceedings of the 8th International Conference on Mobile and Ubiquitous Multimedia MUM ’09, Cambridge, UK, 22–25 November 2009; Association for Computing Machinery: New York, NY, USA, 2009; pp. 1–4. [CrossRef] p g y pp 25. Sundramoorthy, V.; Liu, Q.; Cooper, G.; Linge, N.; Cooper, J. DEHEMS: A User-Driven Domestic Energy Monitnoring System. In Proceedings of the IEEE 2010 Internet of Things (IOT), Tokyo, Japan, 29 November–1 December 2010; pp. 1–8. [CrossRef] 26. Cieslik, W.; Szwajca, F.; Golimowski, W.; Berger, A. Experimental Analysis of Residential Photovoltaic (PV) and Electric Vehicle (EV) Systems in Terms of Annual Energy Utilization. Energies 2021, 14, 1085. [CrossRef] y gy g 27. Geelen, D.; Mugge, R.; Silvester, S.; Bulters, A. The use of apps to promote energy saving: A study feedback in the Netherlands. Energy Efﬁc. 2019, 12, 1635–1660. [CrossRef] 27. Geelen, D.; Mugge, R.; Silvester, S.; Bulters, A. The use of apps to promote energy saving: A study of smart meter–related feedback in the Netherlands. Energy Efﬁc. 2019, 12, 1635–1660. [CrossRef] 28 Schultz PW ; Estrada M ; Schmitt J ; Sokoloski R ; Silva Send N Using in home displays to provide smart meter feedback 28. Schultz, P.W.; Estrada, M.; Schmitt, J.; Sokoloski, R.; Silva-Send, N. Using in-home displays to provide smart meter feedback about household electricity consumption: A randomized control trial comparing kilowatts, cost, and social norms. Energy 2015, 90, 351–358. [CrossRef]
https://openalex.org/W3154445705	https://www.frontiersin.org/articles/10.3389/fimmu.2021.673196/pdf	English	null	Adapting T Cell Receptor Ligand Discrimination Capability via LAT	Frontiers in immunology	2,021	cc-by	10,256	Correspondence: Arthur Weiss Correspondence: Arthur Weiss arthur.weiss@ucsf.edu Adapting T Cell Receptor Ligand Discrimination Capability via LAT Wan-Lin Lo 1 and Arthur Weiss 1,2* 1 Division of Rheumatology, Rosalind Russell and Ephraim P. Engleman Arthritis Research Center, Department of Medicine, University of California, San Francisco, San Francisco, CA, United States, 2 Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, United States 1 Division of Rheumatology, Rosalind Russell and Ephraim P. Engleman Arthritis Research Center, Department of Medicine, University of California, San Francisco, San Francisco, CA, United States, 2 Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, United States Self- and non-self ligand discrimination is a core principle underlying T cell-mediated immunity. Mature ab T cells can respond to a foreign peptide ligand presented by major histocompatibility complex molecules (pMHCs) on antigen presenting cells, on a background of continuously sensed self–pMHCs. How ab T cells can properly balance high sensitivity and high speciﬁcity to foreign pMHCs, while surrounded by a sea of self- peptide ligands is not well understood. Such discrimination cannot be explained solely by the afﬁnity parameters of T cell antigen receptor (TCR) and pMHC interaction. In this review, we will discuss how T cell ligand discrimination may be molecularly deﬁned by events downstream of the TCR–pMHC interaction. We will discuss new evidence in support of the kinetic proofreading model of TCR ligand discrimination, and in particular how the kinetics of speciﬁc phosphorylation sites within the adaptor protein linker for activation of T cells (LAT) determine the outcome of TCR signaling. In addition, we will discuss emerging data regarding how some kinases, including ZAP-70 and LCK, may possess scaffolding functions to more efﬁciently direct their kinase activities. Keywords: LAT, coreceptor scanning, LCK, CD8, PLCg1, T cell receptor ligand discrimination, kinetic proofreading model Specialty section: Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology Received: 27 February 2021 Accepted: 29 March 2021 Published: 16 April 2021 Citation: Lo W-L and Weiss A (2021) Adapting T Cell Receptor Ligand Discrimination Capability via LAT. Front. Immunol. 12:673196. doi: 10.3389/fimmu.2021.673196 Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology Received: 27 February 2021 Accepted: 29 March 2021 Published: 16 April 2021 The T cell antigen receptor (TCR) is the major surface receptor used by every T cell to survey host cells expressing short self-peptides derived from host self-proteins bound to self-MHC molecules (self–pMHC). At the same time, peptides derived from pathogen proteins that are likewise bound to MHC molecules can also be recognized by the TCR and serve as agonists to initiate T cell responses in a highly sensitive and speciﬁc manner. One to ten foreign agonist– pMHCs are sufﬁcient to activate an antigen-speciﬁc T cell (thus, high sensitivity); each TCR clonotype also reacts with one foreign agonist–pMHC (thus, high speciﬁcity) (1, 2). However, a MINI REVIEW published: 16 April 2021 doi: 10.3389/fimmu.2021.673196 Edited by: Anne Spurkland, University of Oslo, Norway Reviewed by: Cosima T. Baldari, University of Siena, Italy David L. Wiest, Fox Chase Cancer Center, United States David J. Williamson, King’s College London, United Kingdom Reviewed by: Cosima T. Baldari, University of Siena, Italy David L. Wiest, Fox Chase Cancer Center, United States David J. Williamson, King’s College London, United Kingdom *Correspondence: Arthur Weiss arthur.weiss@ucsf.edu INTRODUCTION The immune system maintains a homeostatic state within an organism while remaining poised to vigorously respond to life-threatening challenges. T cells, as a major component of the adaptive immune system, contribute to this difﬁcult task by maintaining a level of tonic signaling which is required for their survival under homeostatic conditions. However, these same T cells are capable of rapidly responding to challenges by pathogens or cancerous cells to induce remarkably precise immune responses through the generation of signals that lead to clonal expansion and acquisition of effector functions only by appropriate antigen-speciﬁc T cells. INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY A prevailing hypothesis is that the architecture of the intracellular signaling pathways downstream of the TCR engenders T cells with their remarkable sensitivity and selectivity for speciﬁc antigens (37, 38, 44). This is achieved through a kinetic proofreading mechanism that functions as a molecular timing device to set an activation threshold for T cells (45–47) (Figure 1). T cell ligand discrimination instructs fate decisions at multiple developmental, homeostatic and differentiation stages. The basis for the molecular mechanisms underlying T cell ligand discrimination is an important question, especially in the context of recent advances in T cell-based immune therapies. For example, chimeric antigen receptor (CAR)-induced signaling requires more ligand-binding events and is less sensitive than natural/conventional TCR signaling (21–23). Such therapy can also lead to off-target antigen recognition driven T cell expansion and inﬂammation in healthy tissues (24–26). A better fundamental understanding of how T cells discriminate self- from non-self-ligands undoubtedly will facilitate the improvement of current T cell-directed immunotherapies. g The kinetic proofreading model was ﬁrst proposed by Hopﬁeld (47) and then adapted by McKeithan (45) to explain the remarkable selectivity of T cells. McKeithan envisioned that the TCR interaction with pMHC initiates a series of reversible biochemical reactions, such as phosphorylation, and these multiple steps create a time delay between the input (pMHC recognition) and the output (T cell activation) (45). If these signaling steps are rapidly reversible, the TCR:pMHC interaction must persist for a sufﬁcient duration to allow signaling to reach a “competent state” and induce essentially irreversible signaling steps (such as the ampliﬁcation of second messengers) in order to initiate successful T cell activation (45) (Figure 1). In other words, only those TCR:pMHC interactions with strong enough afﬁnity, a long enough bound half-life, or a stable enough catch- bond formation can sustain the TCR proximal signaling long enough to overcome the temporal threshold to trigger a bona- ﬁde activating signal by a T cell (Figure 1B). Built on the kinetic proofreading model, adaptation to intrinsic signaling events and modiﬁcation of the signaling network (such as up-regulation of PD-1 or other negative regulators) can ﬁne tune the reaction threshold in T cells (17, 48). Therefore, T cell ligand discrimination capability can potentially be engineered to change cellular outcomes. Extrinsic factors (e.g., cell-cell interactions) and intrinsic ones (e.g., cellular signaling proteins) contribute to T cell ligand discriminatory capability. Citation: Lo W-L and Weiss A (2021) Adapting T Cell Receptor Ligand Discrimination Capability via LAT. Front. Immunol. 12:673196. doi: 10.3389/fimmu.2021.673196 April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org T Cell Receptor Ligand Discrimination Lo and Weiss signiﬁcant portion of the naive T cell repertoire is also capable of responding to multiple foreign antigens (thus, cross-reactivity) or to allogeneic-MHC complexes (alloreactivity) (3, 4). bound quality under forces to form a catch bonds (e.g., force prolongs bond lifetime for TCR:agonist–pMHC) versus slip bonds (e.g., force shortens bond lifetime for TCR:self–pMHC) (30, 39–41). However, those reported differences seem insufﬁcient to account for a TCR’s high degree of speciﬁcity and sensitivity (29). Interestingly, by measuring the binding afﬁnities of ultra-low TCR–pMHC afﬁnities (those with a KD in the range of ~1000 µM) at 37°C, a recent study showed that the level of TCR discrimination is lower than the level that was estimated by earlier work (42). Regardless, these models still need to be coupled with activation thresholds set by cellular signaling networks, thereby allowing a T cell to distinguish whether a given TCR–pMHC signal is below, or above, that activation threshold. T CELLS NEED TO PROPERLY DISCRIMINATE AMONG FOREIGN, SELF-, AND ABSENCE OF PEPTIDE–MHC STIMULATION Circulating naive T cells continuously survey their local environments with their TCRs via interactions with self– pMHCs which generates survival signals, so called tonic signaling (5–10). Those naive T cells that fail to engage self– pMHCs rapidly die (11, 12). The levels of tonic signaling received by each individual T cell varies from clone to clone, and are correlated with each TCR’s reactivity towards particular self– pMHCs (13, 14). Importantly, the TCR’s reactivity toward self– pMHCs also inﬂuences the functional potential of T cells during anti-bacterial or anti-viral immune responses (13, 15–19), or dictates regulatory T cell suppressive function (20). These data suggest that tonic signaling plays an active role in modulating or adapting the ability of T cells to mediate effector functions. How T cells can achieve such precise immune regulation while avoiding autoimmune diseases remains unclear. More speciﬁcally, how do T cells properly discriminate a foreign antigen from the sea of self–pMHCs, and discriminate self– pMHC stimulation from the absence of pMHCs? Thus, it is unclear how TCR ligand discrimination ability can be chemically encoded to control the T cell fate decision process and transform the TCR:pMHC signal to a digital activation outcome (1, 43), such as cytokine production or proliferation. Frontiers in Immunology \| www.frontiersin.org INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY The kinetic proofreading model envisions that TCR:pMHC engagement triggers a series of biochemical signaling steps, which eventually lead to activation of T cells. The series of biochemical signaling steps are reversible, allowing for TCR:pMHC disengagement to quickly restore signaling intermediates back to the initial resting stage. These reversible biochemical reactions could be phosphorylation (yellow circle; a)/ dephosphorylation (gray circle; d), or protein-protein interaction (b)/dissociation (c). (A). TCR:self–pMHC interaction is weak with a relatively short bound half life so that signaling does not propagate all the way downstream to an irreversible step before the TCR:pMHC dissociates. (B). In contrast, the TCR interaction with foreign pMHC interaction is sufﬁciently long to reach a terminal irreversible step. Only when the TCR:pMHC interaction time is sustained long enough to engage all of the reversible kinetic proofreading steps and get to the key irreversible step will the T cell be activated. B B FIGURE 1 \| Illustration of kinetic proofreading model. The kinetic proofreading model envisions that TCR:pMHC engagement triggers a series of biochemical signaling steps, which eventually lead to activation of T cells. The series of biochemical signaling steps are reversible, allowing for TCR:pMHC disengagement to quickly restore signaling intermediates back to the initial resting stage. These reversible biochemical reactions could be phosphorylation (yellow circle; a)/ dephosphorylation (gray circle; d), or protein-protein interaction (b)/dissociation (c). (A). TCR:self–pMHC interaction is weak with a relatively short bound half life so that signaling does not propagate all the way downstream to an irreversible step before the TCR:pMHC dissociates. (B). In contrast, the TCR interaction with foreign pMHC interaction is sufﬁciently long to reach a terminal irreversible step. Only when the TCR:pMHC interaction time is sustained long enough to engage all of the reversible kinetic proofreading steps and get to the key irreversible step will the T cell be activated. kinase (SFK) LCK and z chain-associated protein kinase 70 (ZAP-70), as two proximal and essential T cell speciﬁc kinases in TCR signal initiation, have attracted great interest regarding their supportive roles in kinetic proofreading. triggered only when the last kinetic proofreading step is accomplished. The small differences in TCR–pMHC interaction– if coupled to a series of reversible and/or feedback loops that amplify small stimulatory signals and suppress excessive basal signaling– can lead to dramatically different cellular outcomes (38). Frontiers in Immunology \| www.frontiersin.org INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY A TCR typically binds to an agonist foreign–pMHC with a longer half-life than it does to a self– pMHC (20, 27–32). The TCR has higher ligand discrimination capability than B cell receptors (BCR), cytokine receptors, or other growth factor receptors (33). This discrimination capability relies on the intrinsic attributes of T cells to recognize self– pMHCs but not lead to activation, while recognition of foreign agonist–pMHCs should lead to potent T cell activation (34–38). Initial studies focused on the distinct characteristics of TCR binding kinetics toward foreign versus self–pMHC, such as the association on-rates and dissociation off-rates of the binding afﬁnities between TCR and pMHC molecules (32), or the structural and biophysical change underlying a TCR:pMHC A key prediction of the kinetic proofreading model is that through a series of reversible signaling steps, the TCR is April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org 2 T Cell Receptor Ligand Discrimination Lo and Weiss A B FIGURE 1 \| Illustration of kinetic proofreading model. The kinetic proofreading model envisions that TCR:pMHC engagement triggers a series of biochemical signaling steps, which eventually lead to activation of T cells. The series of biochemical signaling steps are reversible, allowing for TCR:pMHC disengagement to quickly restore signaling intermediates back to the initial resting stage. These reversible biochemical reactions could be phosphorylation (yellow circle; a)/ dephosphorylation (gray circle; d), or protein-protein interaction (b)/dissociation (c). (A). TCR:self–pMHC interaction is weak with a relatively short bound half life so that signaling does not propagate all the way downstream to an irreversible step before the TCR:pMHC dissociates. (B). In contrast, the TCR interaction with foreign pMHC interaction is sufﬁciently long to reach a terminal irreversible step. Only when the TCR:pMHC interaction time is sustained long enough to engage all of the reversible kinetic proofreading steps and get to the key irreversible step will the T cell be activated. A B FIGURE 1 \| Illustration of kinetic proofreading model. The kinetic proofreading model envisions that TCR:pMHC engagement triggers a series of biochemical signaling steps, which eventually lead to activation of T cells. The series of biochemical signaling steps are reversible, allowing for TCR:pMHC disengagement to quickly restore signaling intermediates back to the initial resting stage. These reversible biochemical reactions could be phosphorylation (yellow circle; a)/ A B FIGURE 1 \| Illustration of kinetic proofreading model. INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY In such a case, the more downstream in this TCR signal proofreading chain, the stronger difference in the level of signaling intermediates among TCR:pMHC of different half-lives, the greater discrimination is achieved. A recent study showed that it requires an approximate 2.8 sec of proofreading time delay and 2.67 biochemical steps to reach the estimated TCR discrimination capability (42). However, the speciﬁc key signaling steps where a small difference can enact a large change in functional outcome have not been fully identiﬁed or explored. In particular, it is not clear which early or intermediate signaling steps serve solely to pass along or amplify the signal, versus which terminal step(s) plays a more effectively digital role in terms of discriminating which ligands lead to activation versus those ligands that do not (31, 38, 49–53). LCK initiates the immediate signaling step after TCR:pMHC engagement (37, 54, 55). Functionally, LCK can phosphorylate the tyrosine residues of the immunoreceptor tyrosine-based activation motifs (ITAMs) in either the CD3 or z chains. Doubly phosphorylated ITAM motifs then create the docking sites for the tandem SH2 domains of ZAP-70 to bind and thereby be recruited to the engaged TCR complexes (56, 57). This docking step can be induced by the TCR binding to self– pMHC (56, 57). However, full release of ZAP-70 from its autoinhibitory conformation and its activation requires LCK to subsequently phosphorylate and activate the ITAM-bound ZAP- 70 (58, 59). Afterwards, the activated ZAP-70 kinase phosphorylates tyrosine residues in adaptor proteins, including LAT and lymphocyte cytosolic protein 2 (SLP76), to promote the assembly of a LAT-based signalosome to diversify and amplify TCR signals. Of particular importance is the recruitment and activation of phospholipase Cg1 (PLCg1) which is required for second messenger generation leading to calcium increases as well as Ras and protein kinase C (PKC) activation. Signaling beyond this point might be considered an irreversible (or a highly Many efforts have focused on understanding how the activities of protein kinases or phosphatases might provide the framework for the kinetic proofreading model. The Src family April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org 3 T Cell Receptor Ligand Discrimination Lo and Weiss energy-consuming if it was to be reversed) signaling step since at least some T cell responses can be rapidly initiated subsequent to these events. domain, promotes ZAP-70-mediated LAT phosphorylation by bridging ZAP-70 to LAT (Figure 2D). INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY This tri-protein interaction is likely to be a transient, low afﬁnity interaction. We speculate that this higher order of protein-protein interaction might be illustrated by the following sequential intermolecular interactions: 1) the coreceptor-bound LCK uses its SH2 domain to bind to phospho-Y319 ZAP-70 thereby stabilizing its own catalytic function; 2) the coreceptor-bound LCK associates with the proline-rich PIPRSP motif in LAT via its SH3 domain; 3) in a coreceptor-bound form, LCK bridges these signal initiating proteins, ZAP-70 and LAT, to the engaged TCR: pMHC complexes. Like other SFKs, LCK has an SH3 domain, an SH2 domain, a tyrosine kinase domain and a C-terminal inhibitory tyrosine (60). However, the function of LCK is particularly noteworthy with regard to its roles in T cells. LCK’s unique N-terminal region anchors it to the plasma membrane by myristylation and palmitoylation, and allows it to associate with the coreceptors CD4 or CD8 (60–63) (Figure 2A). The coreceptor interaction is relatively weak, so LCK can also exist as a free, coreceptor- unbound form (64) (Figure 2A). Some recent studies suggest that the free form of LCK may be more mobile and more active than the coreceptor-bound form of LCK (38). However, coreceptor-bound LCK has some unique features which could play important roles in supporting TCR ligand discrimination (31, 39, 53, 65, 66) (Figure 2B). As suggested by a recent coreceptor scanning model (31), upon TCR recognition of a foreign pMHC, the engaged TCR may need to scan through many coreceptors to ﬁnd one that is coupled with active LCK kinases to initiate signaling. The time involved in searching for the LCK-bound coreceptors is a potential kinetic proofreading step and can affect the sensitivity and magnitude of T cell responses (Figure 2B). This study also suggests coreceptor- bound LCK may display some function that is unique from the free LCK. This trimolecular protein interaction may facilitate weak TCR:pMHC interactions, especially those at the borderline of T cell activation thresholds, and contribute to the reversible kinetic proofreading signaling steps. In addition, this ZAP-70– LCK–LAT intramolecular coordination also supports TCR signal transduction in the proximity of engaged TCR:pMHC complexes. From the perspective of kinetic proofreading, this may be a key event to allow the internal signaling network to properly discriminate TCR interaction with an agonist or a self– pMHC. Thus, only the TCR:foreign–pMHC interaction would successfully propagate downstream signaling steps. INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY Indeed, these data, combined with computational modeling, further revealed that this higher order of coreceptor-coupled protein-protein interaction is particularly important to endow TCRs with the sensitivity to detect and respond to weak ligands. Elimination of the proline-rich motif in LAT compromised TCR signaling and T cell development (49). Indeed, each of the LCK structural domains are also suggested to play important roles in regulating TCR signaling. The LCK SH2 domain binds to phospho-Y319 of ZAP-70, promoting the open, active conformations of both LCK and ZAP-70 and sustaining coreceptor localization to the stimulated TCR (56, 59, 67, 68). Computational modeling also supports the notion that the interaction of LCK’s SH2 domain can increase the sensitivity of TCR-ligand recognition and the rapidity of TCR- induced activation (69). These observations on the SH2 domain of LCK, as well as previous studies that suggested an important role for the SH3 domain in TCR signaling and T cell development hinted that the SH3 domain of LCK might also contribute to downstream signaling in an important kinetic proofreading step (70–73). The kinetic proofreading model assumes that the signaling intermediates among the proofreading signaling steps can instantly reﬂect the status of TCR:pMHC interaction. Put differently, once the TCR is disengaged from pMHC, all the proofreading steps are quickly reversed (Figure 1A). Although such a reset process might exhibit some time delay and it remains elusive whether all signaling intermediates have to be reversed to the very initial or resting state, this assumption must be valid to a certain extent since it is essential for the kinetic proofreading model to work. We envision that the quick reset of unsuccessful signaling can only be possible if all these signaling intermediates are in one physical complex, allowing for ligand unbinding from the TCR to quickly lead to the reversal of the signaling events (38). Thus, the scaffold function of coreceptor-bound LCK is unique as it enables LAT to be in the same complex with TCR:pMHC, ZAP- 70 and LCK (38) (Figures 2B, D). These recent studies together support the model that coreceptor-bound LCK may have dual roles in supporting TCR signal initiation: as a kinase as well as a scaffold protein to orchestrate a distinct kinetic proofreading step in TCR signaling (Figures 2B, D). We recently found a function of the LCK SH3 domain that had not previously been appreciated (49) (Figures 2C, D). Frontiers in Immunology \| www.frontiersin.org INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY In examining the sequences of LAT among 41 mammalian species, we found that there are sixteen proline residues within the membrane-proximal region of LAT (from P33 to P95 in human LAT). The positions and frequencies of proline residues within this region are highly conserved in all mammalian LAT sequences that were examined. Yet the functionality of this region had not been appreciated. We found one particular proline motif in this region, PIPRSP (P80 −P82−P85), interacted with the LCK SH3 domain and that this interaction was functionally important (Figure 2C). In addition to the coreceptor-bound LCK data (31, 49), we and others also explored other signaling steps that might function as molecular timing devices in a similar manner (50–52). We focused our attention on another key tyrosine kinase involved in proximal TCR signaling, ZAP-70 (50, 74). Most ZAP-70 substrate phosphorylation sites are localized in Using a series of cellular experiments as well as molecular and biochemical approaches, we concluded that LCK, via its SH2 domain interacting with ZAP-70 phospho-Y319 and its SH3 domain by interacting with the identiﬁed LAT proline-rich April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org T Cell Receptor Ligand Discrimination Lo and Weiss A B C D E F FIGURE 2 \| Illustration of kinetic proofreading steps. (A) Forms of LCK. LCK can exist as coreceptor-bound form or free form. The yellow circle represents the phosphorylated tyrosine residues. (B) oreceptor scanning model. After the TCR is engaged with pMHC, the TCR scans through coreceptors (CD4 or CD8) to ﬁnd a coreceptor that is bound to active LCK. Engagement with an LCK-bound coreceptor may promote TCR sensitivity toward the weak ligand stimulation. The time involved to search for a LCK-bound coreceptor may function as a kinetic proofreading step. (C) Two unique features of LAT. Recent studies have revealed two unique features of LAT: 1) the PIPRSP motif in LAT can bind to SH3 domain of LCK (a); 2) LAT Y132 is a very poor substrate for ZAP-70 (b), because its preceding residue is a glycine. In contrast, other three tyrosine residues (Y171, Y191, Y226) are good ZAP-70 substrates because they all have acidic amino acid at the -1 positions. (D) Coreceptor-bound Lck has dual functions. LCK functions as both a kinase and a scaffold protein. As a kinase, LCK phosphorylates tyrosine residues of ITAM motifs in CD3 and z-chains. INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY Doubly-phosphorylated ITAM motifs create the docking site for kinase ZAP-70. The recruited ZAP-70 is further phosphorylated by LCK and becomes activated. As a scaffold protein, the LCK SH2 domain binds to phosphorylated Y319 in ZAP-70 to promote continued LCK activity and ZAP-70 activity. LCK can also interact with LAT to actively recruit LAT to the pMHC engaged TCR complex. This active recruitment is mediated through LCK SH3 domain and LAT PIPRSP motif. The active recruitment of LAT to the proximity of pMHC-engaged TCR supports TCR sensitivity toward weak ligand stimulation. (E) LAT Y132 has a slow phosphorylation kinetics. On the contrary, Y171, Y191, Y226 in LAT have fast phosphorylation rates promoted by ZAP-70. (F) LAT Y132-PLCg1 activation is a kinetic proofreading bottleneck. The adaptor protein LAT has ﬁve tyrosine residues, which are ZAP-70 substrates. Among these tyrosines, Y127, Y171, Y191 and Y226 all have either glutamate or aspartate at the -1 position, providing better binding to ZAP-70 substrate recognition site through an electrostatic selection mechanism. Therefore, these four tyrosine residues have faster phosphorylation kinetics mediated by ZAP-70. In contrast, LAT Y132 has a glycine residue at the -1 position, making Y132 a poor substrate to ZAP-70. This feature causes Y132 to have very slow phosphorylation kinetics relative to the other tyrosine residues in LAT. The slow phosphorylation of Y132 ensures that the PLCg1 pathway is the last to be activated. PLCg1 activation, a key event in T cell signaling events, may represent the last step in the chain of kinetic proofreading steps. The yellow circle represents the phosphorylated tyrosine residues and the gray circle represents the unphosphorylated tyrosine residues. B D A B C D B D E E F F FIGURE 2 \| Illustration of kinetic proofreading steps. (A) Forms of LCK. LCK can exist as coreceptor-bound form or free form. The yellow circle represents the phosphorylated tyrosine residues. (B) oreceptor scanning model. After the TCR is engaged with pMHC, the TCR scans through coreceptors (CD4 or CD8) to ﬁnd a coreceptor that is bound to active LCK. Engagement with an LCK-bound coreceptor may promote TCR sensitivity toward the weak ligand stimulation. The time involved to search for a LCK-bound coreceptor may function as a kinetic proofreading step. (C) Two unique features of LAT. THE SCAFFOLD PROTEIN LAT IS A UNIQUE SIGNALING PLATFORM THAT COORDINATES MULTIPLE SIGNALING- COMPETENT STATES AT DIFFERENTIAL SIGNALING KINETIC RATES g g p p y p The phosphorylation of LAT Y132, leading to the recruitment and activation of PLCg1 pathway, has some unique features. Biochemical and structural analysis of ZAP- 70 revealed that the substrate binding site in its kinase domain is unusually enriched in basic residues (74). This feature creates a microenvironment that allows a tyrosine substrate with surrounding acidic amino acids, to serve as a good ZAP- 70 substrate (74). Particularly important is a negatively charged residue at the -1 position (Figure 2C). Sequence analysis of the majority of known ZAP-70 substrates supported this model, including tyrosine residues in LAT (Y127, Y171, Y191 and Y226 in human LAT) and SLP76 (Y113, Y128, Y145 in human SLP76). These sites have either an aspartate or a glutamate residue preceding these known tyrosine substrates (Figure 2C). However, Y132 in human LAT (and Y136 in mouse LAT) is an exception (74, 96) (Figure 2C). Despite being a bona-ﬁde and long known ZAP-70 substrate, Y132 has a neutral residue, glycine, at the 131 position (74) (Figure 2C). As a consequence, this glycine compromised Y132 phosphorylation by ZAP-70 compared to the other well-characterized tyrosine phosphorylation sites in LAT (Figure 2E). This observation was supported by an in vitro kinase assay which showed that replacement of glycine at 131 in LAT peptides with an aspartate or a glutamate substantially enhanced ZAP-70 kinase-mediated phosphorylation efﬁciency of LAT Y132 (50, 74). In an immunoblot analysis of LAT- deﬁcient Jurkat cells reconstituted with G>D/E mutants of LAT or wild-type LAT, similar results were also observed (50). This slow phosphorylation of LAT Y132 is a feature that had been previously noted (96) but with an unclear basis or signiﬁcance. Scaffold proteins can also function within kinetic proofreading steps but have received less attention than kinases. Together with SLP76, LAT is a scaffold protein capable of amplifying T cell proximal signals upon TCR stimulation. LAT has a total of nine tyrosine residues and four of them have been intensively studied, including human LAT Y132, Y171, Y191 and Y226 (mouse LAT Y136, Y175, Y195 and Y235) (75, 76). The LAT tyrosine phosphorylation sites are important to initiate the assembly of higher order LAT complexes (77–79), which can become separated signaling microclusters in the membrane to facilitate downstream signaling, including actin reorganization, RAS/ mitogen-activated protein kinase (MAPK) signaling, PKC activation, and calcium mobilization. INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org 5 T Cell Receptor Ligand Discrimination Lo and Weiss two adaptor proteins, LAT and SLP76, which are known to be critically important for TCR-dependent T cell development and responses. This made LAT and SLP76 potentially interesting candidates with kinetic proofreading functions. as well as elevation of the levels of intracellular Ca2+ concentration. Evidence provided by optogenetic controllers of TCR signaling has suggested calcium increases and diacylglycerol production are critically involved TCR kinetic proofreading (51, 52). These two studies indicate that the signaling steps that trigger the production of these second messengers may function as ﬁnal signaling checkpoints to allow incoming signals to be properly proofread. THE SCAFFOLD PROTEIN LAT IS A UNIQUE SIGNALING PLATFORM THAT COORDINATES MULTIPLE SIGNALING- COMPETENT STATES AT DIFFERENTIAL SIGNALING KINETIC RATES LAT signalosome- induced signaling is also essential for most TCR induced signaling and transcriptional responses (79–81). Importantly, LAT signalosomes are also coupled to several negative regulators, such as thymocyte-expressed molecule involved in selection (THEMIS) (82–85), the Src homology region 2 domain- containing phosphatase 1 (SHP1) (86–88), and leucine-rich repeats and calponin homology domain containing 1 (LRCH1) (89). INTRACELLULAR SIGNALING PATHWAYS CONTROL THE SENSITIVITY AND SELECTIVITY OF T CELL LIGAND DISCRIMINATION CAPABILITY Recent studies have revealed two unique features of LAT: 1) the PIPRSP motif in LAT can bind to SH3 domain of LCK (a); 2) LAT Y132 is a very poor substrate for ZAP-70 (b), because its preceding residue is a glycine. In contrast, other three tyrosine residues (Y171, Y191, Y226) are good ZAP-70 substrates because they all have acidic amino acid at the -1 positions. (D) Coreceptor-bound Lck has dual functions. LCK functions as both a kinase and a scaffold protein. As a kinase, LCK phosphorylates tyrosine residues of ITAM motifs in CD3 and z-chains. Doubly-phosphorylated ITAM motifs create the docking site for kinase ZAP-70. The recruited ZAP-70 is further phosphorylated by LCK and becomes activated. As a scaffold protein, the LCK SH2 domain binds to phosphorylated Y319 in ZAP-70 to promote continued LCK activity and ZAP-70 activity. LCK can also interact with LAT to actively recruit LAT to the pMHC engaged TCR complex. This active recruitment is mediated through LCK SH3 domain and LAT PIPRSP motif. The active recruitment of LAT to the proximity of pMHC-engaged TCR supports TCR sensitivity toward weak ligand stimulation. (E) LAT Y132 has a slow phosphorylation kinetics. On the contrary, Y171, Y191, Y226 in LAT have fast phosphorylation rates promoted by ZAP-70. (F) LAT Y132-PLCg1 activation is a kinetic proofreading bottleneck. The adaptor protein LAT has ﬁve tyrosine residues, which are ZAP-70 substrates. Among these tyrosines, Y127, Y171, Y191 and Y226 all have either glutamate or aspartate at the -1 position, providing better binding to ZAP-70 substrate recognition site through an electrostatic selection mechanism. Therefore, these four tyrosine residues have faster phosphorylation kinetics mediated by ZAP-70. In contrast, LAT Y132 has a glycine residue at the -1 position, making Y132 a poor substrate to ZAP-70. This feature causes Y132 to have very slow phosphorylation kinetics relative to the other tyrosine residues in LAT. The slow phosphorylation of Y132 ensures that the PLCg1 pathway is the last to be activated. PLCg1 activation, a key event in T cell signaling events, may represent the last step in the chain of kinetic proofreading steps. The yellow circle represents the phosphorylated tyrosine residues and the gray circle represents the unphosphorylated tyrosine residues. Frontiers in Immunology \| www.frontiersin.org LAT SIGNALOSOME MAINTAINS A DYNAMIC INTERACTOME WITH THE CD5 SIGNALOSOME AND THE CD6 SIGNALOSOME TO INTEGRATE POSITIVE AND NEGATIVE REGULATORY SIGNALS SLP76, GRB2, and GRB2 related adaptor protein 2 (GRAP2) can be associated with either the LAT signalosome or CD6 signalosome, whereas SOS, PLCg1 and hematopoietic progenitor kinase 1 (HPK1) can only associate with the LAT signalosome (81). Competition of these shared interactors inﬂuences the relative ratio of active LAT signalosome versus that of the CD6 complex. For example, both CD6 and LAT interacts with SLP76; however, although SLP76 can bind to LAT through an intermediate protein GRB2 related adaptor protein 2 (GRAP2), SLP76’s SH2 domain is required for assembly with CD6 signalosome. This difference in the interaction mechanism allows SLP76 to only activate HPK1 when it resides in the LAT signalosome but not within the CD6 signalosome, thereby inﬂuencing the compositions and dynamics of these signalosomes. This study revealed that dynamic interactions among LAT-, CD5-, and CD6- signalosomes may engage different positive or negative feedback loop to further ﬁne tune TCR signaling. Further work is needed to understand how such interactions are regulated during physiologic TCR responses to antigen. p y g The ﬁnding that nearly all tetrapods have a comparable glycine at the homologous position in LAT which is likely to lead to slow activation of PLCg1 (50), but that other phosphorylation sites in LAT and even within SLP76 are better substrates for ZAP-70 (74), emphasizes the critical importance of Y132 as a critical timer to assess whether T cells should be fully activated to respond to potential threats in most warm-blooded organisms. Surprisingly, sequences conferring more rapid and efﬁcient phosphorylation of LAT Y132 could be found in the LAT homologs of some cold- blooded animals, such as zebraﬁsh. Thus, cold-blooded animals may require more efﬁcient phosphorylation of the Y132 homologous sequence to ensure a strong PLCg1 dependent responses in cold environments. Indeed, we showed that zebraﬁsh thymocytes were less sensitive to cold temperatures than mammalian LAT (50). Expression of G131D in OT-I TCR+ Jurkat human T cell variants was able to endow these cells with the ability to respond to OVA stimulation at room temperature, whereas the wild-type glycine at position 131 in LAT prevented cells from responding at room temperature (50). LAT G131 IS AN APPARENT TCR SIGNALING BOTTLENECK FOR TCR ACTIVATION-INDUCED ACTIVATION OF PLCg1 AND CALCIUM PATHWAYS Interestingly, the simple substitution of an acidic residue, glutamate or aspartate, at the -1 position (i.e., G131E or G131D) increased the phosphorylation rates of LAT Y132, the recruitment, phosphorylation and activation of PLCg1, and the magnitude and the rate of calcium elevation (50, 97). The augmented LAT–PLCg1–calcium pathway resulted in an increase in T cell responses (50). OT-I TCR transgenic CD8 T cells that were virally transduced with the gain-of-activity LAT mutants (G135>D or G135>E in mouse) acquired the ability to react with weak ligands or even self–pMHC which did not activate control CD8 T cells transduced with wild-type LAT (50) (Figure 2F). These hyper-sensitive OT-I TCR+ CD8 T cells expressing either G135>D or G135>E mutant LAT upregulated the expression of activation marker CD69 and increased their production of the proinﬂammatory cytokine IFNg (50). A similar gain-of-activity from the ectopic expression of these two LAT mutants could also be observed in OT-II TCR+ CD4 T As adaptors in TCR signaling, LAT and SLP76 assemble signalosomes to link the ZAP-70-mediated “input” to downstream diverse pathways through individual key tyrosine substrates. Y132 in human LAT (Y136 in mouse) is especially important for the recruitment and activation of PLCg1. Replacement of this key tyrosine with phenylalanine disrupted thymic development of T cells and TCR induced T cell activation (90–95). Importantly, unlike other key tyrosine residues that share redundant interacting partners, Y132 is the one and only tyrosine associated with PLCg1 interaction and function. Activation of the PLCg1 pathway increases the production of the second messengers diacylglycerol and inositol-1,4,5- triphosphate, and eventually increases PKC and Ras activation April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org 6 T Cell Receptor Ligand Discrimination Lo and Weiss cells (50). Such enhanced responsiveness could have negative consequences in vivo since T cells expressing the G135>D/E LAT mutants might enable responses against self-peptides in vivo which could potentially result in autoimmunity or immunopathology. LAT SIGNALOSOME MAINTAINS A DYNAMIC INTERACTOME WITH THE CD5 SIGNALOSOME AND THE CD6 SIGNALOSOME TO INTEGRATE POSITIVE AND NEGATIVE REGULATORY SIGNALS The slow phosphorylation of Y132 in LAT appears to be a key and perhaps the most important rate-determining event in the kinetic proofreading process that assesses the quality of the TCR: pMHC interaction (Figure 2F). The slow phosphorylation rate of LAT Y132 is particularly interesting, for it is a unique kinetic proofreading event within a molecule which has ﬁve key tyrosine-based phosphorylation modiﬁcations which could allow multiple signaling-competent states to be achieved at different rates. However, each of the four other sites conform to preferred ZAP-70 substrates and the individual mutation of each of these other sites did not alter the sensitivity or magnitude of TCR response to pMHC (50). Thus, Y132 in LAT appears to a critically important step in kinetic proofreading, at least within the LAT protein. While Y132 appears to be a signaling bottleneck, it suggests the possibility that PLCg1 activation is either the last step in the kinetic proofreading chain of events; or alternatively, the consequences of activation of the PLCg1 pathway is the ﬁrst signaling step not physically linked to the engaged TCR:pMHC complex. This model then further supports the importance of the LCK–LAT interaction within the stimulated TCR:pMHC complex for ligand discrimination, especially in the case of weak ligands. Interestingly, the mutation of Y132 also led to TCR-independent lymphoproliferation of T cells (94, 98–102). This suggests PLCg1 signal may serve a negative regulatory function to maintain the balanced cell signaling that leads to an appropriate T cell response. Thus, the LAT-assembled signalosome may multitask, with both positive and negative regulatory functions, within a complicated yet intricate signal transduction network. w t a co p cated yet t cate s g a t a sduct o etwo . A recent proteomic study revealed that the LAT signalosome exhibits dynamic time-dependent interaction relationships with the transmembrane receptors CD5- and CD6-assembled signalosomes (81). According to that study, the LAT signalosome mostly plays a dominant role as an activating signaling pathway node, as characterized by its ability to activate SOS-ERK and PLCg1-NFAT pathways (81). However, unlike the LAT signalosome, the CD5 signalosome is enriched with many proteins with inhibitory functions, including Cbl-b and Ubash3A (81). Interestingly, the CD6-induced signalosome functions as a buffering zone, competing interacting proteins with either the inhibitory CD5 signalosome or with the stimulatory LAT signalosome. LAT SIGNALOSOME MAINTAINS A DYNAMIC INTERACTOME WITH THE CD5 SIGNALOSOME AND THE CD6 SIGNALOSOME TO INTEGRATE POSITIVE AND NEGATIVE REGULATORY SIGNALS Thus, our data suggested that the slow phosphorylation kinetics of human Y132 (and mouse Y136) in LAT evolved to be a poor site of phosphorylation in order to serve as a critical signaling step to control kinetic proofreading in mammalian T cells, whereas at least some cold-blooded animals require a more optimal site of phosphorylation at the Y132 homolog due to the constraint on phosphorylation imposed by cold temperature. These data offer insights into a novel mechanism to allow mammals to ﬁne tune their T cell response threshold and contribute to T cell antigen discrimination. Frontiers in Immunology \| www.frontiersin.org REFERENCES Cell Res (2020) 30:948–9. doi: 10.1038/s41422-020-00420-6 7. Lo WL, Allen PM. Self-peptides in TCR repertoire selection and peripheral T cell function. Curr Top Microbiol Immunol (2014) 373:49–67. doi: 10.1007/82_2013_319 22. Gudipati V, Rydzek J, Doel-Perez I, Goncalves VDR, Scharf L, Konigsberger S, et al. Inefﬁcient CAR-proximal signaling blunts antigen sensitivity. Nat Immunol (2020) 21:848–56. doi: 10.1038/s41590-020-0719-0 8. Morris GP, Allen PM. How the TCR balances sensitivity and speciﬁcity for the recognition of self and pathogens. Nat Immunol (2012) 13:121–8. doi: 10.1038/ni.2190 23. Harris DT, Hager MV, Smith SN, Cai Q, Stone JD, Kruger P, et al. Comparison of T Cell Activities Mediated by Human TCRs and CARs That Use the Same Recognition Domains. J Immunol (2018) 200:1088–100. doi: 10.4049/jimmunol.1700236 9. Myers DR, Zikherman J, Roose JP. Tonic Signals: Why Do Lymphocytes Bother? Trends Immunol (2017) 38:844–57. doi: 10.1016/j.it.2017.06.010 10. Paprckova D, Stepanek O. Narcissistic T cells: reactivity to self makes a difference. FEBS J (2020) 288:1778–88. doi: 10.1111/febs.15498 24. Kotter S, Andresen C, Kruger M. Titin: central player of hypertrophic signaling and sarcomeric protein quality control. Biol Chem (2014) 395:1341–52. doi: 10.1515/hsz-2014-0178 11. Sprent J, Surh CD. Normal T cell homeostasis: the conversion of naive cells into memory-phenotype cells. Nat Immunol (2011) 12:478–84. doi: 10.1038/ ni.2018 25. Linette GP, Stadtmauer EA, Maus MV, Rapoport AP, Levine BL, Emery L, et al. Cardiovascular toxicity and titin cross-reactivity of afﬁnity-enhanced T cells in myeloma and melanoma. Blood (2013) 122:863–71. doi: 10.1182/ blood-2013-03-490565 12. Surh CD, Sprent J. Homeostasis of naive and memory T cells. Immunity (2008) 29:848–62. doi: 10.1016/j.immuni.2008.11.002 13. Weber KS, Li QJ, Persaud SP, Campbell JD, Davis MM, Allen PM. Distinct CD4+ helper T cells involved in primary and secondary responses to infection. Proc Natl Acad Sci USA (2012) 109:9511–6. doi: 10.1073/ pnas.1202408109 26. Cameron BJ, Gerry AB, Dukes J, Harper JV, Kannan V, Bianchi FC, et al. Identiﬁcation of a Titin-derived HLA-A1-presented peptide as a cross- reactive target for engineered MAGE A3-directed T cells. Sci Transl Med (2013) 5:197ra103. doi: 10.1126/scitranslmed.3006034 14. Mandl JN, Monteiro JP, Vrisekoop N, Germain RN. T cell-positive selection uses self-ligand binding strength to optimize repertoire recognition of foreign antigens. Immunity (2013) 38:263–74. doi: 10.1016/ j.immuni.2012.09.011 27. Daniels MA, Teixeiro E, Gill J, Hausmann B, Roubaty D, Holmberg K, et al. Thymic selection threshold deﬁned by compartmentalization of Ras/MAPK signalling. Nature (2006) 444:724–9. doi: 10.1038/nature05269 15. REFERENCES T cell response. Proc Natl Acad Sci USA (2006) 103:16846–51. doi: 10.1073/pnas.0607771103 T cell response. Proc Natl Acad Sci USA (2006) 103:16846–51. doi: 10.1073/pnas.0607771103 1. Huang J, Brameshuber M, Zeng X, Xie J, Li QJ, Chien YH, et al. A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4(+) T cells. Immunity (2013) 39:846–57. doi: 10.1016/ j.immuni.2013.08.036 1. Huang J, Brameshuber M, Zeng X, Xie J, Li QJ, Chien YH, et al. A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4(+) T cells. Immunity (2013) 39:846–57. doi: 10.1016/ j.immuni.2013.08.036 16. Hogquist KA, Jameson SC. The self-obsession of T cells: how TCR signaling thresholds affect fate ‘decisions’ and effector function. Nat Immunol (2014) 15:815–23. doi: 10.1038/ni.2938 17. Persaud SP, Parker CR, Lo WL, Weber KS, Allen PM. Intrinsic CD4+ T cell sensitivity and response to a pathogen are set and sustained by avidity for thymic and peripheral complexes of self peptide and MHC. Nat Immunol (2014) 15:266–74. doi: 10.1038/ni.2822 2. Brameshuber M, Kellner F, Rossboth BK, Ta H, Alge K, Sevcsik E, et al. Monomeric TCRs drive T cell antigen recognition. Nat Immunol (2018) 19:487–96. doi: 10.1038/s41590-018-0092-4 3. Morris GP, Ni PP, Allen PM. Alloreactivity is limited by the endogenous peptide repertoire. Proc Natl Acad Sci USA (2011) 108:3695–700. doi: 10.1073/pnas.1017015108 18. Bartleson JM, Viehmann Milam AA, Donermeyer DL, Horvath S, Xia Y, Egawa T, et al. Strength of tonic T cell receptor signaling instructs T follicular helper cell-fate decisions. Nat Immunol (2020) 21:1384–96. doi: 10.1038/s41590-020-0781-7 4. Felix NJ, Allen PM. Speciﬁcity of T-cell alloreactivity. Nat Rev Immunol (2007) 7:942–53. doi: 10.1038/nri2200 19. Fulton RB, Hamilton SE, Xing Y, Best JA, Goldrath AW, Hogquist KA, et al. The TCR’s sensitivity to self peptide-MHC dictates the ability of naive CD8 (+) T cells to respond to foreign antigens. Nat Immunol (2015) 16:107–17. doi: 10.1038/ni.3043 5. Davis MM, Krogsgaard M, Huse M, Huppa J, Lillemeier BF, Li QJ. T cells as a self-referential, sensory organ. Annu Rev Immunol (2007) 25:681–95. doi: 10.1146/annurev.immunol.24.021605.090600 6. Lo WL, Allen PM. Self-awareness: how self-peptide/MHC complexes are essential in the development of T cells. Mol Immunol (2013) 55:186–9. doi: 10.1016/j.molimm.2012.10.028 20. Stadinski BD, Shekhar K, Gomez-Tourino I, Jung J, Sasaki K, Sewell AK, et al. Hydrophobic CDR3 residues promote the development of self-reactive T cells. Nat Immunol (2016) 17:946–55. doi: 10.1038/ni.3491 21. Malissen B. CAR T cells: from tinkering to rational design. ACKNOWLEDGMENTS The authors thank D.L. Donermeyer, P.J. Ebert, B. Au-Yeung, and O. Stepanek for providing feedback on the manuscript. FUNDING AW was supported by NIH P01 AI091580 and 1R37AI114575. AW was supported by NIH P01 AI091580 and 1R37AI114575. CONCLUSION AND FUTURE PERSPECTIVES Recent advances in T cell-targeted immunotherapies, including the development of chimeric antigen receptor (CAR) T cells, have successfully leveraged our knowledge of T cell signal transduction and T cell activation to treat cancers and/or autoimmune diseases. Although CARs can elicit T cell effector functions, these artiﬁcial April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org T Cell Receptor Ligand Discrimination Lo and Weiss AUTHOR CONTRIBUTIONS receptors appear to be much less efﬁcient at detecting foreign antigens than genuine TCRs. Moreover, most CARs lack the ability to fully propagate signals through the canonical TCR signaling pathway and appear to bypass some key signaling steps (22, 103–105). For example, CARs are less efﬁcient at recruiting the kinase ZAP-70, which is responsible for initiating TCR signaling and LAT phosphorylation, and they do not efﬁciently activate Ca2+ signaling. A recent study also shows that CAR-mediated signaling may bypass LAT signals. These differences between TCR- and CAR-mediated signaling may underlie common issues confounding CAR-T therapy, such as diminished in vivo persistence of the therapeutic cells. Thus, further studies engineering more responsive CAR-T cells by enhancing the ability of CARs to tap into intrinsic T cell signaling pathways might offer new insights into better control T cell sensitivity and selectivity. W-LL and AW wrote the manuscript and approved the submitted version. All authors contributed to the article and approved the submitted version. REFERENCES ZAP-70: an essential kinase in T-cell signaling. Cold Spring Harb Perspect Biol (2010) 2:a002279. doi: 10.1101/cshperspect.a002279 34. Davis SJ, van der Merwe PA. The kinetic-segregation model: TCR triggering and beyond. Nat Immunol (2006) 7:803–9. doi: 10.1038/ni1369 35. Lever M, Maini PK, van der Merwe PA, Dushek O. Phenotypic models of T cell activation. Nat Rev Immunol (2014) 14:619–29. doi: 10.1038/nri3728 57. Gascoigne NR, Casas J, Brzostek J, Rybakin V. Initiation of TCR phosphorylation and signal transduction. Front Immunol (2011) 2:72. doi: 10.3389/ﬁmmu.2011.00072 36. Siller-Farfan JA, Dushek O. Molecular mechanisms of T cell sensitivity to antigen. Immunol Rev (2018) 285:194–205. doi: 10.1111/imr.12690 58. Malissen B, Bongrand P. Early T cell activation: integrating biochemical, structural, and biophysical cues. Annu Rev Immunol (2015) 33:539–61. doi: 10.1146/annurev-immunol-032414-112158 37. Chakraborty AK, Weiss A. Insights into the initiation of TCR signaling. Nat Immunol (2014) 15:798–807. doi: 10.1038/ni.2940 59. Au-Yeung BB, Shah NH, Shen L, Weiss A. ZAP-70 in Signaling, Biology, and Disease. Annu Rev Immunol (2018) 36:127–56. doi: 10.1146/annurev- immunol-042617-053335 38. Ganti RS, Lo WL, McAffee DB, Groves JT, Weiss A, Chakraborty AK. How the T cell signaling network processes information to discriminate between self and agonist ligands. Proc Natl Acad Sci USA (2020) 117:26020–30. doi: 10.1073/pnas.2008303117 60. Palacios EH, Weiss A. Function of the Src-family kinases, Lck and Fyn, in T- cell development and activation. Oncogene (2004) 23:7990–8000. doi: 10.1038/sj.onc.1208074 39. Hong J, Ge C, Jothikumar P, Yuan Z, Liu B, Bai K, et al. A TCR mechanotransduction signaling loop induces negative selection in the thymus. Nat Immunol (2018) 19:1379–90. doi: 10.1038/s41590-018-0259-z 61. Norment AM, Salter RD, Parham P, Engelhard VH, Littman DR. Cell-cell adhesion mediated by CD8 and MHC class I molecules. Nature (1988) 336:79–81. doi: 10.1038/336079a0 40. Sibener LV, Fernandes RA, Kolawole EM, Carbone CB, Liu F, McAffee D, et al. Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding. Cell (2018) 174:672–87.e27. doi: 10.1016/j.cell.2018.06.017 62. Doyle C, Strominger JL. Interaction between CD4 and class II MHC molecules mediates cell adhesion. 1987. J Immunol (2010) 184:5935–8. doi: 10.1038/330256a0 41. Hong J, Persaud SP, Horvath S, Allen PM, Evavold BD, Zhu C. Force- Regulated In Situ TCR-Peptide-Bound MHC Class II Kinetics Determine Functions of CD4+ T Cells. J Immunol (2015) 195:3557–64. doi: 10.4049/ jimmunol.1501407 63. Shaw AS, Chalupny J, Whitney JA, Hammond C, Amrein KE, Kavathas P, et al. REFERENCES Short related sequences in the cytoplasmic domains of CD4 and CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase. Mol Cell Biol (1990) 10:1853–62. doi: 10.1128/MCB.10.5.1853 42. Pettmann J, Abu-Shah E, Kutuzov M, Wilson DB, Dustin ML, Davis SJ, et al. T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low afﬁnity peptide-MHC ligands. bioRxiv (2020) 2020.11.14.382630. doi: 10.1101/2020.11.14.382630 64. Casas J, Brzostek J, Zarnitsyna VI, Hong JS, Wei Q, Hoerter JA, et al. Ligand- engaged TCR is triggered by Lck not associated with CD8 coreceptor. Nat Commun (2014) 5:5624. doi: 10.1038/ncomms6624 43. Abu-Shah E, Trendel N, Kruger P, Nguyen J, Pettmann J, Kutuzov M, et al. Human CD8(+) T Cells Exhibit a Shared Antigen Threshold for Different Effector Responses. J Immunol (2020) 205:1503–12. doi: 10.4049/ jimmunol.2000525 65. Jiang N, Huang J, Edwards LJ, Liu B, Zhang Y, Beal CD, et al. Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination. Immunity (2011) 34:13–23. doi: 10.1016/j.immuni.2010.12.017 44. Courtney AH, Lo WL, Weiss A. TCR Signaling: Mechanisms of Initiation and Propagation. Trends Biochem Sci (2018) 43:108–23. doi: 10.1016/ j.tibs.2017.11.008 66. Horkova V, Drobek A, Mueller D, Gubser C, Niederlova V, Wyss L, et al. Dynamics of the Coreceptor-LCK Interactions during T Cell Development Shape the Self-Reactivity of Peripheral CD4 and CD8 T Cells. Cell Rep (2020) 30:1504–14.e7. doi: 10.1016/j.celrep.2020.01.008 45. McKeithan TW. Kinetic proofreading in T-cell receptor signal transduction. Proc Natl Acad Sci USA (1995) 92:5042–6. doi: 10.1073/pnas.92.11.5042 67. Williams BL, Irvin BJ, Sutor SL, Chini CC, Yacyshyn E, Bubeck Wardenburg J, et al. Phosphorylation of Tyr319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-gamma1 and Ras activation. EMBO J (1999) 18:1832–44. doi: 10.1093/emboj/18.7.1832 46. Ninio J. Kinetic ampliﬁcation of enzyme discrimination. Biochimie (1975) 57:587–95. doi: 10.1016/S0300-9084(75)80139-8 47. Hopﬁeld JJ. Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high speciﬁcity. Proc Natl Acad Sci USA (1974) 71:4135–9. doi: 10.1073/pnas.71.10.4135 68. PelosiM,DiBartoloV,Mounier V,MegeD,PascussiJM,Dufour E,etal.Tyrosine 319intheinterdomainBofZAP-70isabindingsitefortheSrchomology2domain of Lck. J Biol Chem (1999) 274:14229–37. doi: 10.1074/jbc.274.20.14229 48. Zinzow-Kramer WM, Weiss A, Au-Yeung BB. Adaptation by naive CD4(+) T cells to self-antigen-dependent TCR signaling induces functional heterogeneity and tolerance. Proc Natl Acad Sci USA (2019) 116:15160–9. doi: 10.1073/pnas.1904096116 69. Thill PA, Weiss A, Chakraborty AK. REFERENCES Anikeeva N, Lebedeva T, Clapp AR, Goldman ER, Dustin ML, Mattoussi H, et al. Quantum dot/peptide-MHC biosensors reveal strong CD8- dependent cooperation between self and viral antigens that augment the 28. Lo WL, Felix NJ, Walters JJ, Rohrs H, Gross ML, Allen PM. An endogenous peptide positively selects and augments the activation and survival of peripheral CD4+ T cells. Nat Immunol (2009) 10:1155–61. doi: 10.1038/ni.1796 April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org 8 T Cell Receptor Ligand Discrimination Lo and Weiss 29. Juang J, Ebert PJ, Feng D, Garcia KC, Krogsgaard M, Davis MM. Peptide- MHC heterodimers show that thymic positive selection requires a more restricted set of self-peptides than negative selection. J Exp Med (2010) 207:1223–34. doi: 10.1084/jem.20092170 51. YouseﬁOS, Gunther M, Horner M, Chalupsky J, Wess M, Brandl SM, et al. Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor. Elife (2019) 8. doi: 10.7554/eLife.42475 52. Tischer DK, Weiner OD. Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling. Elife (2019) 8:e42498. doi: 10.7554/eLife.42498 30. Liu B, Chen W, Evavold BD, Zhu C. Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling. Cell (2014) 157:357–68. doi: 10.1016/j.cell.2014.02.053 53. Trendel N, Kruger P, Gaglione S, Nguyen J, Pettmann J, Sontag ED, et al. Perfect adaptation of CD8+ T cell responses to constant antigen input over a wide range of afﬁnities is overcome by costimulation. Sci Signaling (2021) 14: eaay9363. doi: 10.1126/scisignal.aay9363 31. Stepanek O, Prabhakar AS, Osswald C, King CG, Bulek A, Naeher D, et al. Coreceptor scanning by the T cell receptor provides a mechanism for T cell tolerance. Cell (2014) 159:333–45. doi: 10.1016/j.cell.2014.08.042 54. Iwashima M, Irving BA, van Oers NS, Chan AC, Weiss A. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science (1994) 263:1136–9. doi: 10.1126/science.7509083 32. Stone JD, Chervin AS, Kranz DM. T-cell receptor binding afﬁnities and kinetics: impact on T-cell activity and speciﬁcity. Immunology (2009) 126:165–76. doi: 10.1111/j.1365-2567.2008.03015.x 33. Huhn A, Wilson DB, van der Merwe PA, Dushek O. The discriminatory power of the T cell receptor. bioRxiv (2020) 2020.11.16.384495. doi: 10.1101/ 2020.11.16.384495 55. Weiss A, Littman DR. Signal transduction by lymphocyte antigen receptors. Cell (1994) 76:263–74. doi: 10.1016/0092-8674(94)90334-4 56. Wang H, Kadlecek TA, Au-Yeung BB, Goodfellow HE, Hsu LY, Freedman TS, et al. REFERENCES Aguado E, Richelme S, Nunez-Cruz S, Miazek A, Mura AM, Richelme M, et al. Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science (2002) 296:2036–40. doi: 10.1126/science.1069057 75. Wange RL. LAT, the linker for activation of T cells: a bridge between T cell- speciﬁc and general signaling pathways. Sci STKE (2000) 2000:re1. doi: 10.1126/stke.2000.63.re1 95. Mingueneau M, Roncagalli R, Gregoire C, Kissenpfennig A, Miazek A, Archambaud C, et al. Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor. Immunity (2009) 31:197–208. doi: 10.1016/j.immuni.2009.05.013 76. Balagopalan L, Kortum RL, Coussens NP, Barr VA, Samelson LE. The linker for activation of T cells (LAT) signaling hub: from signaling complexes to microclusters. J Biol Chem (2015) 290:26422–9. doi: 10.1074/ jbc.R115.665869 96. Houtman JC, Houghtling RA, Barda-Saad M, Toda Y, Samelson LE. Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways. J Immunol (2005) 175:2449–58. doi: 10.4049/ jimmunol.175.4.2449 77. Ditlev JA, Vega AR, Koster DV, Su X, Tani T, Lakoduk AM, et al. A composition-dependent molecular clutch between T cell signaling condensates and actin. Elife (2019) 8. doi: 10.7554/eLife.42695 78. Su X, Ditlev JA, Hui E, Xing W, Banjade S, Okrut J, et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science (2016) 352:595–9. doi: 10.1126/science.aad9964 97. Arbulo-Echevarria MM, Vico-Barranco I, Narbona-Sanchez I, Garcia-Cozar F, Miazek A, Aguado E. Increased Protein Stability and Interleukin-2 Production of a LAT(G131D) Variant With Possible Implications for T Cell Anergy. Front Cell Dev Biol (2020) 8:561503. doi: 10.3389/fcell.2020.561503 79. Voisinne G, Kersse K, Chaoui K, Lu L, Chaix J, Zhang L, et al. Quantitative interactomics in primary T cells unveils TCR signal diversiﬁcation extent and dynamics. Nat Immunol (2019) 20:1530–41. doi: 10.1038/s41590-019- 0489-8 98. Genton C, Wang Y, Izui S, Malissen B, Delsol G, Fournie GJ, et al. The Th2 lymphoproliferation developing in LatY136F mutant mice triggers polyclonal B cell activation and systemic autoimmunity. J Immunol (2006) 177:2285–93. doi: 10.4049/jimmunol.177.4.2285 80. Roncagalli R, Hauri S, Fiore F, Liang Y, Chen Z, Sansoni A, et al. Quantitative proteomics analysis of signalosome dynamics in primary T cells identiﬁes the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub. Nat Immunol (2014) 15:384–92. doi: 10.1038/ni.2843 99. Wang Y, Kissenpfennig A, Mingueneau M, Richelme S, Perrin P, Chevrier S, et al. REFERENCES Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells. J Immunol (2008) 180:1565–75. doi: 10.4049/ jimmunol.180.3.1565 81. Mori D, Gregoire C, Voisinne G, Celis-Gutierrez J, Aussel R, Girard L, et al. The T cell CD6 receptor operates a multitask signalosome with opposite functions in T cell activation. J Exp Med (2021) 218. doi: 10.1084/ jem.20201011 100. Archambaud C, Sansoni A, Mingueneau M, Devilard E, Delsol G, Malissen B, et al. STAT6 deletion converts the Th2 inﬂammatory pathology afﬂicting Lat (Y136F) mice into a lymphoproliferative disorder involving Th1 and CD8 effector T cells. J Immunol (2009) 182:2680–9. doi: 10.4049/jimmunol.0803257 82. Fu G, Vallee S, Rybakin V, McGuire MV, Ampudia J, Brockmeyer C, et al. Themis controls thymocyte selection through regulation of T cell antigen receptor-mediated signaling. Nat Immunol (2009) 10:848–56. doi: 10.1038/ ni.1766 101. Chevrier S, Genton C, Malissen B, Malissen M, Acha-Orbea H. Dominant Role of CD80-CD86 Over CD40 and ICOSL in the Massive Polyclonal B Cell Activation Mediated by LAT(Y136F) CD4(+) T Cells. Front Immunol (2012) 3:27. doi: 10.3389/ﬁmmu.2012.00027 83. Lesourne R, Uehara S, Lee J, Song KD, Li L, Pinkhasov J, et al. Themis, a T cell-speciﬁc protein important for late thymocyte development. Nat Immunol (2009) 10:840–7. doi: 10.1038/ni.1768 102. Kortum RL, Rouquette-Jazdanian AK, Miyaji M, Merrill RK, Markegard E, Pinski JM, et al. A phospholipase C-gamma1-independent, RasGRP1-ERK- dependent pathway drives lymphoproliferative disease in linker for activation of T cells-Y136F mutant mice. J Immunol (2013) 190:147–58. doi: 10.4049/jimmunol.1201458 84. Johnson AL, Aravind L, Shulzhenko N, Morgun A, Choi SY, Crockford TL, et al. Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection. Nat Immunol (2009) 10:831– 9. doi: 10.1038/ni.1769 85. Gascoigne NR, Acuto O. THEMIS: a critical TCR signal regulator for ligand discrimination. Curr Opin Immunol (2015) 33:86–92. doi: 10.1016/ j.coi.2015.01.020 103. Wu L, Wei Q, Brzostek J, Gascoigne NRJ. Signaling from T cell receptors (TCRs) and chimeric antigen receptors (CARs) on T cells. Cell Mol Immunol (2020) 17:600–12. doi: 10.1038/s41423-020-0470-3 104. Dong R, Libby KA, Blaeschke F, Fuchs W, Marson A, Vale RD, et al. Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR). EMBO J (2020) 39:e104730. doi: 10.15252/embj.2020104730 86. Choi S, Warzecha C, Zvezdova E, Lee J, Argenty J, Lesourne R, et al. REFERENCES Phosphorylation of a Tyrosine Residue on Zap70 by Lck and Its Subsequent Binding via an SH2 Domain May Be a Key Gatekeeper of T Cell Receptor Signaling In Vivo. Mol Cell Biol (2016) 36:2396–402. doi: 10.1128/MCB.00165-16 49. Lo WL, Shah NH, Ahsan N, Horkova V, Stepanek O, Salomon AR, et al. Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT. Nat Immunol (2018) 19:733–41. doi: 10.1038/s41590-018-0131-1 70. Patel VP, Moran M, Low TA, Miceli MC. A molecular framework for two- step T cell signaling: Lck Src homology 3 mutations discriminate distinctly regulated lipid raft reorganization events. J Immunol (2001) 166:754–64. doi: 10.4049/jimmunol.166.2.754 50. Lo WL, Shah NH, Rubin SA, Zhang W, Horkova V, Fallahee IR, et al. Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination. Nat Immunol (2019) 20:1481–93. doi: 10.1038/s41590-019- 0502-2 71. Denny MF, Kaufman HC, Chan AC, Straus DB. The lck SH3 domain is required for activation of the mitogen-activated protein kinase pathway but April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org 9 T Cell Receptor Ligand Discrimination Lo and Weiss not the initiation of T-cell antigen receptor signaling. J Biol Chem (1999) 274:5146–52. doi: 10.1074/jbc.274.8.5146 not the initiation of T-cell antigen receptor signaling. J Biol Chem (1999) 274:5146–52. doi: 10.1074/jbc.274.8.5146 91. Zhang W, Sommers CL, Burshtyn DN, Stebbins CC, DeJarnette JB, Trible RP, et al. Essential role of LAT in T cell development. Immunity (1999) 10:323–32. doi: 10.1016/S1074-7613(00)80032-1 72. Rudd ML, Tua-Smith A, Straus DB. Lck SH3 domain function is required for T-cell receptor signals regulating thymocyte development. Mol Cell Biol (2006) 26:7892–900. doi: 10.1128/MCB.00968-06 92. Finco TS, Kadlecek T, Zhang W, Samelson LE, Weiss A. LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway. Immunity (1998) 9:617–26. doi: 10.1016/S1074-7613(00)80659-7 73. Rossy J, Williamson DJ, Gaus K. How does the kinase Lck phosphorylate the T cell receptor? Spatial organization as a regulatory mechanism. Front Immunol (2012) 3:167. doi: 10.3389/ﬁmmu.2012.00167 93. Sommers CL, Lee J, Steiner KL, Gurson JM, Depersis CL, El-Khoury D, et al. Mutation of the phospholipase C-gamma1-binding site of LAT affects both positive and negative thymocyte selection. J Exp Med (2005) 201:1125–34. doi: 10.1084/jem.20041869 74. Shah NH, Wang Q, Yan Q, Karandur D, Kadlecek TA, Fallahee IR, et al. An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor. Elife (2016) 5. doi: 10.7554/eLife.20105 94. REFERENCES THEMIS enhances TCR signaling and enables positive selection by selective inhibition of the phosphatase SHP-1. Nat Immunol (2017) 18:433–41. doi: 10.1038/ni.3692 105. Salter AI, Ivey RG, Kennedy JJ, Voillet V, Rajan A, Alderman EJ, et al. Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function. Sci Signal (2018) 11. doi: 10.1126/scisignal.aat6753 87. Paster W, Bruger AM, Katsch K, Gregoire C, Roncagalli R, Fu G, et al. A THEMIS:SHP1 complex promotes T-cell survival. EMBO J (2015) 34:393– 409. doi: 10.15252/embj.201387725 88. Kosugi A, Sakakura J, Yasuda K, Ogata M, Hamaoka T. Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts. Immunity (2001) 14:669–80. doi: 10.1016/S1074-7613(01) 00146-7 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 89. Liu C, Xu X, Han L, Wan X, Zheng L, Li C, et al. LRCH1 deﬁciency enhances LAT signalosome formation and CD8(+) T cell responses against tumors and pathogens. Proc Natl Acad Sci USA (2020) 117:19388–98. doi: 10.1073/ pnas.2000970117 Copyright © 2021 Lo and Weiss. 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. 90. Koonpaew S, Shen S, Flowers L, Zhang W. LAT-mediated signaling in CD4 +CD25+ regulatory T cell development. J Exp Med (2006) 203:119–29. doi: 10.1084/jem.20050903 April 2021 \| Volume 12 \| Article 673196 Frontiers in Immunology \| www.frontiersin.org 10
https://openalex.org/W2955650104	https://www.nature.com/articles/s41598-019-44362-5.pdf	English	null	Author Correction: Transcriptional Regulation on Aneuploid Chromosomes in Diverse Candida albicans Mutants	Scientific reports	2,019	cc-by	488	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Author Correction: Transcriptional Regulation on Aneuploid Chromosomes in Diverse Candida albicans Mutants xx OPEN Published: xx xx xxxx Christopher Tucker1, Soumyaroop Bhattacharya 2, Hironao Wakabayashi1, Christopher Tucker1, Soumyaroop Bhattacharya 2, Hironao Wakabayashi1, Stanislav Bellaousov1,Anatoliy Kravets1, Stephen L.Welle3, Jason Myers4, Jeffrey J. Christopher Tucker1, Soumyaroop Bhattacharya 2, Hironao Wakabayashi1, Stanislav Bellaousov1, Anatoliy Kravets1, Stephen L. Welle3, Jason Myers4, Jeffrey J. Hayes Michael Bulger5 & Elena Rustchenko1 Correction to: Scientific Reports https://doi.org/10.1038/s41598-018-20106-9, published online 26 January 2 The original version of this Article contained an error in the title of the paper, where the word “diverse” was incorrectly given as “divers”. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 1Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, USA. 2Division of Neonatology and Program in Pediatrics Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, USA. 3Department of Medicine and Center for Pediatric Biochemical Research, Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, USA. 4University of Rochester Genomic Research Center, Rochester, New York, USA. 5Center for Pediatric Biochemical Research, Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, USA. Christopher Tucker and Soumyaroop Bhattacharya contributed equally. Correspondence and requests for materials should be addressed to E.R. (email: elena_bulgac@urmc.rochester.edu) 1Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, USA. 2Division of Neonatology and Program in Pediatrics Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, USA. Author Correction: Transcriptional Regulation on Aneuploid Chromosomes in Diverse Candida albicans Mutants xx OPEN 3Department of Medicine and Center for Pediatric Biochemical Research, Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, USA. 4University of Rochester Genomic Research Center, Rochester, New York, USA. 5Center for Pediatric Biochemical Research, Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, USA. Christopher Tucker and Soumyaroop Bhattacharya contributed equally. Correspondence and requests for materials should be addressed to E.R. (email: elena_bulgac@urmc.rochester.edu) Scientific Reports \| (2019) 9:10019 \| https://doi.org/10.1038/s41598-019-44362-5 1
https://openalex.org/W3110668467	https://www.e3s-conferences.org/10.1051/e3sconf/202021707007/pdf	English	null	Modeling the competitive environment of the entrepreneurship cluster	E3S web of conferences	2,020	cc-by	5,405	Modeling the competitive environment of the entrepreneurship cluster Viktoriya Sevka1,, Dmitry Nekhaychuk2, Viktoriia Sihua1, and Andrei Shevchuk2 1Academy of Civil Engineering and Architecture, Derzhavina str., 2, 286123, Donetsk, Ukraine 2Sevastopol Branch of Plekhanov Russian University of Economics, Vakulenchuka Str., 29-4, Sevastopol, Russia Abstract. The paper aims to verify the hypothesis about cooperation expediency of different in scale and similar in activity entities in the form of the entrepreneurship cluster for ensuring energy needs of the region. The integral coefficients of economic development of the innovative component of industrial enterprises on energy activity, the financial component of communal enterprises for heat supply, the readiness of small enterprises operating in the energy industry for innovative transformations has been analysed. Modeling of the competitive environment of the entrepreneurship cluster allowed to reveal logical interrelation between the level of technological changes in industry and technological capabilities of communal and small enterprises and to define their sectoral positions. Corresponding author: v.g.sevka@donnasa.ru E3S Web of Conferences 217, 07007 (2020) ERSME-2020 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 https://doi.org/10.1051/e3sconf/202021707007 © 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 intensification of innovation activities against the background of transformations in the natural, resource, social, economic, cultural and technical spheres requires the adaptation of new organizational forms of activity. Intensification of globalization phenomena, energy independence and redistribution of energy resources for industrial, housing and communal consumption require research of the current condition of the competitive environment of industrial, communal and small enterprises and the possibility of their work as part of the entrepreneurial cluster. Research of problems in regional and spatial development, resource planning, evaluation of financial and innovative components, diagnostics of the enterprise innovation potential, cluster analysis, innovative activity, strategic planning and management of innovative development in a highly competitive environment in the context of regionalization and globalization are devoted to the work of national and foreign scientists. However, the theoretical basis and practical effectiveness evaluation of the cooperation of different scale enterprises that work to supply power to regional consumers, taking into account factors of innovation remain relevant. 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research The aggravation of economic relations in the communal sector, the increase in social tensions and difficulties in adapting foreign experience force enterprises to use innovative ways of cooperation with business structures, financial institutions and organizations that respond quickly and effectively solve complex problems in the industry. Increasing the number of partnerships, innovative approaches, programs, initiatives and projects that contribute to the social, economic and environmental well-being of large industrial cities should be the task of the state, local authorities, heads of communal enterprises. Increased attention to the problem of the interaction of globalization and regionalization causes the need for a specific formal or regional-spatial approach to improve the conditions of entrepreneurship (P.V. Shukanov, 2012). Experts note that it is advisable to use a variety of criteria to assess the impact of globalization and regionalization on the development of both individual and different ethno-cultural groups of the population while studying global socio-geographical processes of transformation of the world economy. Modern research suggests that this thesis can be applied to economic systems (enterprises), as well as to the totality of business entities (cluster and adjacent infrastructure) to identify the reasons for the decrease in work efficiency and to identify promising ways to solve problems (V.S. Bochko, 2015). Management experience of recent years clearly shows that the need for cooperation on the part of communal companies is constantly growing, because joining to the cluster means free access to experts, information and investment. The cluster form implies the creation of such an organizational and economic mechanism of strategic priorities of innovative development of territories, which is aimed at restoring financial stability, strengthening the technical sphere and achieving competitive advantages of communal enterprises in the territories where large business structures presents, taking into account the synergetic effect. V.P. Solovyov notes that «the points of growth» of innovation clusters are small enterprises. The beginning of their development is given by enterprises that are already successfully operating in the market (A.A. Melikhov, 2014). ) Research of transformation phenomena in the innovation environment and the process of evolution of cluster formations allows to determine the essence of the entrepreneurial cluster as an effective mechanism of market self-regulation for the implementation of entrepreneurial innovations to improve the competitiveness of the economy sectors within the framework of the chosen strategy of innovative development. * Corresponding author: v.g.sevka@donnasa.ru © 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/). https://doi.org/10.1051/e3sconf/202021707007 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research The decentralization of management decisions as the condition for the emergence of an entrepreneurial cluster, and the attraction to the priority sectors as a point of growth were identified as the main features of the entrepreneurial cluster. Entrepreneurial clustering is developed on the basis of the business cluster technology. Multidimensional cluster analysis and selective indicators of economic systems development point to the important role of the entrepreneurial cluster in the energy industry. Entrepreneurial cluster in the power industry is a united network of entities that product, supply of power services and provide the accompanying services. It is an economic system aimed to obtain the economic benefits and to achieve qualitative and quantitative indicators of the socially-oriented strategy of the state. At the same time, small entrepreneurship makes the role of integrating the conditions when participants enter into entrepreneurial cluster. Small entrepreneurship is also carry out the methods of direct and indirect state regulation while economic system is organized. y g In order to assess the level of innovative development of industrial, communal and small enterprises in the entrepreneurial cluster, it is proposed to apply the methodology of 2 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 https://doi.org/10.1051/e3sconf/202021707007 selecting the sector. It determines the industrial technological and organizational features of the development of innovation. As a result of the systematization of the analytical control points of the level of innovation activity, we will build the logic of the research according to the presented scheme (Figure 1). selecting the sector. It determines the industrial technological and organizational features of the development of innovation. As a result of the systematization of the analytical control points of the level of innovation activity, we will build the logic of the research according to the presented scheme (Figure 1). Fig. 1. Directions of Complex Assessment of Elements of Innovative Potential and Efficiency of Innovative Activity for the Enterprises in Entrepreneurial Cluster. 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research Group 1 Industrial enterprises for production, distribution and supply of energy resources Scientific and information level, innovation potential Technical and technological potential; technical and economic efficiency of innovative projects Enterprise's ability to implementations of a given function The level of economic adaptation of the complex of innovative measures The level of transfersize of the internal environment for enterprise Level of innovative activity of the enterprise Innovative reserves of reducing tariff for heat supply services Level of development of the financial component of thermal power enterprises The level of readiness of small business to implement pilot innovative projects in communal sector Business modeling relationships between «small business – communal enterprise»with contracting base Group 2 Communal companies providing heat supply services Group 3 Small enterprises providing public services of heat supply The object of estimate Entity of the activity The result of estimate The object of estimate The result of estimate Entity of the activity Level of innovative activity of the enterprise Scientific and information level, innovation potential Group 1 Industrial enterprises for production, distribution and supply of energy resources Group 1 Industrial enterprises for production, distribution and supply of energy resources Technical and technological potential; technical and economic efficiency of innovative projects Enterprise's ability to implementations of a given function Group 2 Communal companies providing heat supply services Level of development of the financial component of thermal power enterprises Business modeling relationships between «small business – communal enterprise»with contracting base The level of readiness of small business to implement pilot innovative projects in communal sector Fig. 1. Directions of Complex Assessment of Elements of Innovative Potential and Efficiency of Innovative Activity for the Enterprises in Entrepreneurial Cluster. 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research The first stage of the methodology involves the evaluation of innovative activities of industrial enterprises (Moskalenko V.P., 2009; Tishchenko O.M., 2008; Statistical Yearbook, 2014) for the production, distribution and supply of energy resources in following areas: g 1) The level of activity of innovative activity of the enterprise, which characterizes the organizational, social, technical and financial components ia R (innovative activity, the development of technosphere, personnel training, the introduction of new (low-waste, resource-saving, waste-free) technological processes, the cost of innovation activities and the development of technosphere, the cost of mastering the production of innovative 1) The level of activity of innovative activity of the enterprise, which characterizes the organizational, social, technical and financial components ia R (innovative activity, the development of technosphere, personnel training, the introduction of new (low-waste, resource-saving, waste-free) technological processes, the cost of innovation activities and the development of technosphere, the cost of mastering the production of innovative 3 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 https://doi.org/10.1051/e3sconf/202021707007 products, the introduction of innovative processes, organizational and marketing innovations); products, the introduction of innovative processes, organizational and marketing innovations); 2) The level of innovation transfersize at the enterprise, which characterizes the financial component tR (costs for external and internal research, acquisition and transfer of new technologies); g ); 3) Economic adaptation of innovative measures at the enterprise, which characteri R 3) Economic adaptation of innovative measures at the enterprise, which characterizes commercialization and financial component a R (market introduction of innovations, implementation of innovative products, financing innovation activities at their own expense, state, local budgets and extra-budgetary funds, domestic and foreign investors, loans and others). The results of the estimate of the system of special indicators of innovation activity of the first group of contractors, which may be part of the entrepreneurial cluster – iR , are presented in Table 1. Table 1. Rates of Innovative Development of Enterprises on Production, Distribution and Supply of Energy Resources (Prepared by the authors on the basis of Scientific and Innovative Activity in Ukraine). Table 1. Rates of Innovative Development of Enterprises on Production, Distribution and Supply of Energy Resources (Prepared by the authors on the basis of Scientific and Innovative Activity in Ukraine). Table 1. Rates of Innovative Development of Enterprises on Production, Distribution and Supply Energy Resources (Prepared by the authors on the basis of Scientific and Innovative Activity in Ukraine). The dynamics of the innovative component of the economic development of industrial enterprises for the production, distribution and supply of energy resources is presented in Figure 2. 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research Period Rates Growth Index ia R tR a R iR ia R tR a R iR P1 0,026 min 0,002 min 0,091 0,040 min Х Х Х Х P2 0,028 0,003 0,103 0,045 1,07 1,17 1,13 1,12 P3 0,038 0,005 0,091 min 0,045 1,39 1,64 0,88 1,00 P4 0,128 max 0,152 0,242 0,174 3,34 max 33,00 max 2,66 max 3,90 max P5 0,106 0,239 max 0,253 max 0,199 max 0,82 1,57 1,05 1,14 P6 0,049 0,080 0,126 0,085 0,46 min 0,34 min 0,50 min 0,43 min P7 0,070 0,096 0,163 0,110 1,43 1,20 1,29 1,29 x 0,064 0,082 0,153 0,010 1,22 5,56 1,07 1,27 The dynamics of the innovative component of the economic development of industrial enterprises for the production, distribution and supply of energy resources is presented in Figure 2. 4 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 https://doi.org/10.1051/e3sconf/202021707007 0.001 0.101 0.201 P1 P2 P3 P4 P5 P6 P7 Innovative activity rate The rate of innovation transfersize The economic adaptation rate The integral rate of the innovative component of the economic development of enterprises for the production and distribution of energy resourses Poly. (The integral rate of the innovative component of the economic development of enterprises for the production and distribution of energy resourses) The integral rate of the innovative component of the economic development of enterprises for the production and distribution of energy resourses Poly. (The integral rate of the innovative component of the economic developmen of enterprises for the production and distribution of energy resourses) The integral rate of the innovative component of the economic development of enterprises for the production and distribution of energy resourses Poly. (The integral rate of the innovative component of the economic developme of enterprises for the production and distribution of energy resourses) Fig. 2. Dynamics of the Innovative Component of the Economic Development of Industrial Enterprises for Production, Distribution and Supply of Energy Resources for the Research Period. Guided by the methodology of diagnostics of innovative potential of the industrial enterprise (Shipulina Yu.S., 2008), the law of proportional development, the principle of balance and proportionality of production placement in the region, signs of complex proportional development of types and branches of activity (Vasilenko V.M., 2014) we defined sufficiency levels of innovative development of the enterprises for production, distribution and supply of energy resources (Table 2). Table 2. 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research Assessment of the Adequacy of Economic Potential for the Implementation of Projects o Innovative Development of Industrial Enterprises for the Production, Distribution and Supply of Energy Resources (Prepared by the authors on the basis of the references (Shipulina Yu.S., 2008 Vasilenko V.M., 2014). Table 2. Assessment of the Adequacy of Economic Potential for the Implementation of Projects of Innovative Development of Industrial Enterprises for the Production, Distribution and Supply of Energy Resources (Prepared by the authors on the basis of the references (Shipulina Yu.S., 2008; Vasilenko V.M., 2014). min Value Indicator max Value Meaning 0.23 ≤ Нgen. ≤ 0.25 Absolute sufficiency 0.17 ≤ Нgen. ≤ 0.23 Normal sufficiency 0.11 ≤ Нgen. ≤ 0.17 Unstable state 0.05 ≤ Нgen. ≤ 0.11 Critical condition 0.00 ≤ Нgen. ≤ 0.05 Crisis condition The value of the integral coefficient of the innovative component of the economic development of industrial enterprises for the production, distribution and supply of energy resources indicates the transformation phenomena from the crisis to the unstable state of the internal environment of the enterprise. In particular, this trend was due to growth ratio of transversal and adaptation. The rate of innovation activity decreased slightly. During research period, energy companies failed to reach a mark that would characterize the current state of innovative development as normal. The polynomial dynamics curve of the integral rate of the innovative component of the economic development in industrial enterprises for the production and distribution of energy resources indicates has the tendency to increase the innovative activity of large 5 https://doi.org/10.1051/e3sconf/202021707007 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 energy enterprises and to the gradual improvement of the innovation macro-climate. Based on the results of the estimate of this indicator, we can make the conclusion about the level of technological changes in the industry. energy enterprises and to the gradual improvement of the innovation macro-climate. Based on the results of the estimate of this indicator, we can make the conclusion about the level of technological changes in the industry. The next step will be the analyzes the main financial results of activities that affect the tariff policy of heat supply enterprises. This will determine the readiness of the communal sector to innovative transformations and will be a stimulating motive for attracting small entrepreneurship on the basis of innovative management. 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research There is noted the existence of a relationship between the level of innovative development potential of the enterprise and financial resources for the implementation of innovative development strategy (V.M. Vasilenko, 2014; A.A. Melikhov, 2014; L.S. Zakharkina, 2011; S. Zahra, R. Sisodia, B. Matherne, 1999) in substantiating the aspects of strategic planning in the management of innovative development of industrial enterprises. Therefore, we define the integral rate that characterizes the readiness for innovative transformations, provided that financial development is sufficient (A.A. Trifilova, 2017). , p p ( , ) Analysis of the financial component involves the evaluation of the final results of the communal enterprises, which give an idea of the optimal ratio between income and expenditure, updating the problem of optimizing the structure of tariffs. There was defined the tendency to increase the costs of heat supply, that reflects the regional situation regarding the rise in price of services by 30.6 % in some industrial cities such as Gorlovka, Donetsk, Kramatorsk, Makeyevka, Mariupol. This fact is contradiction of social policy. In addition, the growth of the material component of the tariffs led to the deterioration of the financial and technical condition of the communal heating enterprises of Donetsk region. Tariffs did not cover the actual cost of heat supply services, and the level of compensation for the price of natural gas was 79.5 %, electricity – 51.6 % (Figure 3). Fig. 3. Structure of Tariffs of the Communal Enterprises of Heat Power Engineering in Donetsk Region (on the Left) in 2011 (P4) and Across the Country (on the Right) in 2014 (P6). The high material intensity of heat supply services in Donetsk region is evidenced by the index of operating costs per 1 mon.unit net income from services, which increased from 1.34 to 1.58 (17.9 %) (Figure4). Material Costs; 72,2% Salary; 16% Social Costs; 5,7% Amortisation; 2,9% Other Costs; 3,2% 53 9.5 3.4 7.9 17.8 6.6 1.8 Fuel (natural gas) Electricity Material Costs Amortisation Salary Social Costs Other Costs Fig. 3. Structure of Tariffs of the Communal Enterprises of Heat Power Engineering in Donetsk Region (on the Left) in 2011 (P4) and Across the Country (on the Right) in 2014 (P6). Fig. 3. Structure of Tariffs of the Communal Enterprises of Heat Power Engineering in Donetsk Region (on the Left) in 2011 (P4) and Across the Country (on the Right) in 2014 (P6). 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research The consumption of thermal energy (С)(line 7: line 5) 0.70 0.69 0.72 -1.43 4.35 2.86 7 https://doi.org/10.1051/e3sconf/202021707007 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 9. Material efficiency (С)(line 5 : line 7) 1.44 1.46 1.38 1.39 -5.48 -4.17 10. The level of compensation rate (average). %. including: 93.77 - 90.60 - - -3.38 for the population 59.3 63.5 51.7 7.08 -18.58 -12.8 for non-industrial consumer groups 98.8 - 86.2 - - -12.8 for industrial consumer groups 123.2 - 133.9 - - 8.69 11. Impact of changes in material costs m Q  . mon.units / GCal - 33.41 81.91 - - 145.2 12. The impact of changes in material efficiency m f Q  . mon.units / GCal - 4.93 18.15 - - 268.2 The results of the assessment show that for 1 mon.unit of giga-calorie’s production of heat accounts for 0.7 mon.unit cost of material costs in average. The results of factor analysis indicate that the increase in material costs leads to an increase in tariff income. As a result of changes in material efficiency tariff income is also increased. The next one we are going to determine the level of development of the financial component of communal enterprises of thermal power, taking into account the analysis of material consumption and material efficiency of thermal energy (Table 4). Table 4. Assessment of Parameters of Financial and Economic Growth for the Communal Enterprises of Heat Supply (Prepared by the authors on the basis of Table 3). Indicator Period P2 P3 P4 1. Relation coefficient between the net income from services’ sales and the costs of ordinary activities in the provision of services 0.72 0.69 0.61 2. Relation coefficient between operating income and operating expenses 0.23 0.09 0.19 3. Relation coefficient between financial income and expenses of ordinary activities 0.026 0.018 0.021 4. Relation coefficient between the financial result (loss) and the cost of the ordinary course of business -0.03 -0.21 -0.19 5. Relation coefficient between the volume of paid services and the costs of ordinary activities 0.86 0.75 0.72 6. Relation coefficient between the cost of maintaining social facilities and operating costs 0.0012 0.00082 0.0007 7. Profitability of services (ratio of financial results from ordinary activities (profit) to costs of ordinary activities) 0.006 0.00016 0.00035 8. Innovative potential of employees 0.20 0.21 0.26 9. Relation coefficient between depreciation costs and normal business expenses 0.03 0.03 0.028 10. 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research The high material intensity of heat supply services in Donetsk region is evidenced by the index of operating costs per 1 mon.unit net income from services, which increased from The high material intensity of heat supply services in Donetsk region is evidenced by the index of operating costs per 1 mon.unit net income from services, which increased from 1.34 to 1.58 (17.9 %) (Figure4). 6 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 https://doi.org/10.1051/e3sconf/202021707007 Fig. 4. Dynamics of Operating Costs per 1 mon.unit of the net Income from Realization of Heat Supply Services by the Communal Enterprises in Some Industrial Cities of Donetsk region. 1.34 1.41 1.58 1.81 1.99 2.32 1 P2 P3 P4 Total in Region Gorlovka Gorlovka2 Donetsk Donetsk3 Donetsk2 Kramatorsk Makeyevka Mariupol 2 per. Mov. Avg. (Total in Region) Poly. (Donetsk3) Fig. 4. Dynamics of Operating Costs per 1 mon.unit of the net Income from Realization of Heat Supply Services by the Communal Enterprises in Some Industrial Cities of Donetsk region. There are presented the analytical assessment of the level and dynamics of operating and material costs of heat supply enterprises (Table 3). There are presented the analytical assessment of the level and dynamics of operating and material costs of heat supply enterprises (Table 3). Table 3. Analysis of Material Consumption and Material Efficiency of Thermal Energy of Heat Supply Enterprises (Prepared by the authors on the basis of the data provided by Donetsk Regional State Administration and Head Department of Housing and Communal Services of Donetsk Region, communal enterprises’ reports). Indicator Period Growth Rate. % P2 P3 P4 P3/P2 P4/P3 P4/P2 1. Thermal energy. thous. GCal 6812.7 7265.9 7690.8 6.65 5.85 12.89 2. Cost (tariff) (C). mon.units / GCal 320.6 358.7 418.8 11.88 16.75 30.63 3. Net income from services’ sales. thous. mon.units 1632794.8 1848850.5 2043031 .8 13.23 10.50 25.12 4. Operating costs for 1 mon.unitof net income. mon.units (line 1 * line 2 : line 3) 1.34 1.41 1.58 5.22 12.06 17.91 5. Tariff revenue (С). thous. mon.units (line 1 * line 2) 2184151.6 2606278.3 3220907 19.33 23.58 47.47 6. Material costs for 1 unit. mon.units / GCal 223.2 246.4 302.5 10.39 22.77 35.53 7. General material costs. thous. mon.units (line 1 * line 6) 1520594.6 1790317.8 2326467 17.74 29.95 53.00 8. E3S Web of Conferences 217, 07007 (2020) ERSME-2020 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 https://doi.org/10.1051/e3sconf/202021707007 Table 4 shows that the integral coefficient of stability of financial and economic growth of the enterprises of heat supply of the Donetsk region was at the level of 0.35. Table 4 shows that the integral coefficient of stability of financial and economic growth of the enterprises of heat supply of the Donetsk region was at the level of 0.35. At the third stage, we will determine the level of readiness of small enterprises for working in the sphere of industry and services and for innovative transformations by the assessment the coefficient of innovation activity ia CSI .The results of the survey of innovation activities of small enterprises working in the field of industry and services, revealed the following levels of innovation activity (Table 5). Table 5. Initial Data for Assessment of the Integral Index of Readiness of Small Enterprises for Innovation Conductor in Communal Sector (Prepared by the authors on the basis of Official data of Donetsk Regional State Administration and Head Department of Housing and Communal Services of Donetsk Region). Indicator Characteristics of innovative activity of small enterprises during the research period Innovation Activity With Technological Innovations With Organizational Innovation In total 1562 (surveyed 24657) 2608 ia CSI Innovative 0.37 (surveyed 0.06) Innovative 0.63 (surveyed 0.11) Industry 885 (surveyed 10430) 983 ia CSI Innovative 0.47 (surveyed 0.08) Innovative 0.53 (surveyed 0.09) Services 677 (surveyed 14227) 1625 ia CSI Innovative 0.29 (surveyed 0.05) Innovative 0.71 (surveyed 0.11) Thus, small enterprises with organizational innovation have a higher rate of innovation than enterprises with technological innovation, especially in the service sector. Generation and consumption of energy resources in a rapidly growing competitive environment requires rapid response of energy enterprises to changes in the external environment to adapt effective innovation strategies. The most effective are economic competitive strategies based on competitive advantages and combining marketing, financial and technological resources. Foreign experience in the organization of interaction between technological and competitive strategies (S. Zahra, R. Sisodia, B. Matherne, 1999; P. L. Chang, Y. C. 2 Theoretical, Informational and Empirical, and Methodological Grounds of the Research Integral coefficient of stability of financial and economic growth feg ІC (taking into account material intensity and heat output) 0.38 0.34 0.34 ment of Parameters of Financial and Economic Growth for the Communal Enterprises of Heat Supply (Prepared by the authors on the basis of Table 3). 8 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 Chen, 1994) indicates the creation of eight strategic zones embodied in the region: 1) Low-tech environment (competition arises due to high-tech changes instead of incremental innovations); 2) Technologically «sleeping» environment (managed by competitor-leader and apply new (improved) products and technologies); 3) Environment with high speed of technological changes in the industry, low technological heterogeneity among key players and weak technological capabilities of the enterprise (emphasis on competitive advantages in the form of new technologies); 4) Increasing industrial environment with competitors differentiated by technological advantages. At the same time, low-tech competitors should have convincing data to deliver competitive competence to the market; 5) Environment is close to the second situation, but the difference lies in the ownership of the enterprise significant technological innovations in the industry, causing small technological changes; 6) Environment that continues the fifth situation (the company may face additional technological competitors in this low-technology market); 7) Environment in which each enterprise is a technologically capable entity; 9 https://doi.org/10.1051/e3sconf/202021707007 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 8) Environment in which strategic options are limited and enterprises unable to innovate at a high level take the position of followers. 3 Results Based on the results of a comprehensive assessment of the efficiency of innovative activity of energy companies in the region, economic and mathematical models are built taking into account the technological and organizational characteristics of the participants of the innovation and industry cluster (Table 6). Table 6. Source Data for Modeling (Prepared by the authors on the basis of Table 5). Table 6. Source Data for Modeling (Prepared by the authors on the basis of Table 5). Table 6. Source Data for Modeling (Prepared by the authors on the basis of Table 5). Technological Capabilities of Enterprises in the Region Depending on the Level of Technological Changes in the Industry Organizational Capabilities of Enterprises in the Region Depending on the Level of Technological Changes in the Industry Value Х Value Y Value Х Value Y 0.611 0.35 0.611 0.35 0.611 0.47 0.611 0.53 0.611 0.29 0.611 0.71 0.611 0.37 0.611 0.53 Sectoral affiliation of the identified characteristics make a logical relationship between the level of technological change in the industry on a scale of X (enterprises in production, distribution and supply of energy resources) and technological capabilities of the region's enterprises on the scale of Y (the type of enterprises 1 – communal enterprise of heat supply, type of enterprises 2 – small enterprises that operate in some field of industry and services) (Figure 5, 6). Fig. 5. Models of Competitive Environment of Enterprises in the Entrepreneurship Cluster – 1. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Technological Capabilities of Regional Enterprises The Level of Technological Changes in the Industry 3 ↓ 2 ↓ 4 ↓ 8 ↓ 1 ↑ 5 ↑ 7 ↓ 6 ↓ 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 The Level of Technological Changes in the Industry Fig. 5. Models of Competitive Environment of Enterprises in the Entrepreneurship Cluster – 1. Fig. 5. Models of Competitive Environment of Enterprises in the Entrepreneurship Cluster – 1. 10 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 https://doi.org/10.1051/e3sconf/202021707007 Fig. 6. Models of Competitive Environment of Enterprises in the Entrepreneurship Cluster – 2. 3 Results 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Organization Opportunities of Regional Enterprises The Level of Technological Changes in the Industry 5 ↑ 6 ↓ 4 ↓ 8 ↓ 7 ↓ 3 ↓ 2 ↓ 1 ↑ 4 Fig. 6. Models of Competitive Environment of Enterprises in the Entrepreneurship Cluster – 2. 4 Conclusions and recommendations Spatial models of regional cooperation of industrial, communal and small enterprises in the entrepreneurship cluster confirm the feasibility of this form of economic activity. When small enterprises are successfully integrated with technological innovation in a cluster, the potential to design activities from sector 3 to sector 4 is released. In the case of the integration of small enterprises with organizational innovation in the cluster design reaches sectors 7 and 8. This indicates the availability of innovative potential in the energy and service sectors and the possibility of its implementation through flexible forms of management. There was defined the basic indicators of developmentof entrepreneurship cluster on the base of main components of the innovation potential and innovation performance of three groups of enterprises. It include: the industrial enterprises on production, distribution and supply of energy resources (integral rate of innovative component of economic development of the industrial enterprises on production, distribution and consumption of energy resources; communal enterprises providing services in heat supply (integral coefficient of its readiness to provide services in heat supply and to innovative transformations out of financial conditions); small enterprises which provide services in heat supply (integral index of functioning of small enterprises in the entrepreneurshipcluster). Based on this, there was graphically built models of competitive environment of enterprises that makethe entrepreneurshipcluster in the regional space. This approach indicatesabout the high role and efficiency of innovative transformation in the communal sector. 11 11 https://doi.org/10.1051/e3sconf/202021707007 E3S Web of Conferences 217, 07007 (2020) ERSME-2020 References References 1. P.V. Shukanov, Geography and Tourism 18, 192-198 (2012) 2. V.S. Bochko, Economics of the Region 1(41), 39-52 (2015) 3. I.I. Umansky, Bulletin of Khmelnitsky National University 2, 132-136 (2011) 4. V. Holodkova, A. Mottaeva, T. Pokrovskaya, E3S Web of Conferences 164, 11043 (2020) https://doi.org/10.1051/e3sconf /202016411043 5. V.P. Moskalenko, Mechanism of Economic Regulation 2, 109-120 (2009) 6. O.M. Tishchenko, Bulletin of National University «Lviv Polytechnic» 640, 406-415 (2008) 7. Yu.S. Shipulina, Mechanism of Economic Regulation 3, 58-63 (2008) 8. V.M. Vasilenko, Regional Economy 184 (2014) 9. A.A. Melikhov, Bulletin of Dnipropetrovsk University 8(3), 29-37 (2014) 10. L.S. Zakharkina, Strategic Planning in the System of Management of Innovative Development of Engineering Enterprises 12, 167 (2011) 11. S. Zahra, R. Sisodia, B. Matherne, European Management Journal 17(2), 188-203 (1999) 12. P.L. Chang, Y.C. Chen, Fuzzy Sets and Systems 63(2), 131-139 (1994) 1. P.V. Shukanov, Geography and Tourism 18, 192-198 (2012) 2. V.S. Bochko, Economics of the Region 1(41), 39-52 (2015) 2. V.S. Bochko, Economics of the Region 1(41), 39-52 (2015) 3. I.I. Umansky, Bulletin of Khmelnitsky National University 2, 132-136 (2011) 4. V. Holodkova, A. Mottaeva, T. Pokrovskaya, E3S Web of Conferences 164, 11043 (2020) https://doi.org/10.1051/e3sconf /202016411043 5. V.P. Moskalenko, Mechanism of Economic Regulation 2, 109-120 (2009) 6. O.M. Tishchenko, Bulletin of National University «Lviv Polytechnic» 640, 406-415 (2008) 7. Yu.S. Shipulina, Mechanism of Economic Regulation 3, 58-63 (2008) 8. V.M. Vasilenko, Regional Economy 184 (2014) 9. A.A. Melikhov, Bulletin of Dnipropetrovsk University 8(3), 29-37 (2014) 10. L.S. Zakharkina, Strategic Planning in the System of Management of Innovative Development of Engineering Enterprises 12, 167 (2011) 11. S. Zahra, R. Sisodia, B. Matherne, European Management Journal 17(2), 188-203 (1999) 12. P.L. Chang, Y.C. Chen, Fuzzy Sets and Systems 63(2), 131-139 (1994) 12
https://openalex.org/W4384626396	https://link.springer.com/content/pdf/10.1007/s10549-023-07040-9.pdf	English	null	Oestrogen receptor low positive breast cancer: associations with prognosis	Breast cancer research and treatment	2,023	cc-by	7,642	Breast Cancer Research and Treatment (2023) 201:535–545 https://doi.org/10.1007/s10549-023-07040-9 Breast Cancer Research and Treatment (2023) 201:535–545 https://doi.org/10.1007/s10549-023-07040-9 ORIGINAL LABORATORY INVESTIGATION Keywords Breast cancer · Oestrogen receptor · ER · ER low positive · Prognosis · Endocrine treatment Keywords Breast cancer · Oestrogen receptor · ER · ER low positive · Prognosis · Endocrine treatment Oestrogen receptor low positive breast cancer: associations with prognosis Anette H. Skjervold1 · Marit Valla1,2 · Anna M. Bofin1 Received: 27 April 2023 / Accepted: 5 July 2023 / Published online: 18 July 2023 © The Author(s) 2023 * Anette H. Skjervold anette.skjervold@ntnu.no 2 Department of Pathology, St. Olav’s Hospital, Trondheim, Norway Abstract Purpose In this study of oestrogen receptor (ER) Low Positive breast cancers (BC) in three large cohorts of BC patients, we assess associations between levels of ER expression and tumour characteristics and prognosis.i Methods Cases were stratified into patients unlikely to have received adjuvant therapy according to treatment guidelines at time of diagnosis (before 1995), and those who could have received adjuvant therapy (diagnosed in 1995 or later). ER status was divided into < 1%; ≥ 1 < 10%; ≥ 10%. Results were correlated with time of diagnosis, histopathological grade, proliferation status, and molecular subtypes, using Pearson’s Chi-square test. For prognosis, hazard ratios and cumulative incidence of death from BC were used. Results Of the 1955 tumours, 65 (3.3%) were ER Low Positive (ER ≥ 1 < 10%). Overall, the highest proportion of ER Low Positive tumours was observed among Luminal B (HER2 +) subtype (9.4%) and grade 3 tumours (4.3%). The risk of death from BC was lower in ER Low Positive and ER ≥ 10% compared to ER-negative cases. Compared to patients diagnosed before 1995, women diagnosed in 1995 or later showed a higher proportion of ER Low Positive BCs, and their tumours were of smaller size, lower grade, and lower proliferative status. There was no significant difference in prognosis compared to those with ER ≥ 10% tumours. Conclusion Women with ER Low Positive tumours diagnosed in a time period when adjuvant therapy was available had tumours of smaller size, lower grade, and lower proliferative status, and similar prognosis to those with ER ≥ 10% compared to women diagnosed earlier. 1 Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway Introduction Breast cancer differs from most tumours because of its dependence on female sex hormones for development and growth [5]. Expression of ER by immunohistochemistry (IHC) is seen in more than 70% of BC tumours [6]. The ASCO/CAP and current national BC guidelines state that BC tumours with ≥ 1% positive staining tumour cell nuclei should be interpreted as ER + , and negative if < 1% of tumour cell nuclei express ER [2, 7]. However, the ASCO/ CAP Expert Panel states that data on the effect of endocrine therapy for cancers with ER ≥ 1 < 10% are limited. They sug- gest that samples with ER ≥ 1 < 10% should be reported as ER Low Positive, with a comment mentioning the limited data available on the therapeutic benefit of anti-hormonal treatment for this group of patients [2]. According to the St. Gallen 2019 Consensus Discussion on The Optimal Primary Breast Cancer Treatment, there is a need for better evalua- tion of ideal cut-off levels for the prescription of endocrine Oestrogen receptor (ER) status plays an essential role in clinical decision-making and predicting outcome and treat- ment response for breast cancer (BC) patients [1]. According to current guidelines [2], patients with ER-positive tumours are considered eligible for endocrine therapy. Patients with ER-negative tumours are more likely to benefit from chemo- therapy and generally have a poorer outcome than patients with ER-positive (ER +) tumours [3, 4]. :(0123 1 3456789) 3 Breast Cancer Research and Treatment (2023) 201:535–545 536 a previous study 909 of these tumours were classified according to histopathological type and grade and divided into molecular subtypes [12]. For one case ER status was missing, and this case was excluded from the present study, leaving 908 cases. After diagnosis, patients were followed until time of death from BC or death from other causes, or until December 31st, 2015. therapy for ER + tumours, particularly for ER Low Positive cases [8–10]. In this study we examined expression levels of ER in BC tumours and associations between ER status and time of diagnosis, and tumour characteristics such as histopathologi- cal grade, molecular subtypes, proliferation and prognosis, with emphasis on ER Low Positive tumours. Cohort 2: The second cohort comprises 34,221 women born between 1897 and 1977 and derives from the HUNT2 Study conducted between 1995 and 1997 in Nord-Trønde- lag County, Norway [13]. Introduction From attendance until Decem- ber 31st, 2009, 728 women were diagnosed with BC. Of these, 157 cases were already included in Cohort 1 and 57 were unavailable for subtyping. The remaining tumours (n = 514) from Cohort 2 were assigned histopathological type and grade and reclassified into molecular subtypes [14]. ER status was available for all 514 cases. After diag- nosis, these patients were followed until time of death from BC or death from other causes, or until December 31st, 2015. Statistical analyses Of the 1955 tumours included in this study, 1669 (85.4%) were classified as one of the luminal subtypes (Luminal A, Luminal B (HER2-), or Luminal B (HER2 +)). Of these, 1640 were ER positive (ER ≥ 1%). Of the 180 cases of Luminal B (HER2 +), seven (3.9%) cases were ER < 1%, 17 (9.4%) were ER Low Positive and 156 (86.7%) were ER ≥ 10% (p < 0.0001). Among the 937 cases with Lumi- nal A subtype, 10 (1.1%) cases were ER < 1%, 29 (3.1%) were ER Low Positive and 898 (95.8%) were ER ≥ 10%. Of the 552 Luminal B (HER2-) cases 12 (2.2%) cases were ER < 1%, 19 (3.4%) were ER Low Positive and 521 (94.4%) were ER ≥ 10%. Twenty-six cases with ER < 1% were classified as Luminal based on progesterone receptor (PR) positivity (Table 2). For the present study, we divided ER expression into three categories (< 1%; ≥ 1 < 10%; ≥ 10%) and studied associa- tions between ER expression and histopathological grade, molecular subtype, proliferation, and prognosis. Pearson’s chi square test was used to compare patient and tumour characteristics across categories of ER. In analyses of prognosis, we distinguished between women diagnosed before 1995 and women diagnosed in 1995 or later. This cut-off was used to approximate the gradual implementa- tion of adjuvant treatment in Norway [14, 16]. Cumulative incidence of death from BC was estimated, with death from other causes as competing events. Gray’s test was used to compare equality between cumulative incidence curves. Cox proportional hazard analyses were used to estimate hazard ratios (HR) of BC death with 95% confidence intervals (CI) within each diagnostic period, censoring at time of death from other causes. We adjusted for age, stage, histopatho- logical grade, and for these variables combined. No clear violations of proportionality were found in log-minus-log plots. Statistical analyses were performed using Stata/MP version 17 (StataCorp LP, College Station, Texas, USA). Study population This study comprises women from three population-based surveys conducted in Trøndelag County, Norway. Informa- tion on breast cancer incidence was obtained from the Can- cer Registry of Norway. Date of death, and/or emigration was obtained from the National Population Register and causes of death from the Norwegian Cause of Death Regis- try. Formalin-fixed, paraffin embedded (FFPE) tumour tis- sue from the primary tumours and corresponding pathology reports were retrieved from the Department of Pathology at St. Olav’s Hospital, Trondheim University Hospital, Norway (Fig. 1). Cohort 3: The third cohort includes 22,931 women born at E.C. Dahl’s Foundation, Trondheim, Norway between 1920 and 1966. During 52 years of follow-up (1961 to the end of 2012), a total of 870 women were diagnosed with BC. Among them, 598 were diagnosed at St Olav’s Hospital, and histopathological typing, grading and molecular subtyping were successful for 537 of these cases [15]. ER status was available for 533 of these cases. After diagnosis, patients were followed until time of death from BC or death from other causes, or until December 31st, 2015. Cohort 1: The cohort includes 25,727 women born 1886–1928 [11] invited to attend a population-based survey for the early detection of breast cancer conducted in Nord-Trøndelag County, Norway, between 1956 and 1959. During 47 years of follow-up (1961 to end of 2008), 1379 new BCs were registered among these women. In Fig. 1 Overview of the three cohorts of breast cancer patients included in the study Fig. 1 Overview of the three cohorts of breast cancer patients included in the study 1 3 3 537 Breast Cancer Research and Treatment (2023) 201:535–545 Results Tissue Microarray (TMA) paraffin blocks were made from the archival tumour tissue using the TissueArrayer Minicore with TMA Designer2 software (Alphelys). Three 1 mm in diameter tissue cylinders from the periphery of the FFPE primary tumours were transferred to TMA recipient blocks. TMA Sections (4 µm) were cut and IHC-staining for ER was carried out within four weeks after sectioning. Between cut- ting and staining, sections were stored at − 20 °C. Staining intensity was not quantified in this study. Molecular subtypes for all cases in all three cohorts were determined using IHC and in situ hybridization in lieu of gene expression analyses, and have been published previously [12, 14, 15]. The IHC markers including ER are shown in Table 1. Patient and tumour characteristics for the 1955 patients included in the present study are shown in Table 2. Mean age at diagnosis was 67.3 years (SD: 12.8) and mean fol- low-up after diagnosis was 9.9 years (SD: 7.3). By end of follow-up, 545 (27.9%) patients had died from BC and 588 (30.1%) died from other causes. Of the 1955 tumours, 315 (16.1%) were ER < 1%, 65 (3.3%) were ER Low Positive (ER ≥ 1 < 10%) and 1575 (80.6%) were ER ≥ 10%. Of the 545 deaths from BC, 129 (23.7%) cases were ER < 1%, 16 (2.9%) were ER Low Positive and 400 (73.4%) were ER ≥ 10%. ER categories, histopathological grade, proliferation, and histopathological type In this study, 287 (14.7%) tumours were grade 1, 1015 (51.9%) were grade 2 and 653 (33.4%) were grade 3. The highest proportion of ER Low Positive (28/653 (4.3%)) was observed among grade 3 tumours (p < 0.0001). Of the 1057 cases with Ki-67 < 15%, 74 (7.0%) were ER < 1%, 31 (2.9%) were ER Low Positive, and 952 (90.1%) were ER ≥ 10%. Of the 898 cases with Ki-67 ≥ 15%, 241 (26.8%) were ER < 1%, 34 (3.8%) were ER Low Positive, and 623 (69.4%) were ER ≥ 10% (p < 0.0001). Similarly, of the 459 cases with ≤ 2 mitoses/10 High power fields (HPF) (p25), 23 (5.0%) were ER < 1%, 9 (2.0%) were ER Low Positive and 427 (93.0%) were ER ≥ 10% (p < 0.0001). Whereas, of the 875 cases with > 13 mitoses/10 HPF (p75), 215 (24.6%) were ER < 1%, 40 (4.5%) were ER Low Positive, and 620 (70.9%) were ER ≥ 10% (p < 0.0001). Of the 65 ER Low Positive cases, 50/1507 (3.3%) were invasive Table 1 Algorithm for reclassification of breast cancers into molecu- lar subtypes [12] ER Oestrogen receptor, PR Progesterone receptor, HER2 Human epi- dermal growth factor receptor 2, CK5 Cytokeratin 5, EGFR Epider- mal growth factor receptor 1 Molecular subtype Classified by Luminal A ER + and/or PR + , HER2-, Ki-67 < 15% Luminal B (HER2-) ER + and/or PR + , HER2-, Ki-67 ≥ 15% Luminal B (HER2 +) ER + and/or PR + , HER2 + HER2 type ER-, PR-, HER2 + Five-negative phenotype ER-, PR-, HER2-, CK5-, EGFR- Basal phenotype ER-, PR-, HER2-, CK5 + and/or EGFR + Table 1 Algorithm for reclassification of breast cancers into molecu- lar subtypes [12] 1 3 Breast Cancer Research and Treatment (2023) 201:535–545 538 Table 2 Patient and tumour characteristics according to ER categories a NOS Not otherwise specified Total study population ER categories < 1% ≥ 1 < 10% ≥ 10% p value (χ2) N (%) 1955 315 (16.1) 65 (3.3) 1575 (80.6) Mean age at diagnosis, years (SD) 67.3 (12.8) 65.4 (14.0) 63.3 (13.9) 67.9 (12.4) Mean follow-up, years (SD) 9.9 (7.3) 8.4 (7.6) 10.3 (6.9) 10.2 (9.0) Alive Dec. a NOS Not otherwise specified ER categories and prognosis A total of 774 cases were diagnosed before 1995, and 1181 were diagnosed in 1995 or later. The distribution of cases according to time of diagnosis are shown in Table 3. Of women diagnosed before 1995, 352/774 (45.5%) died from BC during follow-up, as opposed to 193/1181 (16.3%) of those diagnosed in 1995 or later. Among women diagnosed before 1995, 152/774 (19.6%) tumours were ER < 1%, fall- ing to 163/1181 (13.8%) among women diagnosed in 1995 or later. Similarly, 16/774 (2.1%) tumours were ER Low Pos- itive before 1995, rising to 49/1181 (4.2%) in 1995 or later, and 606/774 (78.3%) cases diagnosed before 1995 were ER ≥ 10%, rising to 969/1181 (82.1%) among women diag- nosed in 1995 or later. Furthermore, we found that 310/774 (40.1%) of tumours diagnosed before 1995 were ≤ 2 cm in diameter, rising to 725/1181 (61.4%) for tumours diagnosed in 1995 or later (p < 0.0001) (Table 3). Cumulative incidence of death by BC according to ER status is shown in Fig. 2. The risk of death from BC for all cat- egories of ER expression was lower for women diagnosed in 1995 or later compared to women diagnosed before 1995 (Table 5). The cumulative risk of death from BC after 5 years, for women diagnosed before 1995, was 47.4% among cases with ER < 1%, 37.5% for cases with ER Low Positive and 20.8% for cases with ER ≥ 10%. Among women diagnosed with breast cancer in 1995 or later the cumulative risk of death from BC was 22.3% after 5 years for ER < 1%, and 8.3% for both the ER Low Positive and ER ≥ 10% group (Table 5). Thus, among patients diagnosed in 1995 or later, there was no clear difference in risk of death from BC between cases with ER Low Positive and ER > 10%. Cox regression analyses showed that the risk of death was lower among patients with ER ≥ 10%, compared to those with ER < 1%, both among patients diagnosed before 1995, and among patients diagnosed in 1995 or later. The Cox analysis shows a lower relative risk of death from BC among patients with ER ≥ 10% tumours, compared to ER < 1% both before and after 1995. We observed a tendency towards a lower relative risk of death from BC among ER Low Posi- tive, compared to ER < 1%. ER categories, histopathological grade, proliferation, and histopathological type Positive tumours, the proportion of Luminal A subtype rose from 25% in ER Low tumours diagnosed before 1995, to 51% when diagnosed in 1995 or later. The proportion of Luminal B (HER2 +) tumours was lower among the women diagnosed in 1995 or later (18.4%), than the women diag- nosed before 1995 (50%) (p = 0.037) (Table 4). ER categories, histopathological grade, proliferation, and histopathological type 31st 2015 (%) 822 (42.1) 102 (32.5) 34 (51.5) 686 (43.6) < 0.001 Deaths from breast cancer (%) 545 (27.9) 129 (41.0) 16 (24.6) 400 (25.4) Deaths from other causes or by the end of 2015 (%) 588 (30.1) 84 (26.7) 15 (23.1) 489 (31.1) Histopathological grade (%) I 287 (14.7) 13 (4.1) 6 (9.2) 268 (17.0) < 0.001 II 1015 (51.9) 73 (23.2) 31 (47.7) 911 (57.8) III 653 (33.4) 229 (72.7) 28 (43.1) 396 (25.1) Tumour size (%) ≤ 2 cm 1035 (52.9) 124 (39.4) 33 (50.8) 878 (55.8) < 0.001 > 2 cm, ≤ 5 cm 391 (20.0) 75 (23.8) 15 (23.1) 301 (19.1) > 5 cm 24 (1.2) 9 (2.9) 3 (4.6) 12 (0.8) Uncertain, but > 2 cm 161 (8.2) 44 (14.0) 7 (10.8) 110 (7.0) Uncertain 344 (17.6) 63 (20.0) 7 (10.8) 274 (17.4) Stage (%) I 881 (45.1) 113 (35.9) 25 (38.5) 743 (47.2) 0.010 II 708 (36.2) 137 (43.5) 26 (40.0) 545 (34.6) III 98 (5.0) 23 (7.3) 3 (4.6) 72 (4.6) IV 72 (3.7) 14 (4.4) 2 (3.1) 56 (3.6) Unknown 196 (10.0) 28 (8.9) 9 (13.9) 159 (10.1) Molecular subtype (%) Luminal A 937 (47.9) 10 (3.2) 29 (44.6) 898 (57.0) < 0.001 Luminal B (HER2-) 552 (28.2) 12 (3.8) 19 (29.2) 521 (33.1) Luminal B (HER2 +) 180 (9.2) 7 (2.2) 17 (26.2) 156 (9.9) HER2 type 108 (5.5) 108 (34.3) 0 (0.0) 0 (0.0) 5NP 53 (2.7) 53 (16.8) 0 (0.0) 0 (0.0) BP 125 (6.4) 125 (39.7) 0 (0.0) 0 (0.0) Histopathological subtype (%) Invasive carcinoma (NOSa) 1507 (77.1) 218 (69.2) 50 (76.9) 1239 (78.7) < 0.001 Lobular carcinoma 210 (10.7) 17 (5.4) 8 (12.3) 185 (11.8) Tubular carcinoma 6 (0.3) 0 (0.0) 0 (0.0) 6 (0.4) Mucinous carcinoma 65 (3.3) 2 (0.6) 0 (0.0) 63 (4.0) Medullary carcinoma 60 (3.1) 38 (12.1) 4 (6.2) 18 (1.1) Papillary carcinoma 39 (2.0) 5 (1.6) 0 (0.0) 34 (2.2) Metaplastic 18 (0.9) 15 (4.8) 1 (1.5) 2 (0.1) Other 50 (2.6) 20 (6.4) 2 (3.1) 28 (1.8) Ki-67 low/high (%) Ki-67 < 15% 1057 (54.1) 74 (23.5) 31 (47.7) 952 (60.4) < 0.001 Ki-67 ≥ 15% 898 (45.9) 241 (76.5) 34 (52.3) 623 (39.6) Mitoses/10 HPF, median (IQR p25, p75) 5 (2,13) 15 (7,29) 8 (4,17) 4 (1,10) Mitoses/10 HPF, quartiles (%) ≤ 2 459 (23.5) 23 (7.3) 9 (13.9) 427 (27.2) < 0.001 > 2, ≤ 5 275 (14.1) 23 (7.3) 6 (9.2) 246 (15.7) > 5, ≤ 13 342 (17.5) 54 (17.1) 10 (15.4) 278 (17.7) > 13 875 (44.9) 215 (68.3) 40 (61.5) 620 (39.5) Table 2 Patient and tumour characteristics according to ER categories 1 3 539 Breast Cancer Research and Treatment (2023) 201:535–545 carcinoma NOS, 8/210 (3.8%) were lobular carcinoma, 4/60 (6.6%) were medullary carcinoma, and 1/18 (5.5%) was metaplastic carcinoma (Table2). ER categories and prognosis However, these findings were not statistically significant (Table 5). Characteristics of ER low positive tumours The distribution of tumour characteristics in patients with ER Low tumours are shown in Table 4. There was a total of 65 (3.3%) ER Low Positive tumours in this study. Of these, 16 were diagnosed before 1995, and 49 was diag- nosed in 1995 or later. Among the ER Low Positive tumours diagnosed before 1995, 8/16 (50%) died from BC during follow-up, as opposed to 8/49 (16.3%) of those diagnosed in 1995 or later. Among ER Low tumours, the proportion of tumours < 2 cm, rose from 31% in patients diagnosed before 1995 to 57% in those diagnosed in 1995 or later (p < 0.0001). For all cases, there was a higher proportion of grade 1 tumours (17.2%), and a lower proportion of tumours with grade 3 (29.6%) among women diagnosed in 1995 or later, compared to women diagnosed before 1995 (Grade 1: 10.9%, Grade 3: 39.1% (p < 0.0001)). Among ER Low Posi- tive cases, there was a higher proportion of grade 1 (12.2%) and 2 (53.1%) tumours among women diagnosed in 1995 or later, compared to the women diagnosed before 1995 (grade 1: 0%, grade 2: 31.2%). For grade 3 tumours the proportion of ER low tumours was lower when diagnosed in 1995 or later (p = 0.04) (Table 4). The distribution of tumour characteristics in patients with ER Low tumours are shown in Table 4. There was a total of 65 (3.3%) ER Low Positive tumours in this study. Of these, 16 were diagnosed before 1995, and 49 was diag- nosed in 1995 or later. Among the ER Low Positive tumours diagnosed before 1995, 8/16 (50%) died from BC during follow-up, as opposed to 8/49 (16.3%) of those diagnosed in 1995 or later. Among ER Low tumours, the proportion of tumours < 2 cm, rose from 31% in patients diagnosed before 1995 to 57% in those diagnosed in 1995 or later (p < 0.0001). Discussion For all cases, there was a higher proportion of grade 1 tumours (17.2%), and a lower proportion of tumours with grade 3 (29.6%) among women diagnosed in 1995 or later, compared to women diagnosed before 1995 (Grade 1: 10.9%, Grade 3: 39.1% (p < 0.0001)). Among ER Low Posi- tive cases, there was a higher proportion of grade 1 (12.2%) and 2 (53.1%) tumours among women diagnosed in 1995 or later, compared to the women diagnosed before 1995 (grade 1: 0%, grade 2: 31.2%). For grade 3 tumours the proportion of ER low tumours was lower when diagnosed in 1995 or later (p = 0.04) (Table 4). In this study of 1955 primary BC tumours, we found that 65 (3.3%) tumours fell under the ER Low Positive category. We found the highest proportion of ER Low Positive among Luminal B (HER2 +) tumours (9.4%). Among cases diag- nosed before 1995, 2.1% were ER Low Positive rising to 4.2% among cases diagnosed in 1995 or later. We found an association between ER Low Positive and high histo- pathological grade, high Ki-67 levels and high mitotic count. However, the results did not show a significant association with prognosis. Breast cancer survival in Norway has increased since the mid-1990s as seen in the present and other studies [17]. This may be ascribed to earlier detection [18, 19] and improved treatment [6, 20]. The reduced risk of death observed between the two time-periods for all categories of ER expression, probably reflects earlier diagnosis with the For all cases, the proportion of Luminal A subtype was higher for women diagnosed in 1995 or later (52.5%) com- pared to those diagnosed before 1995 (41.0%). The propor- tion of Luminal B (HER2-) and HER2 subtypes was lower for women diagnosed in 1995 or later (p < 0.0001) (Table 3), compared to those diagnosed before 1995. Discussion Among ER Low 1 3 Breast Cancer Research and Treatment (2023) 201:535–545 540 Table 3 Patient and tumour characteristics among women diagnosed before 1995, or in 1995 and later Women diagnosed with BC before 1995 (%) p-value Women diagnosed with BC in 1995 or later (%) p-value Total cases (n) 774 1181 Cohort 1 (n = 908) 661 (72.7) 248 (27.3) Cohort 2 (n = 514) 0 (0.0) 514 (100.0) Cohort 3 (n = 533) 113 (21.2) 420 (78.8) Mean age at diagnosis (SD) 69.5 (10.4) 65.4 (14.3) Mean follow-up-time (SD) 10.9 (9.7) 9.2 (5.0) Deaths by BC (%) 352 (45.5) 0.104 193 (16.3) 0.001 Deaths from other causes or by the end of 2015 (%) 364 (47.0) 224 (19.0) Alive at end of follow-up (31st Dec 2015) 58 (7.5) 764 (64.7) Oestrogen receptor (%) < 1% (%) 152 (19.6) < 0.001 163 (13.8) < 0.001 ≥ 1 < 10% (%) 16 (2.1) 49 (4.2) ≥ 10% (%) 606 (78.3) 969 (82.1) Tumour size ≤ 2 cm (%) 310 (40.1) 0.023 725 (61.4) < 0.001 > 2 ≤ 5 cm (%) 64 (8.3) 327 (27.7) Tumour size > 5 cm (%) 3 (0.4) 21 (1.8) Uncertain, but > 2 cm (%) 148 (19.1) 13 (1.1) Uncertain (%) 249 (32.2) 95 (8.0) Stage 1 346 (44.7) 0.002 535 (45.3) 0.001 2 257 (33.2) 451 (38.2) 3 47 (6.1) 51 (4.3) 4 39 (5.0) 33 (2.8) Unknown 85 (11.0) 111 (9.4) Histopathological grade 1 84 (10.9) < 0.001 203 (17.2) < 0.001 2 387 (50.0) 628 (53.2) 3 303 (39.1) 350 (29.6) Histopathological type Invasive carcinoma (NOS) 566 (73.1) < 0.001 941 (79.7) < 0.001 Lobular carcinoma 96 (12.4) 114 (9.7) Mucinous carcinoma 27 (3.5) 38 (3.2) Medullary carcinoma 27 (3.5) 33 (2.8) Papillary carcinoma 21 (2.7) 18 (1.5) Metaplastic carcinoma 8 (1.0) 10 (0.9) Tubular carcinoma 2 (0.3) 4 (0.3) Other 27 (3.5) 23 (2.0) Molecular subtypes Luminal A 317 (41.0) < 0.001 620 (52.5) < 0.001 Luminal B (HER2-) 243 (31.4) 309 (26.2) Luminal B (HER2 +) 69 (8.9) 111 (9.4) HER2 type 63 (8.1) 45 (3.8) Five-negative phenotype 25 (3.2) 28 (2.4) Basal phenotype 57 (7.4) 68 (5.8) Mitoses/10 HPF, median (IQR p25, p75) 2 (7, 15) 4 (1, 10) Mitoses /10 HPF (%) ≤ 2 203 (26.2) < 0.001 256 (21.8) < 0.001 > 2, ≤ 5 140 (18.1) 135 (11.5) Table 3 Patient and tumour characteristics among women diagnosed before 1995, or in 1995 and later W di d ith BC l W di d Table 3 Patient and tumour characteristics among women diagnosed before 1995, or in 1995 and later 1 Breast Cancer Research and Treatment (2023) 201:535–545 541 NOS = Not otherwise specified, HPF = High Power Field Table 3 (continued) Women diagnosed with BC before 1995 (%) p-value Women diagnosed with BC in 1995 or later (%) p-value > 5, ≤ 13 202 (26.1) 140 (11.9) > 13 229 (29.6) 646 (54.9) Ki-67 < 15% 377 (48.7) < 0.001 680 (57.6) < 0.001 ≤ 15% 397 (51.3) 501 (42.4) NOS = Not otherwise specified, HPF = High Power Field lower proliferation compared to ER Low Positive tumours diagnosed before 1995. Discussion Thus, the improved prognosis may be attributed to factors other than endocrine treatment, such as earlier diagnosis due to the introduction of mammography screening and greater BC awareness among women. Deter- mining endocrine treatment for patients with a diagnosis of ER Low Positive BC should be carefully considered in light of the potential risks and benefits of the treatment [24]. introduction of mammography screening and the introduc- tion of adjuvant treatment therapies in the mid-1990s. The change in prognosis observed across time for patients with ER Low Positive tumours may also be attributed to adju- vant therapy other than antihormonal treatment in addition to changing tumour characteristics such as smaller tumour size and lower histopathological grade. However, a draw- back of the present study was lack of availability of disease- free survival data. i In the present study, the proportion of Luminal A tumours was higher among women diagnosed in the time period dur- ing which adjuvant treatment and earlier diagnosis became available, a finding previously observed by our group in an analysis of cohorts 1 and 2 [14]. It has been suggested that BC patients with ER Low Positive are more similar to the ER-negative group, and therefore may not profit from endo- crine therapy [9]. Thus, it has been suggested that cut-off levels should be further investigated in order to offer BC patients personalized endocrine treatment [22, 29, 30]. In the present study we found that among cases diagnosed in 1995 or later, ER Low Positive cases showed a prognosis similar to that of ER ≥ 10% cases. However, the impact of hormonal therapy could not be assessed in this study, due to lack of individual information on treatment.i ER status is an important indicator of prognosis and a predictor of the effect of endocrine treatment. ER signalling is a main driver of proliferation in ER Positive BCs, and inhibition of ER signalling has improved survival among ER Positive BC patients [6, 21]. Studies suggest that selec- tion of patients for endocrine therapy may need to be further personalized [9, 22, 23]. While most ER + BCs have high IHC scores, about 2–3% of cases are ER Low Positive [10, 24, 25]. In the present study, 3.3% of the total number of cases were ER Low Positive. Discussion It has been shown that antigenicity is, for the most part, preserved in 1 3 1 Breast Cancer Research and Treatment (2023) 201:535–545 542 Table 4 Patient and tumour characteristics among patients with ER Low Positive (≥ 1 < 10%) diagnosed before 1995, and in 1995 or later Women diagnosed with BC 5 % Women diagnosed with BC in 5 % p-value paraffin blocks over decades but may decrease in sections Norway, the Cause of Death Registry and the Norwegian Table 4 Patient and tumour characteristics among patients with ER Low Positive (≥ 1 < 10%) diagnosed before 1995, and in 1995 or later Women diagnosed with BC before 1995 (%) Women diagnosed with BC in 1995 or later (%) p-value Total cases (n) 16 49 Mean age at diagnosis (SD) 66.9 (12.8) 62.2 (14.2) Mean follow-up-time (SD) 10.8 (11.5) 10.2 (4.7) Deaths from breast cancer (%) 8 (50.0) 8 (16.3) < 0.001 Deaths from other causes or by the end of 2015 (%) 7 (43.7) 8 (16.3) Alive at end of follow-up 1 (6.3) 33 (67.4) Tumour size ≤ 2 cm (%) 5 (31.2) 28 (57.1) < 0.001 > 2 ≤ 5 cm (%) 1 (6, 3) 14 (28.6) Tumour size > 5 cm (%) 0 (0.0) 3 (6.1) Uncertain, but > 2 cm (%) 6 (37.5) 1 (2.0) Uncertain (%) 4 (25.0) 3 (6.1) Stage 1 5 (31.3) 20 (40.8) 0.001 2 2 (12.5) 24 (49.0) 3 2 (12.5) 1 (2.0) 4 2 (12.5) 0 (0.0) Unknown 5 (31.3) 4 (8.2) Histopathological grade 1 0 (0.0) 6 (12.2) 0.041 2 5 (31.2) 26 (53.1) 3 11 (68.8) 17 (34.7) Molecular subtypes Luminal A 4 (25.0) 25 (51.0) 0.037 Luminal B (HER2-) 4 (25.0) 15 (30.6) Luminal B (HER2 +) 8 (50.0) 9 (18.4) HER2 type 0 (0.0) 0 (0.0) 5NP 0 (0.0) 0 (0.0) BP 0 (0.0) 0 (0.0) Mitoses/10 HPF, median (IQR p25, p75) 9.5 (5, 16.5) 8 (2, 17) Mitoses /10 High power field (HPF) p25 = 4, p50 = 8, p75 = 17 (ER Low) ≤ 4/10 HPF 4 (25.0) 8 (16.3) 0.047 > 4 ≤ 8/10 HPF 3 (18.7) 5 (10.2) > 8 ≤ 17/10 HPF 5 (31.3) 5 (10.2) > 17/10 HPF 4 (25.0) 31 (63.3) Ki-67 < 15% 5 (31.2) 26 (53.1) 0.129 ≤ 15% 11 (68.8) 23 (46.9) Norway, the Cause of Death Registry and the Norwegian Patient register [36, 37] thus enabling comparability within the study population across time. 1 3 Discussion While these tumours are clas- sified within the ER + category, their risk profile appears to be more like that of ER-negative breast cancers [24]. A recent study found no benefit of endocrine therapy in the ER < 10% group compared to the ER > 10% group [25]. The lack of benefit of endocrine therapy in patients with low ER expression has recently been shown in a meta-analysis, including more than 16,000 patients [26]. The meta-analysis indicated that primary BC patients with ER 1–9% gained no significant survival benefit from endocrine therapy, but manifested better overall prognosis than patients with can- cers expressing ER < 1% [26]. In the present study, among patients diagnosed in 1995 or later, the ER Low Positive patient group had similar survival to those with ER ≥ 10%. The patients included in this study were diagnosed with BC between 1961 and 2012, and the ER > 1% cut-off level for endocrine treatment was first introduced in Norway in 2011 after recommendations from ASCO/CAP [27]. There- fore, the improved prognosis seen among ER Low Positive patients diagnosed in 1995 or later, can most likely not be attributed to endocrine treatment [28]. Among women diag- nosed in 1995 or later, we found a greater proportion of ER Low Positive tumours with smaller size, lower grade, and Similar to our findings, a recent study showed that ER Low Positive tumours were more frequently grade 3 and had a higher expression of Ki-67, compared to BCs with intermediate or high expression of ER [31]. Furthermore, they found that the expression of immune-related biomark- ers in ER Low Positive was similar to that of ER-negative tumours. We observed four cases of medullary carcinoma and one metaplastic carcinoma among the ER Low Posi- tive cases. When determining treatment for patients with ER Low Positive BC, it may be useful to consider including a panel of immune-related biomarkers. The FFPE tumour tissue included in this study covered a diagnostic timespan of several decades, and preanalytical conditions may have varied over time. Many of the tumours were diagnosed at a time when ER IHC was not done in the diagnostic setting. However, valuable information can be drawn from archival tissue blocks [32, 33]. Discussion paraffin blocks over decades but may decrease in sections stored over time, resulting in weaker staining [33–35]. We observed no apparent trend towards a negative result among the older specimens but felt it would be unwise to attempt to quantify staining intensity due to the varying preanalytical conditions over which we had no control. Norway, the Cause of Death Registry and the Norwegian Patient register [36, 37] thus enabling comparability within the study population across time. Using TMA sections enables us to stain hundreds of tumour samples at the same time, under the same condi- tions. The samples comprise a small amount of the original tumour tissue samples, compared to full-face sections. Thus, some important information from the tumour may be lost. However, it has been shown that IHC for ER carried out Other strengths of this study include reliable information on BC incidence and follow-up data that were available from high-quality national registries like the Cancer Registry of 1 3 Breast Cancer Research and Treatment (2023) 201:535–545 543 Fig. 2 Cumulative incidence of death from breast cancer according to oestrogen receptor (ER) levels. A Women diagnosed with BC before 1995. B Women diagnosed with BC in 1995 or later. Gray’s test: p < 0.0001 Fig. 2 Cumulative incidence of death from breast cancer according to oestrogen receptor (ER) levels. A Women diagnosed with BC before 1995. B Women diagnosed with BC in 1995 or later. Discussion Gray’s test: p < 0.0001 Table 5 Absolute and relative risk of death from breast cancer according to ER levels, and breast cancer diagnosis ater Table 5 Absolute and relative risk of death from breast cancer according to ER levels, and breast cancer diagnosis before 1995 and in 1995 or later ER Oestrogen receptor, HR Hazard ratio, CI confidence interval ER levels, diagnosis before 1995 ER levels, diagnosis in 1995 or later < 1% ≥ 1 < 10% ≥ 10% < 1% ≥ 1 < 10% ≥ 10% Cumulative risk after 5 years (%) (95% CI) 47.4 (39.8–55.6) 37.5 (18.9–65.1) 20.8 (17.8–24.3) 22.3 (16.6–29.5) 8.3 (3.2–20-5) 8.3 (6.8–10.3) Cumulative risk after 10 years (%) (95% CI) 51.3 (43.7–59.5) 43.8 (23.8–70.5) 31.4 (27.8–35.2) 28.5 (22.1–36.3) 16.7 (8.2–32.2) 13.8 (11.7–16.3) HR unadjusted (95% CI) 1.0 0.8 (0.4–1.6) 0.6 (0.5–0.7) 1.0 0.5 (0.2–1.0) 0.5 (0.3–0.6) HR adjusted for age (95% CI) 1.0 0.7 (0.3–1.8) 0.6 (0.4–0.8) 1.0 0.6 (0.3–1.3) 0.4 (0.3–0.6) HR adjusted for stage (95% CI) 1.0 0.8 (0.3–1.9) 0.6 (0.4–0.7) 1.0 0.6 (0.3–1.2) 0.4 (0.3–0.6) HR adjusted for grade (95% CI) 1.0 0.7(0.4–1.6) 0.7 (0.5–0.9) 1.0 0.6 (0.3–1.2) 0.6 (0.4–0.8) HR adjusted for age, stage, and grade (95% CI) 1.0 0.7 (0.3–1.8) 0.7 (0.5–1.0) 1.0 0.9 (0.4–1.9) 0.5 (0.3–0.8) e and relative risk of death from breast cancer according to ER levels, and breast cancer diagnosis before 1995 and more frequently Luminal A Women with ER Low Positive tumours had similar prognosis to patients with ER ≥ 10% when diagnosed in 1995 or later. on sections from TMAs can provide equivalent information regarding clinical endpoint when compared to IHC on full- face tissue Sections [38, 39]. Immunohistochemistry for ER on full-face tissue sections was not carried out in the present study. Acknowledgements The authors would like to thank the Department of Pathology at St. Olav´s Hospital, Trondheim University Hospital for making the diagnostic archives available for this project and the Cancer Registry of Norway for supplying the corresponding patient data. Conclusion Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by all authors. The first draft of the manuscript was written by Anette H Skjervold. All authors commented on previous versions of the manuscript, and read and approved the final manuscript. Overall, ER Low Positive BCs exhibited many character- istics similar to ER-negative tumours and were frequently Luminal B (HER2 +). Among women diagnosed in 1995 or later, the proportion of ER Low Positive BCs was higher than among women diagnosed before 1995 and ER Low Positive tumours diagnosed in 1995 or later were of smaller size, lower grade, lower proliferative status, and were Funding Open access funding provided by NTNU Norwegian Univer- sity of Science and Technology (incl St. Olavs Hospital - Trondheim University Hospital). This present study has received funding from the Department of Clinical and Molecular Medicine, Norwegian University 1 3 3 Breast Cancer Research and Treatment (2023) 201:535–545 544 av pasienter med brystkreft https://www.helsedirektoratet.no/ retningslinjer/brystkreft-handlingsprogram: Helsedirektoratet, avdeling spesialisthelsetjenester; [updated 08/2020. IS-2945]. https://www.helsedirektoratet.no/retningslinjer/brystkreft-handl ingsprogram. of Science and Technology, Trondheim, Norway. Data included in this study received financial support from the Liaison Committee between the Central Norway Regional Health Authority and the Norwegian University of Science and Technology and The Research Council of Norway. of Science and Technology, Trondheim, Norway. Data included in this study received financial support from the Liaison Committee between the Central Norway Regional Health Authority and the Norwegian University of Science and Technology and The Research Council of Norway. 8. 8. Balic M, Thomssen C, Würstlein R, Gnant M, Harbeck N (2019) St Gallen/Vienna 2019: a brief summary of the consensus dis- cussion on the optimal primary breast cancer treatment. Breast Care 14(2):103–110 Data availability The datasets generated and/or analysed during this study are not publicly available due to issues of sensitivity and limita- tions determined in the conditions for approval by the Regional Com- mittee for Medical and Health Research Ethics. However, the data may be made available from the corresponding author on reasonable request. 9. Yu KD, Cai YW, Wu SY, Shui RH, Shao ZM (2021) Estrogen receptor-low breast cancer: biology chaos and treatment para- dox. Cancer Commun (Lond) 41(10):968–980 dox. Cancer Commun (Lond) 41(10):968–980 10. Fei F, Siegal GP, Wei S (2021) Characterization of estrogen receptor-low-positive breast cancer. Breast Cancer Res Treat 188(1):225–235 Declarations 11. Kvâle G, Heuch I, Eide GE (1987) A prospective study of repro- ductive factors and breast cancer: I—parity. Am J Epidemiol 126(5):831–841 Competing interest The authors declare that they have no competing interests. 12. Engstrom MJ, Opdahl S, Hagen AI, Romundstad PR, Akslen LA, Haugen OA et al (2013) Molecular subtypes, histopathological grade and survival in a historic cohort of breast cancer patients. Breast Cancer Res Treat 140(3):463–473 Ethical approval This study and publication of its results was granted approval by the Regional Committee for Medical and Health Research Ethics, Central Norway (REK 836-09). The approval includes dispen- sation from the usual requirement of patient consent. 13. Holmen J (2011) The Nord-Trøndelag health study 1995–97 (HUNT 2). Norsk Epidemiologi. https://doi.org/10.5324/nje. v13i1.305 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 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/. 14. Valla M, Vatten LJ, Engstrom MJ, Haugen OA, Akslen LA, Bjorngaard JH et al (2016) Molecular subtypes of breast cancer: long-term incidence trends and prognostic differences. Cancer Epidemiol Biomarkers Prev 25(12):1625–1634j 15. Sandvei MS, Opdahl S, Valla M, Lagiou P, Vesterfjell EV, Rise TV et al (2021) The association of women’s birth size with risk of molecular breast cancer subtypes: a cohort study. BMC Cancer 21(1):299 16. Research-based evaluation of The Norwegian Breast Cancer Screening Program [press release] (2015). Oslo, Norway. 17. Norway CRo (2022) Cancer in Norway 2021—cancer incidence, mortality, survival and prevalence in Norway. Oslo. 18. Hofvind S, Ursin G, Tretli S, Sebuodegard S, Moller B (2013) Breast cancer mortality in participants of the Norwegian breast cancer screening program. Cancer 119(17):3106–3112 References 19. Weedon-Fekjaer H, Romundstad PR, Vatten LJ (2014) Modern mammography screening and breast cancer mortality: population study. BMJ 348:g3701f 1. Regan MM, Viale G, Mastropasqua MG, Maiorano E, Golouh R, Carbone A et al (2006) Re-evaluating adjuvant breast cancer tri- als: assessing hormone receptor status by immunohistochemical versus extraction assays. J Natl Cancer Inst 98(21):1571–1581 20. Early Breast Cancer Trialists’ Collaborative G (1988) Effects of adjuvant tamoxifen and of cytotoxic therapy on mortality in early breast cancer: an overview of 61 randomized trials among 28,896 women. N Engl J Med 319(26):1681–1692 2. Allison KH, Hammond MEH, Dowsett M, McKernin SE, Carey LA, Fitzgibbons PL et al (2020) Estrogen and progesterone receptor testing in breast cancer: ASCO/CAP guideline update. J Clin Oncol 38(12):1346–1366 21. ATAC The (Arimidex TAoiCTG) (2002) Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early breast cancer: first results of the ATAC randomised trial. Lancet 359(9324):2131–2139 3. Lippman ME, Allegra JC, Thompson EB, Simon R, Barlock A, Green L et al (1978) The relation between estrogen receptors and response rate to cytotoxic chemotherapy in metastatic breast cancer. N Engl J Med 298(22):1223–1228 22. Scabia V, Ayyanan A, De Martino F, Agnoletto A, Battista L, Laszlo C et al (2022) Estrogen receptor positive breast cancers have patient specific hormone sensitivities and rely on progester- one receptor. Nat Commun 13(1):3127 g 4. Barrios CH, Sampaio C, Vinholes J, Caponero R (2009) What is the role of chemotherapy in estrogen receptor-positive, advanced breast cancer? Ann Oncol 20(7):1157–1162 23. Schrodi S, Braun M, Andrulat A, Harbeck N, Mahner S, Kiechle M et al (2021) Outcome of breast cancer patients with low hor- mone receptor positivity: analysis of a 15-year population-based cohort. Ann Oncol 32(11):1410–1424 5. Clemons M, Goss P (2001) Estrogen and the risk of breast can- cer. N Engl J Med 344(4):276–285 6. Early Breast Cancecr Trialists’ Collaborative G, Davies C, Godwin J, Gray R, Clarke M, Cutter D et al (2011) Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet 378(9793):771–784 24. Fusco N, Ragazzi M, Sajjadi E, Venetis K, Piciotti R, Morganti S et al (2021) Assessment of estrogen receptor low positive status in breast cancer: implications for pathologists and oncologists. Histol Histopathol 36(12):1235–1245 ( ) 7. References Helsedirektoratet NBCGN (2020) Nasjonalt handlingsprogram med retningslinjer for diagnostikk, behandling og oppfølging 1 3 545 Breast Cancer Research and Treatment (2023) 201:535–545 research studies using immunohistochemistry. Mod Pathol 17(11):1414–1420 25. Kim MC, Park MH, Choi JE, Kang SH, Bae YK (2022) Charac- teristics and prognosis of estrogen receptor low-positive breast cancer. J Breast Cancer 25(4):318–326f 33. Grillo F, Bruzzone M, Pigozzi S, Prosapio S, Migliora P, Fiocca R et al (2017) Immunohistochemistry on old archival paraffin blocks: is there an expiry date? J Clin Pathol 70(11):988–993 ( ) 26. Chen T, Zhang N, Moran MS, Su P, Haffty BG, Yang Q (2018) Borderline ER-positive primary breast cancer gains no significant survival benefit from endocrine therapy: a systematic review and meta-analysis. Clin Breast Cancer 18(1):1–8 34. Dowsett T, Verghese E, Pollock S, Pollard J, Heads J, Hanby A et al (2014) The value of archival tissue blocks in understanding breast cancer biology. J Clin Pathol 67(3):272–275f y 27. Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S et al (2010) American society of clinical oncology/col- lege of American pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone recep- tors in breast cancer. Arch Pathol Lab Med 134(6):907–922 35. Karlsson C, Karlsson MG (2011) Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides. J Histochem Cytochem 59(12):1113–1121 36. Larsen IK, Smastuen M, Johannesen TB, Langmark F, Parkin DM, Bray F et al (2009) Data quality at the cancer registry of Norway: an overview of comparability, completeness, validity and timeli- ness. Eur J Cancer 45(7):1218–1231 28. Ogawa Y, Moriya T, Kato Y, Oguma M, Ikeda K, Takashima T et al (2004) Immunohistochemical assessment for estrogen recep- tor and progesterone receptor status in breast cancer: analysis for a cut-off point as the predictor for endocrine therapy. Breast Cancer 11(3):267–275 37. Bakken IJ, Ellingsen CL, Pedersen AG, Leistad L, Kinge JM, Ebbing M et al (2015) Comparison of data from the cause of death registry and the norwegian patient register. Tidsskr Nor Laege- foren 135(21):1949–1953 29. Iwamoto T, Booser D, Valero V, Murray JL, Koenig K, Esteva FJ et al (2012) Estrogen receptor (ER) mRNA and ER-related gene expression in breast cancers that are 1% to 10% ER-positive by immunohistochemistry. J Clin Oncol 30(7):729–734 38. References Torhorst J, Bucher C, Kononen J, Haas P, Zuber M, Köchli OR et al (2001) Tissue microarrays for rapid linking of molecular changes to clinical endpoints. Am J Pathol 159(6):2249–2256 30. Fujii T, Kogawa T, Dong W, Sahin AA, Moulder S, Litton JK et al (2017) Revisiting the definition of estrogen receptor posi- tivity in HER2-negative primary breast cancer. Ann Oncol 28(10):2420–2428 39. Rosen DG, Huang X, Deavers MT, Malpica A, Silva EG, Liu J (2004) Validation of tissue microarray technology in ovarian carcinoma. Mod Pathol 17(7):790–797 31. Voorwerk L, Sanders J, Keusters MS, Balduzzi S, Cornelissen S, Duijst M et al (2023) Immune landscape of breast tumors with low and intermediate estrogen receptor expression. NPJ Breast Cancer 9(1):39 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 32. Mirlacher M, Kasper M, Storz M, Knecht Y, Durmuller U, Simon R et al (2004) Influence of slide aging on results of translational 1 3 1 3 3
https://openalex.org/W2123351013	https://findresearcher.sdu.dk/ws/files/129581069/The_potential_role_of_Alu_Y_in_the_development_of_resistance_to_SN38_Irinotecan_or_oxaliplatin_in_colorectal_cancer.pdf	English	null	The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer	BMC genomics	2,015	cc-by	12,812	Download date: 24. Oct. 2024 University of Southern Denmark RESEARCH ARTICLE Open Access Abstract Background: Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown. Results: We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of ‘DNA methylation entropy’ to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely re- sults from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene re- gions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples. Conclusion: Our findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy. Keywords: Alu, DNA methylation, Colorectal cancer, SN38 and oxaliplatin resistance, Diversity, Cell fate dynamics * Correspondence: cla@ki.au.dk; jili@biomed.au.dk; bolund@biomed.au.dk; nbr@sund.ku.dk †Equal contributors 3Department of Molecular Medicine, Aarhus University Hospital, Brendstrupgårdsvej 100, DK-8200 Aarhus N, Denmark 1Department of Biomedicine, University of Aarhus, the Bartholin Building, DK-8000 Aarhus C, Denmark 2Department of Veterinary Disease Biology, Section of Molecular Disease Biology, Faculty of Health and Medical Sciences, Copenhagen University, Strandboulevarden 49, Copenhagen, Denmark Full list of author information is available at the end of the article 3Department of Molecular Medicine, Aarhus University Hospital, © 2015 Lin et al.; licensee BioMed Central. The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer Lin, Xue; Stenvang, Jan; Rasmussen, Mads Heilskov; Zhu, Shida; Jensen, Niels Frank; Tarpgaard, Line S; Yang, Guangxia; Belling, Kirstine; Andersen, Claus Lindbjerg; Li, Jian; Bolund, Lars; Brünner, Nils Citation for pulished version (APA): Lin, X., Stenvang, J., Rasmussen, M. H., Zhu, S., Jensen, N. F., Tarpgaard, L. S., Yang, G., Belling, K., Andersen, C. L., Li, J., Bolund, L., & Brünner, N. (2015). The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer. BMC Genomics, 16, Article 404. https://doi.org/10.1186/s12864-015-1552-y Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: g y y y Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only y y p g • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk Lin et al. BMC Genomics (2015) 16:404 DOI 10.1186/s12864-015-1552-y The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer Xue Lin1, Jan Stenvang2, Mads Heilskov Rasmussen3, Shida Zhu4, Niels Frank Jensen2, Line S Tarpgaard5, Guangxia Yang4, Kirstine Belling6, Claus Lindbjerg Andersen3, Jian Li1,4,7, Lars Bolund1,4† and Nils Brünner2† Background 2000-3000 active Alus in the human genome per individ- ual [13,14]. Mobilization of Alus mainly occurs during the production of gametes or at early stages of embryo de- velopment [5]. In contrast to germ line retrotranspo- sition, the activity of Alus and other mobile genetic elements in somatic cells is mostly silenced by DNA methylation and post-transcriptional mechanisms medi- ated by piwi-interacting RNAs, siRNAs, miRNAs and AID/APOBEC gene family members [5,15-18]. However, genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation [19]. Ac- cordingly, Alu and L1 have been shown to display DNA methylation alterations in colorectal cancers compared with matched normal tissues [20,21]. Additionally, Alu elements pose the largest transposon-based mutagenic threat to the human genome [14]. A recent study, which intensively sequenced 43 cancer and matched germ line genomes, revealed that colorectal cancers and other cancers of epithelial cell origin show activity of somatic L1 and Alu transpositions [22]. Colorectal cancer is a common and often lethal disease [1]. FOLFIRI (folinic acid, 5-fluorouracil and irinotecan) [2], FOLFOX (folinic acid, 5-fluorouracil and oxaliplatin) [3] and XELOX (capecitabine and oxaliplatin) [4] are commonly used chemotherapeutic combinations used to treat colorectal cancer. However, a considerable subpop- ulation of patients will experience disease recurrence due to acquired resistance to treatment. The molecular mechanisms underlying acquired resistance to these drugs remain elusive. Cancer cells usually harbour numerous genomic and epigenomic aberrations, thereby presenting high diver- sities of genotypes and phenotypes as well as cell fate dynamics. For somatic cells, cell fate is well defined and stably maintained by epigenetic mechanisms. DNA methy- lation is a long-term stable epigenetic mechanism. Additionally, DNA methylation represses the activity of mobile genetic elements and maintains genome integrity. p Among Alu sequences, the Alu Y subfamily is the youngest Alu sequence [8] with an evolutionary age of ~15-20 million years (Mya) [23]. Even though the copy number of Alu Y (~125,000 copies) is less than that of Alu S (550,000 copies, at evolutionary age ~40-50 Mya [23]) and Alu J (~160,000 copies, at evolutionary age ~55 Mya [23]), the Alu Y subfamily harbours the largest number of functionally intact Alu core elements that are more active than the older Alus [14,24]. Background Acti- vation of Alus can have many important biological con- sequences: Alus can reshuffle the genome, generating transposon-mediated mutagenesis [25], inducing gen- omic instability [26], and increasing recombination between elements [8], thereby contributing to genetic population diversity [8,27] as well as to heterogeneity in tumorigenesis. Alus can also remodel the epigenome and alter gene expression patterns by changing epigen- etic marks of neighbouring genes at new insertion sites, introducing ectopic promoters of transcription factor binding sites, and generating novel alternative splicing. Integration of Alu sequences and subsequent remodel- ling of DNA methylation might lead to epigenetic re- programming [28] as well as pluripotency induction and maintenance by A-to-I RNA editing of Alu sequences [29]. Whether Alu retrotransposition occurs during chemotherapy with SN38 or oxaliplatin, and thereby plays a potential role in the development of chemother- apy resistance, remains unknown. The concept ‘eukaryotic genomes are dynamic’ has been well accepted [5] since mobile genetic elements were first discovered in maize [6]. One remarkable fea- ture in the human genome is that the DNA consists of at least 45% mobile genetic elements, including short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) [7]. LINE-1 (L1) is a predominant member of LINEs and Alu is the largest family of SINEs in the human genome [7]. Moreover, L1s are the only autono- mous retrotransposable element in the human genome. Alus are non-autonomous retrotransposable elements, which depend on an L1 coded protein ORP2 (endo- nuclease and reverse transcriptase) to mediate their mobility. Alus are primate-specific sequences sharing a typical 282-nucleotide consensus sequence and a char- acteristic structure [8]. There are more than one million Alu family members, constituting 11% of human DNA [8]. The members are ubiquitously dispersed through- out the genome but preferentially overrepresented in GC-rich and high gene density regions [8]. It is thought that about 75% of the total number of genes in the genome are associated with Alus [9]. The presence of Alu sequences is strongly correlated with multifractality in human genome sequences [10]. Alu elements are also associated with more than 25% of all the simple repeti- tive sequences in primate genomes, including microsa- tellites [11]. It is reported that Alu and L1 initiate the spread of CpG methylation, and the length of CpG islands is associated with the distribution of Alu and L1 retrotransposons [12]. Abstract 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. Lin et al. BMC Genomics (2015) 16:404 Page 2 of 17 Global methylome and non-CpG methylation in the cell line models and clinical samples Global methylome and non-CpG methylation in the cell line models and clinical samples We applied the QDMR software [31] with a concept of ‘DNA methylation entropy’ adopted from the ‘Shannon entropy’ [32], to identify differentially methylated cytosines (DMCs) by estimating variability of DNA methylation states between all colorectal cancer cells and clinical samples. DMCs in all samples in the context of CpG as well as CHG and CHH (where H means A, T or C), were identified. Unsupervised clustering using DMCs in the context of CpG, CHG and CHH were performed (Additional file 1: Figure S1A, 1B and 1C). The drug- resistant cell lines clustered with their parental cell ori- gin in the dendrogram representing the methylome profiles. This clustering represents the phenomenon “somatic memory” and is in accord with data from gene expression profiles from the cell lines [30]. Thus, the somatic memory leads to clustering of the resistant and parental cells rather than clustering according to spe- cific drug-resistance. Also, all cell lines merged in a big cluster separated from the clinical samples in unsuper- vised clustering, suggesting that the colorectal cancer cell lines might show similar features of DNA methy- lome, whereas sporadic clinical samples show high diversity between individual methylomes. In the cell line studies, analyses were performed after chemotherapy- induced resistance while in the clinical cancers the samples for analyses were obtained prior to any chemo- therapy. This could also explain separation between the cell line samples and clinical samples in the unsuper- vised cluster analysis. An additional factor distinguish- ing the clinical samples from the cell lines is the clinical samples contain a mixture of cells including cancer cells, stromal cells and endothelial cells whereas the cell lines are much less heterogeneous. Furthermore, we investigated the distribution of the DMCs in different genomic components. The DMCs (composed of CpG, CHH and CHG) were mainly located in intergenic and intronic regions. Notably, even though the majority of CpG loci harboured more frequently in in- trons and promoters than in intergenic regions, the largest number of DMCs in CpG context were found in inter- genic regions (Figure 1A). Compared with the DMCs in a CpG context, DMCs in non-CpG contexts were less fre- quent, residing mainly in introns, intergenic regions and promoters (Figure 1B and Figure 1C). This result suggests that CpG DMCs contribute predominantly to the DNA methylation difference in intergenic regions. Background Moreover, Alu elements are supposed to act as global modifiers of gene expression through changes in their own methylation state [8]. It is estimated that there are about 80-100 active L1s and We hypothesized that development of drug resistance in colorectal cancer follows a linear step-wise progres- sive model and in the present study, we applied reduced representation bisulfite sequencing (RRBS) assay to analyse the DNA methylome from 3 established SN38- resistant and 3 established oxaliplatin-resistant human Page 3 of 17 Lin et al. BMC Genomics (2015) 16:404 colorectal cancer cell line models [30]. Our results indicate a potential role of Alu elements, especially the Alu Y sub- family, in the resistance to SN38 and oxaliplatin. To valid- ate the findings from the cell line models, we extended our RRBS analysis to 14 clinical colorectal cancer samples. Based on the analyses of the cell lines and clinical samples, we have attempted to delineate the influence of altered DNA methylation on activation of retrotransposons as a model for colorectal cancer chemotherapy resistance. methylation modification. From the identified DMCs, we selected the non-CpG cytosine loci (CHH and CHG) shared by both the cell line models and the 14 clinical samples with a methylation level of at least 50% or higher in every sample. There were totals of 19 and 29 cytosine loci identified, in CHG and CHH formats respectively. Among the CHG methylated cytosine loci, 12 loci were located in gene bodies (exon, intron, pro- moter and TSS (transcription start site)) and 7 loci in intergenic regions. Among the CHH methylated cytosine loci, 15 loci were located in coding genes, 1 locus in a microRNA gene, and 13 in intergenic regions. For the CHG and CHH loci located in intergenic regions, most of them were in repeat sequences. For example, there were 3 and 6 loci harbouring in Alu elements in CHG and CHH formats, respectively. Interestingly, 2 of these (out of 3) and 5 (out of 6), respectively, belonged to the Alu Y subfamily. The information concerning the identi- fied CHG and CHH methylated loci is available in the Additional file 2: Table S1A and 1B, respectively. The cytosine loci uniquely presented in the different drug-sensitive or drug-resistant phenotypes enrich Alu elements A shows the distribution of total cytosines (blue bars) and DMCs (red bars) in the context of CpG in different components of the human genome. B and C show the distribution of total cytosines (blue bars) and DMCs (red bars) in the context of CHH and CHG in different components of the human genome, respectively. The height of the left vertical axis indicates the number of total cytosines in the context of CpG (A), CHG (B) and CHH (C), respectively. The height of the right vertical axis indicates the number of the DMCs in the context of CpG (A), CHG (B) and CHH (C) for the given genomic components, respectively. Figure 1 The distribution of differentially methylated cytosines (DMCs) in different genomic components. A shows the distribution of total cytosines (blue bars) and DMCs (red bars) in the context of CpG in different components of the human genome. B and C show the distribution of total cytosines (blue bars) and DMCs (red bars) in the context of CHH and CHG in different components of the human genome, respectively. The height of the left vertical axis indicates the number of total cytosines in the context of CpG (A), CHG (B) and CHH (C), respectively. The height of the right vertical axis indicates the number of the DMCs in the context of CpG (A), CHG (B) and CHH (C) for the given genomic components, respectively. We found that sets P, O and S are highly enriched in Alu elements, which accounted for 48.8%, 60.1% and 53.3% of all identified cytosine loci, respectively. Notably, many identified Alu elements belong to the youngest Alu subfamily – Alu Y – accounting for 32.1%, 35.8% and 34.3% in the identified Alu elements in the set P, O and S, respectively. Subsequently, we performed RRBS analysis for the 14 clinical samples, and then selected the cytosine loci commonly found in these clinical sam- ples, defined as set C. We further selected the cytosine loci by only keeping the loci that were commonly found set S, which contains only these SN38-resistant sub-line loci (including the three formats of CpG, CHG and CHH). The numbers of the identified loci in sets P, O and S are shown in Figure 2A. The definitions of the dif- ferent sets in this study are available in the Additional file 3: Table S2. The cytosine loci uniquely presented in the different drug-sensitive or drug-resistant phenotypes enrich Alu elements By analysing RRBS data for the colorectal cancer cell line models, we identified the loci unique to the three parental cell lines (cell line A (HCT-116 parental), D (HT-29 par- ental) and G (LoVo parental)) and defined a set P, which contains only these parental cell line loci (including the three formats of CpG, CHG and CHH). We then identi- fied loci unique to the three OxPt-resistant sub-lines (cell line B (HCT-116 OxPt resistant), E (HT-29 OxPt resistant) and H (LoVo OxPt resistant)) as uniquely representing OxPt-resistant DNA methylation features and defined a set O, which contains only these OxPt- resistant sub-line loci (including the three formats of CpG, CHG and CHH). Finally, we identified the loci unique to the three SN38-resistant sub-lines (cell line C (HCT-116 SN38 resistant), F (HT-29 SN38 resistant) and I (LoVo SN38 resistant)) as uniquely representing SN38-resistant DNA methylation features and defined a There were a certain number of non-CpG cytosine (CHH and CHG) methylations in all colorectal cancer cell lines and sporadic colorectal cancer samples. Clus- tering based on non-CpG cytosine methylation data was largely consistent with that based on CpG cytosine methylation data (Additional file 1: Figure S1B and 1C). This suggests that both CpG methylation and non-CpG methylation reflect the general somatic memory of DNA Page 4 of 17 Lin et al. BMC Genomics (2015) 16:404 set S which contains only these SN38-resistant sub-line We found that sets P O and S are highly enriched in Figure 1 The distribution of differentially methylated cytosines (DMCs) in different genomic components. A shows the distribution of total cytosines (blue bars) and DMCs (red bars) in the context of CpG in different components of the human genome. B and C show the distribution of total cytosines (blue bars) and DMCs (red bars) in the context of CHH and CHG in different components of the human genome, respectively. The height of the left vertical axis indicates the number of total cytosines in the context of CpG (A), CHG (B) and CHH (C), respectively. The height of the right vertical axis indicates the number of the DMCs in the context of CpG (A), CHG (B) and CHH (C) for the given genomic components, respectively. Figure 1 The distribution of differentially methylated cytosines (DMCs) in different genomic components. The cytosine loci uniquely presented in the different drug-sensitive or drug-resistant phenotypes enrich Alu elements Taking the identified loci in the context of CpG as an example, there were 505,147, 337,242 and 359,770 cytosine loci identified as uniquely representing drug-sensitive DNA methylation features in the parental (P), OxPt-resistant (O), and SN38-resistant (S) cell-lines respectively. set S, which contains only these SN38-resistant sub-line loci (including the three formats of CpG, CHG and CHH). The numbers of the identified loci in sets P, O and S are shown in Figure 2A. The definitions of the dif- ferent sets in this study are available in the Additional file 3: Table S2. Taking the identified loci in the context of CpG as an example, there were 505,147, 337,242 and 359,770 cytosine loci identified as uniquely representing drug-sensitive DNA methylation features in the parental (P), OxPt-resistant (O), and SN38-resistant (S) cell-lines respectively. Page 5 of 17 Lin et al. BMC Genomics (2015) 16:404 Figure 2 Alu Y subfamily enrichment. A: the number of selected loci in the sets P, O and S (see text). B: the percentage of the Alu Y subfamily out of the identified Alu elements in set E. C: the percentage of Alu elements in all cytosine loci and the percentage of the Alu Y subfamily in the identified Alu elements in either sliding windows of fixed size or extended size in RRBS in-silico simulation. Figure 2 Alu Y subfamily enrichment. A: the number of selected loci in the sets P, O and S (see text). B: the percentage of the Alu Y subfamily out of the identified Alu elements in set E. C: the percentage of Alu elements in all cytosine loci and the percentage of the Alu Y subfamily in the identified Alu elements in either sliding windows of fixed size or extended size in RRBS in-silico simulation. Figure 2 Alu Y subfamily enrichment. A: the number of selected loci in the sets P, O and S (see text). B: the percentage of the Alu Y subfamily out of the identified Alu elements in set E. C: the percentage of Alu elements in all cytosine loci and the percentage of the Alu Y subfamily in th identified Alu elements in either sliding windows of fixed size or extended size in RRBS in-silico simulation. in both set C and the united set of P, O and S and defined a new set E, contains only the commonly shared cytosine loci. The cytosine loci uniquely presented in the different drug-sensitive or drug-resistant phenotypes enrich Alu elements We identified 48,944 loci in set E. Informa- tion about these set E cytosine loci is available in the Additional file 4: Table S3. Notably, the percentage of Alu elements among the identified loci in set E was 53.4%, and the percentage of the Alu Y subfamily in all identified Alu elements in the set E was increased to 46.3% (Figure 2B). Subsequently, we performed DMCs analysis for the identified loci and found that the Alu Y subfamily accounted for 48.4% of the identified DMCs Alu loci. from the gel selection by keeping the proper size of the digested DNA fragments in the in-silico stimulation. According to our experimental protocol of RRBS library generation, there was a maximum of about 28% se- quences from Alu repeats in the in-silico stimulation, of which 39.5% belonged to the Alu Y subfamily. Further- more, we compared the number of Alu Y loci in the data set P, O, S and the number of Alu Y loci in the simulated data set, which demonstrated that the Alu Y enrichment in the set of P, O, S was statistically significant (Fisher’s exact test, p-value < 2.2e-16). In consideration of the potential variance of cutting sections of agarose gel from the smear of the digested genomic DNA, we applied a sliding window by moving a fixed size selection section from the simulated smear of the digested genomic DNA in both directions (towards smaller selection size or bigger selection size) and we also extended the selected section by extending additional To exclude the possibility that the enriched Alu Y sequences came from a possible bias of the RRBS tech- nique, we performed an in-silico simulation. We used the same reference genome (GRCh37) that was used for RRBS mapping as the virtual test genome, subjecting it to MspI digestion and recovery of the resulting DNA Lin et al. BMC Genomics (2015) 16:404 Page 6 of 17 50 bp towards the bigger size or towards the smaller size. Neither sliding the fixed size selection section nor extending selection size showed significant variance in the amount of Alu sequences (Figure 2C). We could not further simulate the alignment because it is hard to esti- mate potential DNA methylation state for the simulated cytosines. The cytosine loci uniquely presented in the different drug-sensitive or drug-resistant phenotypes enrich Alu elements Since Alus are repetitive sequences, align- ment of simulated digested genomic DNA to the human reference genome could lead to further decrease in the proportion of Alu sequences. This is due to problems with low mapping quality, which results from repetitive sequences mapping to multiple locations in the human genome reference and/or the low priority for annotation (the priority order is exon, intron, promoter, intergenic region, and finally, repetitive). range of the percentages of Alu elements and the Alu Y subfamily in the above simulations and compared these variances to the variance of both the percentage of Alu elements and the Alu Y subfamily among all the RRBS data, including all colorectal cancer cell models and the 14 clinical colorectal cancer samples. Clearly, the variance of the Alu Y subfamily in all the RRBS samples was much higher than that in the simulations, which in- dicates that bias from RRBS technology cannot explain the observed Alu Y enrichment in the sets P, O and S. The information of variance in all the RRBS samples and variance in the simulations are available in Additional file 5: Table S4. Subsequently, we compared the identified loci in the sets P, O and S with the loci in the RRBS simulation (selection section window size is 40-300 bp), respectively. We found that 54.7%, 42.4% and 42.1% loci in the sets P, O and S overlapped with the loci in the simulation (Figure 3A). Moreover, we identified the common loci shared by all nine colorectal cancer cell lines and defined a new set A. When we compared the loci in the set A, we found that 87.7% of the loci in set A overlapped with the Moreover, we calculated the percentage of the Alu subfamilies (Alu Y, Alu J and Alu S) in the selected Alu elements according to the use of sliding selection win- dow and extending selection windows with different size in the simulations. The percentages of the Alu sub- families were generally consistent in all simulations (Figure 2C). We also calculated the variance in the Figure 3 A shows the percentage of loci overlapping with the simulation in sets A, P, O and S. B shows the percentage of Alu elements, and Alu subfamilies in sets P, O and S, which are not overlapping with the simulation. The cytosine loci uniquely presented in the different drug-sensitive or drug-resistant phenotypes enrich Alu elements ws the percentage of loci overlapping with the simulation in sets A, P, O and S. B shows the percentage of Alu elements, and Al Figure 3 A shows the percentage of loci overlapping with the simulation in sets A, P, O and S. B shows the percentage of Alu elements, and Alu subfamilies in sets P, O and S, which are not overlapping with the simulation. Lin et al. BMC Genomics (2015) 16:404 Lin et al. BMC Genomics (2015) 16:404 Page 7 of 17 To further validate our findings, we compared RRBS reads that can be uniquely mapped to the human repeat sequence between the drug-resistant cell lines and their parental cell lines. We made a linear normalization of the RRBS data across all the cell lines by making the total amount of mapped RRBS reads of each sample equal. Then we extracted the reads annotated as Alu se- quence and compared the number of the reads uniquely mapped to Alu sequences between the drug-resistant cell lines and their parental cell lines. In general, all OxPt and SN38-resistant cell lines consistently show higher number of reads from Alu Y subfamilies than that in their parental cell line. There is only one exception. The SN38-resistant HT-29 cell line showed almost equal number of the reads to its parental cell line. We trans- formed the read number into log2 format to make the distribution of read number fit a normal distribution. Then we performed a paired t-test, and the statistical re- sult showed that the number of Alu Y reads in the drug- resistant cell lines was significantly higher than that in their matched parental cell lines (p-value = 0.0039 for the SN38 resistant cells vs. the parental cells; p-value = 2.13 × 10−12 for the OxPt resistant cells vs. the parental cells, respectively). Moreover, we calculated the percent- age of the reads of Alu subfamily members (Alu Y, Alu J and Alu S) for all the cell line samples. The percentage of the Alu Y subfamily in the OxPt and SN38-resistant cell lines was higher than that in the parental cell lines (Figure 4A). The percentages of Alu J and Alu S sub- families in all cell lines are shown in Figure 4B and Figure 4C, respectively. loci in the simulation (Figure 3A). The cytosine loci uniquely presented in the different drug-sensitive or drug-resistant phenotypes enrich Alu elements A small portion of the overlapping loci reflects the difference of the genomes of the nine cell lines from the human genome reference used in the simulation. This difference might reflect that the colorectal cancer cell lines harbour a certain number of genomic aberrations and these genomic aberrations could lead to some of the difference between the sequenced genomic loci and the loci in the simulation. Notably, set P from the three parental cell lines showed a lower pro- portion of overlapping loci with the simulation com- pared with set A, suggesting set P contained more loci related to genomic aberrations in individual cell lines than set A. Interestingly, set P showed higher portion of overlapping loci with the simulation than sets O or S, suggesting that the genomes of OxPt-resistant and SN38-resistant cell lines had larger extents of difference from the human genome reference than the genomes of their parental cell lines. This implies that changes in genomic structure in drug-resistant cell lines might occur during drug treatment. We further analysed the constitution of the non-overlapping loci in sets P, O and S. As in the previous analyses, a considerable amount of Alu elements contributed to the proportion of the non- overlapping loci in sets P, O and S. More impressively, the percentages of Alu elements in sets O and S were in- creased compared with that in set P. Among Alu sub- family members, we further confirmed that the Alu Y subfamily mainly contributed to the increased propor- tion of Alu elements in the total non-overlapping loci in the drug-resistant cell lines (Figure 3B). Thus, based on our observation and analysis, we found a correlation be- tween the drug-resistant phenotypes and the increment of Alu Y elements. Identifying flanking sequence motif of Alu sequences Identifying flanking sequence motif of Alu sequences We applied WebLogo 3.3 [34] to extract flanking se- quence (up- and down-stream 20 bp) motif from the Alu elements shared by all the RRBS samples, including both the cell lines and the clinical samples in our study. We identified a symmetric sequence in both flanking sequences of the Alu elements (Figure 5A). Further- more, we extracted flanking sequence motifs from the identified Alu elements in set E, which presented the Alu elements shared by the 14 clinical samples and the united sets of P, O and S (Figure 5B). To see whether the above Alu elements had unique features (motif sequences differ- ing from the other Alu elements in the human genome), we extracted the flanking sequence motifs for all Alu se- quences in the human genome reference (Figure 5C). Interestingly and in general, the flanking sequence of the Alus identified in all the RRBS samples and the Alus iden- tified in set E both showed highly similar motif sequence to that of all Alus in the human genome reference, which is also consistent with the typical Alu target site duplica- tion (TSD) sequence. Alu insertion depends on L1-coded ORP2, and the target site sequence of ORP2 for insertion Alu sequences can be activated and propagate into new loci of the human genome triggered by genotoxic stress [5,33]. Especially, Alu Y sequences are the biggest active subfamily of Alu elements in the human genome. The most likely explanation of our observation in the cell lines is that Alu Y elements were reactivated and spread their copies in the genome when triggered by genotoxic stress of OxPt or SN38. In the RRBS library generation, MspI digested the genomic DNA of the drug-resistant cell line that carried many insertions of Alus, mainly Alu Y elements. The newly inserted Alus in the drug-resistant cell lines will change the constitution of digested genomic fragments. When the digested gen- omic DNA underwent gel selection, the portion of the digested DNA that can be finally selected by the selec- tion section window (40-300 bp) will be changed. Conse- quently, the sequenced part of the genome in the RRBS libraries from the parental cell lines and the drug resist- ant cell lines will be different. Taken together, the results from RRBS might reflect Alu Y subfamily retrotransposi- tion in the drug-resistant cell lines. Lin et al. Identifying flanking sequence motif of Alu sequences BMC Genomics (2015) 16:404 Page 8 of 17 Figure 4 The percentage of Alu subfamilies in all colorectal cancer cell lines. A shows that the OxPt and SN38-resistant cell lines consistently show higher percentage of the Alu Y subfamily than their parental cell lines. B and C show the percentages of Alu J and Alu S subfamilies in the cell line models, respectively. igure 4 The percentage of Alu subfamilies in all colorectal cancer cell lines. A shows that the OxPt and SN38-resistant cell lines consistently show igher percentage of the Alu Y subfamily than their parental cell lines. B and C show the percentages of Alu J and Alu S subfamilies in the cell line Figure 4 The percentage of Alu subfamilies in all colorectal cancer cell lines. A shows that the OxPt and SN38-resistant cell lines consistently show higher percentage of the Alu Y subfamily than their parental cell lines. B and C show the percentages of Alu J and Alu S subfamilies in the cell line models, respectively. Page 9 of 17 Lin et al. BMC Genomics (2015) 16:404 Figure 5 The motif of the flanking sequence of Alu elements. A shows the motif of the flanking sequence of the identified Alu elements in all the RRBS samples; B shows the motif of the flanking sequence of the identified Alu elements in set E; C shows the motif of the flanking sequence of all Alu elements in the human genome reference. D shows the motif of the flanking sequence of identified Alu Y subfamily in set E. Figure 5 The motif of the flanking sequence of Alu elements. A shows the motif of the flanking sequence of the identified Alu elements in all the RRBS samples; B shows the motif of the flanking sequence of the identified Alu elements in set E; C shows the motif of the flanking sequence of all Alu elements in the human genome reference. D shows the motif of the flanking sequence of identified Alu Y subfamily in set E. Figure 5 The motif of the flanking sequence of Alu elements. A shows the motif of the flanking sequence of the identified Alu elements in all the RRBS samples; B shows the motif of the flanking sequence of the identified Alu elements in set E; C shows the motif of the flanking sequence of all Alu elements in the human genome reference. D shows the motif of the flanking sequence of identified Alu Y subfamily in set E. Figure 5 The motif of the flanking sequence of Alu elements. A shows the motif of the flanking sequence of the identified Alu elements in all the RRBS samples; B shows the motif of the flanking sequence of the identified Alu elements in set E; C shows the motif of the flanking sequence of all Alu elements in the human genome reference. D shows the motif of the flanking sequence of identified Alu Y subfamily in set E. Figure 5 The motif of the flanking sequence of Alu elements. A shows the motif of the flanking sequence of the identified Alu elements in all the RRBS samples; B shows the motif of the flanking sequence of the identified Alu elements in set E; C shows the motif of the flanking sequence of all Alu elements in the human genome reference. D shows the motif of the flanking sequence of identified Alu Y subfamily in set E. the additional Alu elements and Alu Y subfamily elements in this study preferably locate in GC-rich region. Since GC-rich regions are also gene-dense regions, our result implies their activity might have an effect on gene function. is typically ‘TTAAAA’ [19]. Our results showed that the identified Alu sequences, either in all the RRBS samples or in set E, typically rely on L1-coded ORP2 for their inser- tion, which is in accordance with other observations [19]. Additionally, the ranking of the first flanking sequence (on the left flanking sequence) of the Alus in all the RRBS samples from the top to the bottom was A, C, T and G, according to the probability scale. Moreover, according to the probability scale, the ranking of the first flanking se- quence of the Alus in set E, from the top to the bottom, was A, C, G and T. By contrast, the ranking of the first flanking sequence of the Alus in the whole human gen- ome was A, T, C and G. We also extracted the flanking sequence motifs for the Alu Y subfamily in set E. The order of ranking the first flanking sequence from the top to the bottom was A, C, T and G, according to the prob- ability scale (Figure 5D). These observations suggest that The identified cytosine loci in Set E highlight the high diversity of DNA methylation in SOX1 in the clinical sample The identified cytosine loci in Set E highlight the high diversity of DNA methylation in SOX1 in the clinical samples We identified 48,944 loci in set E, which were related to a total of 5,816 genes. Thus, many genes harbour more than one identified cytosine locus. For example, the TSPYL2 gene harbours 90 cytosine loci, ranking first among the identified total of 5,816 genes. This gene en- codes a testis specific protein, Y-encoded-like 2 (TSPYL2), which is a nucleosome assembly protein and plays a role in chromatin remodelling to determine gene ex- pression, cell proliferation, and terminal differentiation Lin et al. BMC Genomics (2015) 16:404 Page 10 of 17 Page 10 of 17 [35]. Notably, SOX1 harbours 35 of the identified cytosine loci, ranking fifteenth among the 5,816 genes. In addition to SOX1, other SOX gene members including SOX2, SOX3, SOX4, SOX6, SOX8, SOX11, SOX14, SOX18, SOX21 and SOX30 have been identified to harbour at least one identified cytosine locus. Thus there was a clear en- richment of the SOX gene family in this data set. The gene list and the number of harboured loci are available in Additional file 6: Table S5. [35]. Notably, SOX1 harbours 35 of the identified cytosine loci, ranking fifteenth among the 5,816 genes. In addition to SOX1, other SOX gene members including SOX2, SOX3, SOX4, SOX6, SOX8, SOX11, SOX14, SOX18, SOX21 and SOX30 have been identified to harbour at least one identified cytosine locus. Thus there was a clear en- richment of the SOX gene family in this data set. The gene list and the number of harboured loci are available in Additional file 6: Table S5. patients according to good or poor outcome to treat- ment (complete response (CR) and partial response (PR) versus no change (NC) and progressive disease (PD)). However, we did not obtain a clearly distinguishable grouping according to good and poor phenotypes (data not shown). In the next step, we performed a prediction analysis for individual patients to see whether the identified ‘OxPt resistant phenotype associated methylation loci’ could correctly predict the outcome for each patient. We used the DNA methylation information of the iden- tified loci extracted from all the 14 patients as a training data set to select key features and then build a predictor using K-Nearest Neighbour (KNN) [36]. Then, we per- formed a leave-one-out validation to estimate the accur- acy of the prediction. The identified cytosine loci in Set E highlight the high diversity of DNA methylation in SOX1 in the clinical sample For the ‘OxPt resistant phenotype associated loci’ in the context of CpG, we got an accur- acy of 35.7% for the clinical samples to be predicted as good or poor outcome group correctly (Fisher exact test, p-value = 0.59). The OxPt resistant phenotype associated CHH and CHG loci showed 35.7% (p-value = 0.3) and 64.3% (p-value = 0.5804) accuracies, respectively. These results show that it is hard to precisely predict the out- come for individual patients simply based on the DNA methylation state in certain regions identified by the lim- ited number of cell line models. This suggests that DNA methylomes of sporadic clinical samples may show a large diversity in epigenetic reprogramming during the development of drug resistance. The high variability of inter-individual epigenomic profiles poses a big chal- lenge for the selection of useful epigenetic markers for clinical practice. We extracted the DNA methylation information of the 14 clinical samples on the basis of the cytosine loci in set E. Furthermore, we estimated the diversity of DNA methylation states across the 14 clinical samples by measuring the DNA methylation entropy using QDMR [31]. Interestingly, many cytosine loci located in the SOX1 gene region showed high diversity of DNA methy- lation states across all the 14 clinical samples (Figure 6A and B). The cytosine loci harboured in the TSPYL2 gene and their DNA methylation level and entropy are shown in Additional file 7: Figure S2A and B. Identifying differentially methylated cytosines (DMCs) commonly presenting in both the parental cell lines and the OxPt-resistant cell lines In addition to the analysis of the uniquely presenting loci in the parental cell lines (set P) or in drug-resistant cell lines (sets O and S), we extracted the loci that were com- monly presented in the OxPt-resistant cell lines and their parental cell lines. DNA methylation entropy ana- lysis [31] was applied to identify the differentially meth- ylated cytosines that presented high diversity of DNA methylation state across all the cell line samples. There were 1,089,634, 2,105,795 and 726,658 cytosine loci identified as OxPt-resistant phenotype associated methy- lation loci in the context of CpG, CHH and CHG, re- spectively. We hypothesized that colorectal cancer cells either from in vitro samples (the cell line models) or from in vivo samples (the clinical samples) share com- mon epigenetic alterations, which are epigenetic changes responsible for the development of an OxPt-resistant phenotype. Thus, we transferred the extracted OxPt- resistant phenotype associated methylation loci from the analysis of the cell line models to the clinical samples. Discussion Chemotherapeutic agents triggering genotoxic stress Irinotecan is activated by hydrolysis to SN38 which is a topoisomerase I inhibitor [37]. Inhibition of topoisom- erase I by SN38 can result in repression of both DNA replication and transcription [37]. Oxaliplatin is a platinum-based chemotherapeutic agent [38], which ex- erts its effects by interfering with the DNA replication and transcription machinery through nuclear DNA ad- duct formation [39]. In the clinic, oxaliplatin’s efficacy depends on combined use with 5-fluorouracil (5-FU) [40]. Capecitabine is a prodrug, that is enzymatically converted to 5-fluorouracil [41], which inhibits the production of nucleotide thymidine by inhibiting the enzyme thymidylate synthase [42]. These chemothera- peutic agents are able to kill the bulk of cancer cells by introducing stress. However, in some cases, stress also can reactivate retrotransposition in somatic cells. For instance, Hagan et al., reported that Alu retrotransposi- tion can be induced by exposure to a variety of geno- toxic stressors including the topoisomerase II inhibitor Because the methylomes of the patient samples repre- sented the DNA methylation profiles prior to drug treat- ment, we tested whether the identified ‘OxPt resistant phenotype associated methylation loci’ in the cell line models could classify the patients into good or poor out- come groups correctly. Briefly, we extracted the ‘OxPt resistant phenotype- associated methylation loci’ from the 14 RRBS clinical samples. Subsequently, we performed unsupervised clus- tering analysis based on the extracted loci to see whether the identified loci in the cell line models could group the Page 11 of 17 Lin et al. BMC Genomics (2015) 16:404 Figure 6 The DNA methylation states (A) and the diversity of DNA methylation (entropy) (B) of the SOX1 gene for the 14 clinical samples. Lin et al. BMC Genomics (2015) 16:404 Page 11 of Figure 6 The DNA methylation states (A) and the diversity of DNA methylation (entropy) (B) of the SOX1 gene for the 14 clinical samples. Lin et al. BMC Genomics (2015) 16:404 Page 12 of 17 observations support a correlation between alterations of DNA methylation of retrotransposons and colorectal cancers [20,21,47,48]. etoposide [19]. In addition, non-genotoxic stress such as hypoxia, contributing to the cancer phenotype in- cluding drug resistance and genomic instability, can increase transcription of SINEs (mainly Alu elements) and LINEs by global demethylation [43]. Correlation between Alu retrotransposition and cell stemness An increasing body of evidence indicates that integra- tion of L1 and Alu elements occurs in germ cells or during early embryonic development [5]. Furthermore, it is reported that the most expressed Alu elements are enriched for the youngest subfamily Y in hESCs [49], which is also in agreement with their recent evolution- ary amplification in humans [8]. Additionally, non-CpG methylation has been reported to occur in an asymmet- ric, strand-specific manner in SINEs and LINEs in hESCs and iPSCs, which is a characteristic property of pluripotent cells [50]. At the molecular level, cancers are complex diseases attributed to the accumulation of multiple risk factors, from genetic predisposition to environmental factors such as diet, lifestyle and exposure to toxic compounds [44,45]. Epidemiological studies suggest that the envir- onment influences cancer aetiology far more decisively than genetics in many types of cancers [44,45]. DNA methylation, as an important and long-term stable epi- genetic mechanism, defines cell fate by maintaining gene expression patterns and stabilizing genetic mobile ele- ments. During development, germ line cells and embry- onic stem cells show high cell fate dynamics and activity of mobile genetic elements. Accordingly, DNA methyla- tion also shows dynamic change. In somatic cells, cell fate shows a stable differentiated state and mobile gen- etic elements present in silent states, partly due to DNA methylation locks. However, DNA methylation, as a reversible chemical modification of DNA sequences can also be changed according to environmental changes involving endogenous or exogenous (bio)chemical mol- ecules. DNA methylation changes could lead to instabil- ity of cell fate and reactivation of retrotransposons. At the cell level, somatic cells can become dedifferentiated and heterogeneous, through reshuffling of the genome and remodelling of the epigenome by reactivation of retrotransposons. Through the analysis of DNA methy- lomes from 421 individuals, ranging in age from 14 to 94 year old, Johansson et al., recently demonstrated that aging at least affects DNA methylation of 29% of inves- tigated sites, of which 60.5% are hypomethylated and 39.6% are hypermethylated [46]. Notably, they also found that a higher fraction of sites in repetitive regions is not affected by the process of aging [46]. Correlation between Alu retrotransposition and cell stemness This obser- vation suggests that reactivation of Alu retrotransposi- tion presented in our study is not a passive outcome of aging but reflecting a response from mobile genetic ele- ments to environmental stress in line with the finding that the expression of Alu RNAs is shown to increase in response to cellular stress, viral and translational inhibition [8]. Of particular interest is that many prior The SOX1 and other SOX gene family members being representative of stemness-related genes were identified loci in set E. Furthermore, high diversity of DNA methy- lation states of SOX1 and other SOX genes presented in the clinical samples suggests that the cancer cells from different patients are variably dedifferentiated. Notably, SOX1, SOX2 and SOX3 compose the SOXB1 gene sub- family, which shares more than 90% amino acid identity with respect to the DNA binding high-mobility group (HMG) box (a key characteristic sequence feature for defining SOX gene family) and also a high degree of se- quence similarity outside the HMG box [51]. Moreover, SOX1 or SOX3 can substitute for SOX2 to produce iPS cells [51]. The SOXB1 genes are frequently co-expressed in development and exhibit high biological redundancy. In our previous studies, SOX2 and other SOX gene family members were implicated in the development of resistance to the anti-cancer drug tamoxifen [52]. More- over, the tamoxifen-resistant breast cancer cell lines shared some common features with iPSCs (Li et al., in preparation). By sequencing small cell lung cancers, Rudin et al., reported that a considerable portion of mutations occurring in SOX2 and other SOX genes indi- cated a correlation between lung cancers and cell stem- ness [53]. Our observations in the colorectal cancers and in the tamoxifen-resistant breast cell line models ([52]; Li et al., in preparation) also suggest that the SOX gene family might play an important role in tumorigenesis and drug resistance. Discussion Through the analysis of RRBS data for the cell line models, we found the enrichment of Alu sequences, especially the Alu Y subfamily, in the SN38- and oxaliplatin-resistant cell lines, which provides evidence of reactivation of Alu retrotransposition during the development of drug re- sistance in colorectal cancer cells. This finding sheds light on the potential role of mobility of Alu elements in colorectal cancer chemotherapeutic resistance by pre- senting a genomic response to environmental stress. Alu retrotransposition increases uncertainty for cancer progression In the present study, we initially hypothesized that de- velopment of drug resistance in colorectal cancer follows a linear step-wise progressive model. In this hy- pothesis, we assume that all colorectal cancer cells undergo a common path to develop drug resistance, Page 13 of 17 Lin et al. BMC Genomics (2015) 16:404 Page 13 of 17 single mechanism - reactivation of retrotransposition - will bring an ‘unpredictable’ outcome. Because specificity of target insertion of retrotransposons is weak and individual host genomes and their modifications (epigenomes) are different, spread of Alu copies in the genome will generate a diversity of reshaped genomes and epigenomes. Somatic retrotransposition as genomic response to environmental stress occurs in a discernible but initially unforeseen de- velopment [33]. Here, the two features of the non-linear model are dependent on individual genomes and epigen- omes (i.e., sensitive dependence of initial condition) and initially in an unforeseen circumstance (i.e., dynamics and evolution). These two features are two key characteristics of complex systems. Therefore, system theory and meth- odology, applied for interpreting complex systems might be useful for cancer research. In clinical practice, sensitive dependence of initial condition emphasizes the concept of personalized medicine. Following the same simple mechanism (retrotransposition), during tumor develop- ment or acquiring resistance to cancer therapy, individ- ual cancers could show different landscapes of genomes and epigenomes. From intensive large scale cancer gen- ome sequencing, even single types of cancers show high diversity and it is quite hard to find individual muta- tions that can commonly explain all cases. Some genetic or epigenetic features, such as CIMP (CpG island meth- ylator phenotype) [54,55], can match some subtypes of cancers, but every cancer is unique. In addition to the emphasis on individuality, dynamics are important points to focus on in cancer therapy, because mobile genetic elements are not simple genomic parasites but they also contribute to fitness phenotypes of host ge- nomes, fuelling evolution. thereby presenting recurrent landmarks of epigenetic alterations. Based on this hypothesis, we should be able to use the selected ‘OxPt resistant phenotype-associated methylation loci’ from the cell line models to predict the outcome of response to oxaliplatin for the clinical samples based on the information of DNA methylation from the primary tumors. However, we could not find such statistically significant predictors to precisely pre- dict the outcome of treatment for the patients. Cell culture and generation of drug resistant cell lines g g The cell lines HCT116 and HT29 were obtained from the NCI/Development Therapeutics Program, while LoVo was obtained from the American Tissue Culture Collec- tion. Cells were maintained at 37°C, 5% CO2 in RPMI 1640 + Glutamax growth medium (Invitrogen, Naerum, Denmark) supplemented with 10% foetal calf serum (Invitrogen). Oxaliplatin or SN-38 resistant cell lines were generated in our laboratory over a period of 8-10 months by continuous exposure to gradually increasing concentrations of drug [30]. The cell lines were pas- saged three times at each drug concentration and cell vials were frozen at each increase in drug concentration. Prior to subsequent experiments, the cells were maintained in Alu retrotransposition increases uncertainty for cancer progression An alter- native hypothesis is that drug resistance may follow a non-linear model, in which changes in genomes, epi- genomes and cell fate could happen as part of the same mechanism, i.e., retrotransposition, but individual patients could show a diversity of reshaped genome and epige- nome and high dynamics of cell fate states because of their different initial conditions and potential stochastic events during the development of drug resistance. Alus act as endogenous genomic parasites using a ‘copy-and-paste’ mechanism to spread their copies to new locations in the human genome, which can bring three main biological consequences: reshuffling ge- nomes, remodelling epigenomes and reprogramming cell fates. All of these will contribute to heterogeneity of can- cers, posing a big challenge for cancer therapy. A given chemotherapy can kill many, even most, of the cancer cells. On the other hand, cancer chemotherapy using certain molecules targeting a given single target or path- way might lose some of their effectiveness if the cancer cells have acquired increased genetic diversity and chan- ged cell fate. In the clinic, one failed therapeutic protocol will be replaced by another one with new chemotherapeu- tic agent(s). This strategy usually is effective at the begin- ning, because a new environmental stress is introduced to the cancer cells. However, retrotransposition might act as a genomic response to environmental stress again and eventually lead to resistance to the second treatment as well. If a tumor can be detected at a very early phase, the number of malignant cells is still limited. The probability of development of fitness phenotypes by retrotransposi- tion from the limited number of cells is lower than that from large number of cells in a late phase tumor during treatment. This non-linear model thus fits the clinical notion that ‘earlier detection leads to better outcome’. Additionally, this model also fits the McClintock doc- trine, that increases in mobile genetic element tran- scription that are caused by environmental stress lead to higher levels of mobile genetic element integration and these insertions have an impact on host phenotypes and/or survival [5,33]. Consequently, our study underscores the uniqueness of individual cancers, dynamic tracking of cancer pro- gression and new therapy strategies targeting the entire cell system. Conclusion We have summarized the potential role of Alu ele- ments in colorectal cancer chemotherapy resistance in Figure 7. From this model, one can envision that a Lin et al. BMC Genomics (2015) 16:404 Page 14 of 17 Figure 7 The potential role of Alu mobility in development of drug resistance in colorectal cancer cells. A typical model Alu element is approximately 300 bp in length and has a dimeric structure. The elements are composed of two similar but not-equivalent monomers (7SL-derived left monomer and 7SL-derived right monomer) joined by an A-rich linker. In the 7SL-derived left monomer, there are Box A and Box B, serving as internal Pol III promoter elements, which are helped by an up-stream Pol III enhancer for efficient transcription. The right monomer is followed by a short poly(A) tail and the both terminal sequences typically are Target Site Duplication (TSD) sequences (typically, AA\TTTT). In somatic cells, Alu elements are silent. During tumorigenesis and cancer therapy, in response to the environmental stress induced by carcinogen(s) or chemotherapeutic drug(s), the mobility of Alu elements is activated as a genomic response. Alus propagate using a ‘copy and paste’ mechanism. In the ‘copy’ phase, Alus are typically transcribed by RNA polymerase III. For the ‘paste’ phase, Alus use a ribonucleoprotein complex composed of an endonuclease and a reverse-transcriptase encoded by L1. The endonuclease initially cleaves one DNA strand, and the reverse-transcriptase copies an Alu transcript into a single strand of DNA at that genomic location. The second DNA strand is cleaved by an unknown mechanism, and then the DNA repair mechanism generates the strand complimentary to the novel Alu insertion. The process is named Target-Primed Reverse Transcription (TPRT). Because of the two distinct single-strand breaks, the final DNA sequence contains a TSD, which is a sequence of 4 ~ 25 bp repeated just before and just after the new Alu element. dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at -20°C. Drugs were diluted in growth medium immediately prior to use. drug-free growth medium for at least 1 week. The strategy to establish the three drug resistant cell line models is pre- sented in Additional file 8: Figure S3. The cell line identity of parental and resistant cell lines were confirmed using a short tandem repeat DNA analysis (IdentiCell – Cell Line Authentication Service, Aarhus University Hospital, Aarhus, Denmark). Conclusion In addition, all cell lines were confirmed to be mycoplasma-free (Mycoplasma PCR Detection Kit, Minerva Biolabs, Berlin, Germany). The clinical colorectal cancer samples The timeline of the 14 patients under medical care is shown in Additional file 9: Figure S4. Fresh frozen tumor samples were obtained from a previously pub- lished cohort [56], and were collected prior to any chemotherapy. The clinicopathological information from the 14 colorectal cancer patients has been displayed in Additional file 10: Table S6. According to outcome of the therapy (CR and PR versus NC and PD), the 14 patients were divided into a ‘benefited’ and a ‘not benefited’ group RRBS library generation and sequencing RRBS was performed as previously described [52]. Briefly, 5 μg genome DNA from the cell line models and the clin- ical samples was digested by restriction enzyme, MspI (New England BioLabs) over night at 37°C and QIAGEN Mini Purification kit was used to purify the digested prod- ucts. End repair was performed, adding A and adaptors in which the cytosines in the paired end adaptor sequence were methylated. The ligated product was subjected to size selection in 2% agarose gel (Bio-RAD) at 100 V for 2 hours. Agarose gel bands with the inserted genomic DNA size 40 ~ 110 bp and the inserted genomic DNA size 110 ~ 220 bp were excised, so that two libraries were gen- erated from each of the MOMA3, MOMA4, MOMA5, MOMA7, MOMA8 and MOMA9 samples (one consisting of 40 ~ 110 bp target sequences and the other of 110 ~ 220 bp target sequences). The rest of the clinical samples and all cell line samples were generated with a single library with inserted DNA fragments of 40 ~ 300 bp length. The DNA from the excised gel pieces was recovered with the QIAGEN Gel Extraction Purifi- cation Kit, followed by bisulfite treatment using ZYMO EZ DNA Methylation-Gold kit. The resulting converted DNA was amplified by PCR and purified. The RRBS libraries were subjected to paired-end 50 nt sequencing with HiSeq 2000 (Illumina). Competing interests h h d l h The authors declare that they have no competing interests. Additional files Additional file 1: Figure S1. Methylome profiles of the colorectal cancer cell line models and the clinical colorectal cancer samples. Unsupervised clustering profiles of differentially methylated cytosines (DMCs) in the RRBS data for the three colorectal cancer cell line models and the clinical 14 colorectal cancer patients in the context of CpG (Supplementary Fig. 1A), CHG (Supplementary Fig. 1B), and CHH (Supplementary Fig. 1C). The DNA methylation level is shown as percentage. Full green color means 100 percent DNA methylation, whereas full red color means 0 percent DNA methylation. The intermediate DNA methylation levels are shown in gradient color between full green and full red according to the DNA methylation level (percentage). Additional file 2: Table S1. The information of non-CpG cytosine methylation (CHG in Supplementary Table 1A and CHH in Supplementary Table 1B) in the colorectal cancer cell line models and the sporadic colorectal cancer samples. Additional file 3: Table S2. The definitions of the sets in this study. Additional file 3: Table S2. The definitions of the sets in this study. Additional file 3: Table S2. The definitions of the sets in this study. Additional file 4: Table S3. Information on the identified cytosine loci in set E. Additional file 5: Table S4. Information on variance of the percentage of Alu Y in Alu elements in all RRBS samples and variance of the percentage of Alu Y in Alu elements in the simulations. Additional file 6: Table S5. List of involved genes and the number of their harboured cytosine loci identified in set E. Bioinformatic analysis The adaptor sequences were filtered out before the sub- sequent analysis and the resulting reads were aligned using Bismark software [57]. Only uniquely mapped reads, which had the restriction enzyme cutting site at the 5’ end were used in the subsequent analysis. The se- quencing depth and the percentages of methylated cyto- sines/total investigated cytosines for each C location were calculated. The genomic annotation information was based on the hg19 human genome (http://geno- me.ucsc.edu). Differentially methylated regions (DMRs) were identified by quantitative differentially methylated regions (QDMR) [31]. The QDMR is a quantitative approach to quantify methylation difference and identify DMRs from genome-wide methylation profiles with a concept of ‘DNA methylation entropy’ [31]. The ‘DNA methylation entropy’ adapting Shannon entropy was Additional file 7: Figure S2. The DNA methylation state (Supplementary Fig. 2A) and the diversity of DNA methylation (entropy) (Supplementary Fig. 2B) of the TSPYL2 gene between the 14 clinical sample. Additional file 8: Figure S3. The strategy of establishing the three drug-resistant cell line models. Additional file 8: Figure S3. The strategy of establishing the three drug-resistant cell line models. Additional file 9: Figure S4. The timeline of the 14 colorectal patients in medical treatment. Additional file 9: Figure S4. The timeline of the 14 colorectal patients in medical treatment. Additional file 10: Table S6. The clinicopathological information of the 14 sporadic colorectal cancer patients. Availability of supporting data The data set supporting the results of this article is avail- able in the NCBI Gene Expression Omnibus database accession number (for the raw data and metadata of RRBS for the three colorectal cancer drug-resistant cell line models and the 14 sporadic clinical colorectal can- cer samples) is GSE56269. http://www.ncbi.nlm.nih.gov/ gds/?term=GSE56269. Chemotherapeutic drugs Oxaliplatin (Eloxatin, 5 mg/ml, Sanofi-Aventis, Paris, France) was stored at 4°C protected from light. SN-38 (Sigma-Aldrich, Copenhagen, Denmark) was dissolved in Page 15 of 17 Lin et al. BMC Genomics (2015) 16:404 Page 15 of 17 (Additional file 10: Table S6). By histological examination, the percentage of tumor cells were evaluated to account for more than 70% (except MOMA5 and MOMA22 with 60% tumor cells) of the cells in each sample. The genomic DNA was isolated from the samples and passed the quality control for construction of RRBS libraries. Written informed consent was obtained from all patients and was approved by The Regional Ethics Committee (DK: 1999/4678). (Additional file 10: Table S6). By histological examination, the percentage of tumor cells were evaluated to account for more than 70% (except MOMA5 and MOMA22 with 60% tumor cells) of the cells in each sample. The genomic DNA was isolated from the samples and passed the quality control for construction of RRBS libraries. Written informed consent was obtained from all patients and was approved by The Regional Ethics Committee (DK: 1999/4678). used to estimate diversity (or variety) of DNA methylation states for a given locus across samples [31]. We applied WebLogo 3.3 [34] to extract the flanking sequence (up- and down- stream 20 bp) motifs for the investigated genomic sequences. We applied GeneCluster 2.0 [36] to perform the supervised cluster analysis and prediction analysis. References Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921. 34. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res. 2004;14:1188–90. 8. Batzer MA, Deininger PL. Alu repeats and human genomic diversity. Nat Rev Genet. 2002;3:370–9. 35. Tao KP, Fong SW, Lu Z, Ching YP, Chan KW, Chan SY. TSPYL2 is important for G1 checkpoint maintenance upon DNA damage. PLoS One. 2011;6:e21602. 9. Grover D, Mukerji M, Bhatnagar P, Kannan K, Brahmachari SK. Alu repeat analysis in the complete human genome: trends and variations with respect to genomic composition. Bioinformatics. 2004;20:813–7. 36. Reich M, Ohm K, Angelo M, Tamayo P, Mesirov JP. GeneCluster 2.0: an advanced toolset for bioarray analysis. Bioinformatics. 2004;20:1797–8. 37. Hsiang YH, Liu LF. Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res. 1988;48:1722–6. 10. Moreno PA, Velez PE, Martinez E, Garreta LE, Diaz N, Amador S, et al. The human genome: a multifractal analysis. BMC Genomics. 2011;12:506. 11. Jurka J, Pethiyagoda C. Simple repetitive DNA sequences from primates: compilation and analysis. J Mol Evol. 1995;40:120–6. 38. Raymond E, Faivre S, Woynarowski JM, Chaney SG. Oxaliplatin: mechanism of action and antineoplastic activity. Semin Oncol. 1998;25:4–12. compilation and analysis. J Mol Evol. 1995;40:120–6. 12. Kang MI, Rhyu MG, Kim YH, Jung YC, Hong SJ, Cho CS, et al. The length of CpG islands is associated with the distribution of Alu and L1 retroelements. Genomics. 2006;87:580–90. 39. Woynarowski JM, Chapman WG, Napier C, Herzig MC, Juniewicz P. Sequence- and region-specificity of oxaliplatin adducts in naked and cellu DNA. Mol Pharmacol. 1998;54:770–7. 13. Brouha B, Schustak J, Badge RM, Lutz-Prigge S, Farley AH, Moran JV, et al. Hot L1s account for the bulk of retrotransposition in the human population. Proc Natl Acad Sci U S A. 2003;100:5280–5. , , g , gg , y , , Hot L1s account for the bulk of retrotransposition in the human population. Proc Natl Acad Sci U S A. 2003;100:5280–5. 40. Alian OM, Azmi AS, Mohammad RM. Network insights on oxaliplatin anti-cancer mechanisms. Clin Transl Med. 2012;1:26. 14. Bennett EA, Keller H, Mills RE, Schmidt S, Moran JV, Weichenrieder O, et al. 14. Bennett EA, Keller H, Mills RE, Schmidt S, Moran JV, Weichenrieder O, et al. Active Alu retrotransposons in the human genome. Genome Res. 2008;18:1875–83. 41. References 1. GLOBOCAN 2012: Estimated cancer incidence, mortality and prevalence worldwide in 2012. http://globocan.iarc.fr/Pages/fact_sheets_population.aspx. 1. GLOBOCAN 2012: Estimated cancer incidence, mortality and prevalence worldwide in 2012. http://globocan.iarc.fr/Pages/fact_sheets_population.aspx. 29. Germanguz I, Shtrichman R, Osenberg S, Ziskind A, Novak A, Domev H, et al. A-to-I RNA editing of Alu sequences is involved in the regulation of pluripotency induction and maintenance. Stem Cells Dev. 2014 Mar 1;23(5):443-56. doi:10.1089/scd.2013.0206. Epub 2013 Dec 14. 2. Van Cutsem E, Kohne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med. 2009;360:1408–17. 2. Van Cutsem E, Kohne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med. 2009;360:1408–17. 30. Jensen NF, Stenvang J, Beck MK, Hanáková B, Belling KC, Do KN, et al. Establishment and characterization of models of chemotherapy resistance in colorectal cancer: Towards a predictive signature of chemoresistance. Mol Oncol. 2015 Feb 24. doi:10.1016/j.molonc.2015.02.008. [Epub ahead of print] PMID: 25759163. 3. Goldberg RM, Sargent DJ, Morton RF, Fuchs CS, Ramanathan RK, Williamson SK, et al. A randomized controlled trial of fluorouracil plus leucovorin, irinotecan, and oxaliplatin combinations in patients with previously untreated metastatic colorectal cancer. J Clin Oncol. 2004;22:23–30. 4. Cassidy J, Tabernero J, Twelves C, Brunet R, Butts C, Conroy T, et al. XELOX (capecitabine plus oxaliplatin): active first-line therapy for patients with metastatic colorectal cancer. J Clin Oncol. 2004;22:2084–91. 4. Cassidy J, Tabernero J, Twelves C, Brunet R, Butts C, Conroy T, et al. XELOX (capecitabine plus oxaliplatin): active first-line therapy for patients with metastatic colorectal cancer. J Clin Oncol. 2004;22:2084–91. 31. Zhang Y, Liu H, Lv J, Xiao X, Zhu J, Liu X, et al. QDMR: a quantitative method for identification of differentially methylated regions by entropy. Nucleic Acids Res. 2011;39:e58. 5. Levin HL, Moran JV. Dynamic interactions between transposable elements and their hosts. Nat Rev Genet. 2011;12:615–27. 2. Shannon CE. The mathematical theory of communication. 1963. M 32. Shannon CE. The mathematical theory of communication. 1963. MD Comput. 1997;14:306–17. 6. McClintock B. The origin and behavior of mutable loci in maize. Proc Natl Acad Sci U S A. 1950;36:344–55. 33. McClintock B. The significance of responses of the genome to challenge. Science. 1984;226:792–801. 7. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Authors’ contributions XL and JL analysed the RRBS data, interpreted the results, and drafted the manuscript. JS, NFJ and NB contributed the colorectal cancer cell line models. MHR, LST and CLA collected and prepared the clinical colorectal cancer samples. SDZ, GXY, JL and LB organized sequencing the colorectal Page 16 of 17 Page 16 of 17 Lin et al. BMC Genomics (2015) 16:404 cancer cell lines and clinical samples. SDZ and GXY assisted in RRBS data analysis. KB joined in the discussion. NB, CLA and LB planned and designed this study. MHR, JS, LST and SDZ contributed to the revision of the manuscript. NB and LB supervised the work and contributed to the revision of the manuscript. All authors have approved the manuscript for publication. 16. Slotkin RK, Martienssen R. Transposable elements and the epigenetic regulation of the genome. Nat Rev Genet. 2007;8:272–85. 17. Yang N, Kazazian Jr HH. L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells. Nat Struct Mol Biol. 2006;13:763–71. 18. Chiu YL, Greene WC. The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu Rev Immunol. 2008;26:317–53. Received: 9 August 2014 Accepted: 17 April 2015 27. Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10:691–703. 28. Estecio MR, Gallegos J, Dekmezian M, Lu Y, Liang S, Issa JP. SINE retrotransposons cause epigenetic reprogramming of adjacent gene promoters. Mol Cancer Res. 2012;10:1332–42. Acknowledgements This work was supported by the Danish Council for Strategic Research (09-065177/DSF), the Danish Cancer Society (R72-A-4566-B214 and R20-A-1087-B214), the Vigo and Kathrine Skovgaard Foundation, Sawmill-owner Jeppe Juul and Wife Foundation, Director Ib Henriksens Foundation, the John and Birthe Meyer Foundation, and the IMK Foundation. 19. Hagan CR, Sheffield RF, Rudin CM. Human Alu element retrotransposition induced by genotoxic stress. Nat Genet. 2003;35:219–20. 20. Rodriguez J, Vives L, Jorda M, Morales C, Munoz M, Vendrell E, et al. Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells. Nucleic Acids Res. 2008;36:770–84. 21. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Chan AT, Schernhammer ES, et al. A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J Natl Cancer Inst. 2008;100:1734–8. Author details 1 1Department of Biomedicine, University of Aarhus, the Bartholin Building, DK-8000 Aarhus C, Denmark. 2Department of Veterinary Disease Biology, Section of Molecular Disease Biology, Faculty of Health and Medical Sciences, Copenhagen University, Strandboulevarden 49, Copenhagen, Denmark. 3Department of Molecular Medicine, Aarhus University Hospital, Brendstrupgårdsvej 100, DK-8200 Aarhus N, Denmark. 4BGI (Beijing Genomics Institute), Shenzhen 518083, China. 5Department of Oncology, Odense University Hospital, Sdr. Boulevard 29, DK-5000 Odense C, Denmark. 6Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, 2800 Lyngby, Denmark. 7The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of f S S h Ch 22. Lee E, Iskow R, Yang L, Gokcumen O, Haseley P, Luquette 3rd LJ, et al. Landscape of somatic retrotransposition in human cancers. Science. 2012;337:967–71. 23. Wagstaff BJ, Kroutter EN, Derbes RS, Belancio VP, Roy-Engel AM. Molecular reconstruction of extinct LINE-1 elements and their interaction with nonautonomous elements. Mol Biol Evol. 2013;30:88–99. 24. Mills RE, Bennett EA, Iskow RC, Devine SE. Which transposable elements are active in the human genome? Trends Genet. 2007;23:183–91. 25. Iskow RC, McCabe MT, Mills RE, Torene S, Pittard WS, Neuwald AF, et al. Natural mutagenesis of human genomes by endogenous retrotransposons. Cell. 2010;141:1253–61. Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, China. 26. Ade C, Roy-Engel AM, Deininger PL. Alu elements: an intrinsic source of human genome instability. Curr Opin Virol. 2013;3:639–45. Received: 9 August 2014 Accepted: 17 April 2015 Lin et al. BMC Genomics (2015) 16:404 Lin et al. BMC Genomics (2015) 16:404 43. Pal A, Srivastava T, Sharma MK, Mehndiratta M, Das P, Sinha S, et al. Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia. J Cell Mol Med. 2010;14:2646–54. 44. Lichtenstein P, Holm NV, Verkasalo PK, Iliadou A, Kaprio J, Koskenvuo M, et al. Environmental and heritable factors in the causation of cancer–analyses of cohorts of twins from Sweden, Denmark, and Finland. N Engl J Med. 2000;343:78–85. 45. Sorensen TI, Nielsen GG, Andersen PK, Teasdale TW. Genetic and environmental influences on premature death in adult adoptees. N Engl J Med. 1988;318:727–32. 46. Johansson A, Enroth S, Gyllensten U. Continuous Aging of the Human DNA Methylome Throughout the Human Lifespan. PLoS One. 2013;8:e67378. 47. Baba Y, Huttenhower C, Nosho K, Tanaka N, Shima K, Hazra A, et al. Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors. Mol Cancer. 2010;9:125. 48. Ogino S, Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, et al. LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer. 2008;122:2767–73. 49. Macia A, Munoz-Lopez M, Cortes JL, Hastings RK, Morell S, Lucena-Aguilar G, et al. Epigenetic control of retrotransposon expression in human embryonic stem cells. Mol Cell Biol. 2011;31:300–16. 50. Guo W, Chung WY, Qian M, Pellegrini M, Zhang MQ. Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells. Nucleic Acids Res. 2014 Mar;42(5):3009-16. doi:10.1093/nar/gkt1306. Epub 2013 Dec 16. 51. Miyagi S, Kato H, Okuda A. Role of SoxB1 transcription factors in development. Cell Mol Life Sci. 2009;66:3675–84. 51. Miyagi S, Kato H, Okuda A. Role of SoxB1 transcription factors in development. Cell Mol Life Sci. 2009;66:3675–84. development. Cell Mol Life Sci. 2009;66:3675–84. 52. Lin X, Li J, Yin G, Zhao Q, Elias D, Lykkesfeldt AE, et al. Integrative analyses of gene expression and DNA methylation profiles in breast cancer cell line models of tamoxifen-resistance indicate a potential role of cells with stem-like properties. Breast Cancer Res. 2013;15:R119. 53. Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS, et al. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer. Nat Genet. 2012;44:1111–6. gene in small-cell lung cancer. Nat Genet. 2012;44:1111–6. 54. Xu Y, Hu B, Choi AJ, Gopalan B, Lee BH, Kalady MF, et al. References Tabata T, Katoh M, Tokudome S, Hosakawa M, Chiba K, Nakajima M, et al. Bioactivation of capecitabine in human liver: involvement of the cytosolic enzyme on 5’-deoxy-5-fluorocytidine formation. Drug Metab Dispos. 2004;32:762–7. Active Alu retrotransposons in the human genome. Genome Res. 2008;18:1875–83. 15. Maksakova IA, Mager DL, Reiss D. Keeping active endogenous retroviral-like elements in check: the epigenetic perspective. Cell Mol Life Sci. 2008;65:3329–47. 42. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003;3:330–8. Page 17 of 17 Lin et al. BMC Genomics (2015) 16:404 Unique DNA methylome profiles in CpG island methylator phenotype colon cancers. Genome Res. 2012;22:283–91. 55. Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787–93. 56. Rasmussen MH, Jensen NF, Tarpgaard LS, Qvortrup C, Romer MU, Stenvang J, et al. High expression of microRNA-625-3p is associated with poor response to first-line oxaliplatin based treatment of metastatic colorectal cancer. Mol Oncol. 2013;7:637–46. 57. Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics. 2011;27:1571–2. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review
https://openalex.org/W4361871764	https://figshare.com/articles/journal_contribution/Supplementary_Methods_Figure_Legends_1-4_from_STK33_Kinase_Activity_Is_Nonessential_in_KRAS-Dependent_Cancer_Cells/22389270/1/files/39834765.pdf	English	null	Supplementary Methods, Figure Legends 1-4 from STK33 Kinase Activity Is Nonessential in KRAS-Dependent Cancer Cells	null	2,023	cc-by	1,628	Expression and purification of full-length STK33 Expression and purification of full-length STK33 STK33 (NM_030906) was cloned incorporating a TEV-cleavable, N-terminal His7–tag into a pFastBac vector and expressed using the Bac-to-Bac baculovirus/insect cell expression system (Invitrogen). This pFastBac construct, MAH7-TEV-STK33 (amino acids 2-514), was generated by PCR using an Incyte Collection cDNA clone (LIFESEQ90205785, Open Biosystems) as template. The resulting recombinant baculovirus stock was used to infect Trichoplusia ni cells (Orbigen) in ExCell 405 Medium (Sigma) with incubation at 27°C for 50 hours post-infection before the cells were harvested by centrifugation. The protein was purified by passing the cell lysate through a Ni-NTA column, subjecting the eluate to TEV protease cleavage, Ni-NTA subtraction, and Superdex 75 size-exclusion chromatography (SEC). Two STK33 peaks were fractionated on the SEC column consistent with the 60 kDa monomer and 120 kDa dimer. The peak containing the 60 kDa STK33 was used for the high throughput screen to identify small molecule STK33 kinase inhibitors. Supplementary Methods Supplementary Methods High throughput siRNA screens SiRNAs from Qiagen Inc. (Valencia, CA) or from Thermo Scientific (Dharmacon Products, Lafayette CO) were used to create libraries with 4-8 siRNAs for each gene (there were 8 siRNAs for KRAS and for STK33). Each siRNA was individually transfected into cells using Lipofectamine RNAiMAX transfection reagent (Life Technologies, Carlsbad CA). SiRNAs from a library plate were diluted in serum-free media to a volume of 6 µl. Transfection reagents diluted in serum-free media to a volume of 5 µl were added to each well using a BiomekFx Robot (Beckman Coulter). After a 20-minute room temperature incubation, cells were added to the plates using a Multidrop (ThermoScientific). After 96 or 120 hours, cell viability was determined with CellTiterGlo™ (Promega, Madison, WI) and luminescence was measured on a luminometer according to the manufacturer’s instructions. The final siRNA concentrations (10-30 nM) and RNAiMAX volume used per well (0.02-0.1 µl) and plating cell density (500-1500 cells/well) varied by cell line. Most cell lines were screened using multiple transfection conditions. Results from the viability assays were processed through Screener® (Genedata, Basel Switzerland). Viability measurements for the siRNAs for each gene were compared to the remainder of the siRNA using Stouffer’s method (details below) in order to generate a p-value for each gene. Calculation of gene-based p-values Genes were evaluated for their role in cell viability/proliferation by comparing the siRNA targeting the gene of interest (e.g. STK33 or KRAS) to a reference set of siRNA targeting predominantly non-essential genes (generated as described below). P-values were calculated using the Inverse normal method of Stouffer (Hedges 1985). Briefly, each siRNA’s effect on viability was ranked against the reference set, and this ranking was converted to a fraction by dividing by (N+1), where N is the number of siRNA in the reference set. Then, the fractions for all the siRNA targeting a given gene were combined using Stouffer’s method, resulting in a p-value for each gene. The Stouffer method converts the fractions to Z scores and pools the Z scores using the formula where k is the number of siRNA targeting the gene of interest, Zi = Φ−1(fi), where Φ−1 is the inverse of the standard normal cumulative distribution function, fi is the rank of siRNAi targeting the gene of interest expressed as a fraction, and Z is the Z score for the gene. The Z score for each gene is then converted to a p-value using the standard normal cumulative distribution function p = Φ(Z). In large scale siRNA screens, the entire siRNA library is typically used as the reference set, since the number of true hits in the library is normally expected to be low. However, if a library is enriched for essential genes, the statistical power will be diminished. To generate a reference set with fewer true positives, we preferentially removed siRNA targeting essential genes from the reference set as follows. For each siRNA in the library, we assigned what we termed an orthogonal gene average (OGA) p-value. The OGA p-value is identical to the gene p-value described above, except that it is assigned to a siRNA rather than a gene, and instead of being calculated using every siRNA against the gene of interest, it uses only the siRNA other than the siRNA under consideration. For example, to calculate the OGA p-value for a siRNAx that targets geney, every siRNA targeting geney is used in the calculation except for siRNAx itself. This results in the OGA p-value for siRNAx being correlated with the on-target effects of siRNAx on geney, while being completely uncorrelated with the off-target effects of siRNAx. Calculation of gene-based p-values Removal of siRNA from the reference set based on OGA p-value can therefore be used to preferentially remove essential genes without significantly changing the distribution of siRNA targeting non-essential genes in the reference set. In the experiments shown, the OGA p-values were used to calculate a false discovery rate (FDR) by the Benjamini- Hochberg method, and siRNA were removed from the reference set if FDR < 0.5. The top 0.5n siRNA with the highest OGA p-values were also removed, where n is the number of siRNA with FDR < 0.5, so that siRNA were removed from both the high and low OGA p-value tails. The OGA method was also used to generate a reference set on each individual plate in the library for plate normalization purposes. Quantitation of RNA knockdown and cell viability for HCT-116 and MDA-MB-231 cells Cells were transfected in reverse, in duplicate 96-well plates, using a Bravo robotic work station (Agilent Technologies). Briefly, 3µl 1 µM siRNA (Qiagen, Valencia, CA) diluted with 7 µl serum-free media was incubated with either 0.1 µl/well or 0.2 µl/well RNAiMAX (Life Technologies, Carlsbad CA) in an equal volume of serum-free media for 20 min in a 96-well plate. Four thousand cells per well in 80 µl volume were added using a Multidrop™ (ThermoScientific) giving a final siRNA concentration of 30 nM. RNA expression levels were quantified from one of the two 96-well plates 24 hours after transfection using the QuantiGene branched DNA assay as noted in the Materials and Methods section. Viability was quantified from the other 96-well plate 96 hours after transfection using CellTiterGlo™ (Promega, Madison, WI) according to the manufacturer’s protocol. The STK33 probe set detects RNA spanning nucleotides 738-1408 (relative GenBank REFSEQ NM_030906). The KRAS probe set detects RNA spanning nucleotides 668- 1521 (relative GenBank REFSEQ NM_004985). The HPRT probe set detects RNA spanning nucleotides 530-1048 (relative GenBank REFSEQ NM_000194). The cyclophilin B (PPIB) probe set detects RNA spanning nucleotide 69-629 (relative GenBank REFSEQ NM_000942). Supplementary Table 1. siRNA Information Gene symbol (Entrez Gene ID#) Name Vendor (product ID) Target sequence KRAS (3845) KRAS_1Q Qiagen (custom) CCCGGTCCTTAGGTAGTGCTA KRAS_2Q Qiagen (custom) ACCTATGGTCCTAGTAGGAAA KRAS_3Q Qiagen (custom) TTACATAGACTTAGGCATTAA KRAS_4Q Qiagen (custom) AAGAATAGTCATAACTAGATT KRAS_5Q Qiagen (SI03101903) GACGATACAGCTAATTCAGAA KRAS_6Q Qiagen (SI03106824) GTGGACGAATATGATCCAACA KRAS_7Q Qiagen (SI02634191) CTCCTAATTATTGTAATGTAA KRAS_8Q Qiagen (SI02662051) AAGGAGAATTTAATAAAGATA KRAS_238.ST Invitrogen (HSS105871, Stealth) GACGATACAGCTAATTCAGAATCAT STK33 (65975) STK33_1Q Qiagen (SI00139741) CAGTGGTTAACAGGCAATAAA STK33_2Q Qiagen (SI00139748) ATGTGTCTAGTGCATCCTTAA STK33_6Q Qiagen (SI02660203) TCCATAAGTGACTGTGCTAAA STK33_7Q Qiagen (SI02660210) GAGCATAGGCGTCGTAATGTA STK33_3D Dharmacon (D-005383-03) CTAAGGAACTACTAGATAA STK33_4D Dharmacon (D-005383-04) TCACAGACATCAAGCATTG STK33_5D Dharmacon (D-005383-05) ACATAAAGGTGACTGATTT STK33_6D Dharmacon (D-005383-06) GAGCATAGGCGTCGTAATG PLK1 (5347) PLK1_6 Qiagen (SI02223837) CCGGATCAAGAAGAATGAATA PLK1-4 Qiagen (SI00071638) CCCGAGGTGCTGAGCAAGAAA EIF4A3 (9775) DDX48 Qiagen (custom) AAAGAGCAGATTTACGATGTA PPIB (5479) Cyclophilin B Qiagen (custom) TGGTGTTTGGCAAAGTTCT n/a Random1_4 Qiagen (custom) AAGAGGGACATAAAGCTTTCA n/a Random2_4 Qiagen (custom) AACGCAGAGTTCGACCGTTTA n/a Random6_1 Qiagen (custom) AAGGGCAACATCAAGGTTTAT n/a M13_4 Qiagen (custom) AATGCGCTTCCCTGTTTTTAT n/a Luciferase Qiagen (custom) ACGTACGCGGAATACTTCG n/a Scrambled Invitrogen (12935-300, Stealth) Proprietary n/a, not applicable n/a, not applicable Supplementary Figure Legends Supplementary Figure Legends Figure S1. SiRNA oligonucleotides STK33_1Q and STK33_7Q siRNAs Reduce STK33 RNA in NOMO-1 and SKM-1 cells. STK33 RNA levels were determined in each group by bDNA assay 24 hours after transfection (same transfection as Figure 1B and 1D – see methods) of control siRNAs (scrambled - scram, cyclophilin B – cycl B, luciferase - lucif) or with siRNAs against STK33, KRAS or PLK1. Cyclophilin B RNA levels were also determined as a control. The data are expressed as a ratio of STK33 levels over cyclophilin B levels (arbitrary units ± standard deviation, n=2). Figure S2. Efficient Knockdown of STK33 and KRAS with siRNAs in HCT-116 Cells. A, STK33 and KRAS RNA levels were determined 24h after siRNA transfection in HCT-116 cells using a bDNA assay. Data are expressed as a ratio of STK33 levels over cyclophilin B levels. B, In a replicate 96-well plate, the effects of the siRNA on cell viability were quantified using a homogeneous luminescent assay for ATP (CellTiterGlo™). Arrows indicate off-target activities of some STK33 siRNAs. All experiments were done in triplicate and the data are expressed as mean ± standard deviation. Figure S3. Efficient Knockdown of STK33 and KRAS with siRNAs in MDA-MB-231 Cells. A, STK33 and KRAS RNA levels were determined 24h after siRNA transfection in MDA-MB-231 cells using a bDNA assay. Data are expressed as a ratio of STK33 levels over cyclophilin B levels. B, In a replicate 96-well plate, the effects of the siRNA on cell viability were quantified using a homogeneous luminescent assay for ATP (CellTiterGlo™). Arrows indicate off-target activities of some STK33 siRNAs. All experiments were done in triplicate and the data are expressed as mean ± standard deviation. Figure S4. STK33 RNA and Protein Levels are Variable across Cell Lines. A, STK33 levels were determined by western blotting. The same cell lysates were used to determine the levels of S6K (p85 and p70) and RPS6 phosphorylation on a separate gel. B, STK33 RNA expression levels in breast, pancreas and colon cancer cell lines. STK33 RNA levels were determined by a bDNA assay from cell lysates. For each line, the expression of STK33 was normalized to the level of HPRT and the ratio is reported as arbitrary units ± standard deviation (n=2). Cell lines that carry a KRAS mutation are marked with an asterisk.
https://openalex.org/W2914312460	https://leicester.figshare.com/articles/journal_contribution/A_Randomized_Trial_Directly_Comparing_Ventral_Capsule_and_Anteromedial_Subthalamic_Nucleus_Stimulation_in_Obsessive-Compulsive_Disorder_Clinical_and_Imaging_Evidence_for_Dissociable_Effects_/10232945/1/files/18464252.pdf	English	null	A Randomized Trial Directly Comparing Ventral Capsule and Anteromedial Subthalamic Nucleus Stimulation in Obsessive-Compulsive Disorder: Clinical and Imaging Evidence for Dissociable Effects	Biological psychiatry	2,019	cc-by	8,188	Archival Report Archival Report Biological Psychiatry A Randomized Trial Directly Comparing Ventral Capsule and Anteromedial Subthalamic Nucleus Stimulation in Obsessive-Compulsive Disorder: Clinical and Imaging Evidence for Dissociable Effects Himanshu Tyagi, Annemieke M. Apergis-Schoute, Harith Akram, Tom Foltynie, Patricia Limousin, Lynne M. Drummond, Naomi A. Fineberg, Keith Matthews, Marjan Jahanshahi, Trevor W. Robbins, Barbara J. Sahakian, Ludvic Zrinzo, Marwan Hariz, and Eileen M. Joyce ABSTRACT ABSTRACT BACKGROUND: Deep brain stimulation (DBS) is an emerging treatment for severe obsessive-compulsive disorder (OCD). We compared the efﬁcacy of ventral capsule/ventral striatal (VC/VS) and anteromedial subthalamic nucleus (amSTN) DBS in the same patients and tested for mechanistic differences on mood and cognitive ﬂexibility and associated neural circuitry. The possible synergistic beneﬁt of DBS at both sites and cognitive behavioral therapy was explored. METHODS: Six patients with treatment-refractory OCD (5 men; Yale-Brown Obsessive Compulsive Scale score .32) entered double-blind counterbalanced phases of 12-week amSTN or VC/VS DBS, followed by 12-week open phases when amSTN and VC/VS were stimulated together, in which optimal stimulation parameters were achieved and adjunctive inpatient cognitive behavioral therapy was delivered. OCD and mood were assessed with standardized scales and cognitive ﬂexibility with the Cambridge Neuropsychological Test Automated Battery Intra-Extra Dimensional Set-Shift task. Diffusion-weighted and intraoperative magnetic resonance imaging scans were performed for tractography from optimally activated electrode contacts. p g p y p y RESULTS: DBS at each site signiﬁcantly and equivalently reduced OCD symptoms with little additional gain following combined stimulation. amSTN but not VC/VS DBS signiﬁcantly improved cognitive ﬂexibility, whereas VC/VS DBS had a greater effect on mood. The VC/VS effective site was within the VC. VC DBS connected primarily to the medial orbitofrontal cortex, and amSTN DBS to the lateral orbitofrontal cortex, dorsal anterior cingulate cortex, and dorsolateral prefrontal cortex. No further improvement followed cognitive behavioral therapy, reﬂecting a ﬂoor effect of DBS on OCD. CONCLUSIONS: Both the VC/VS and amSTN are effective targets for severe treatment-refractory OCD. Differential improvements in mood and cognitive ﬂexibility and their associated connectivity suggest that DBS at these sites modulates distinct brain networks. Keywords: Anteromedial subthalamic nucleus, DBS, Deep brain stimulation, Obsessive-compulsive disorder, OCD, Ventral internal capsule Keywords: Anteromedial subthalamic nucleus, DBS, Deep brain stimulation, Obsessive-compulsive disorder, OCD, Ventral internal capsule https://doi.org/10.1016/j.biopsych.2019.01.017 https://doi.org/10.1016/j.biopsych.2019.01.017 (ventral capsule/ventral striatum [VC/VS]). Earlier, small studies found that relatively few patients responded to VC/VS DBS (5,6), but more recent, larger studies found level 1 evidence in favor of DBS (7,8). Neurosurgical targeting in these studies changed over time (9), and there were also differences in the choice of DBS electrodes, all of which had four contacts at their tip but variable distances between them. It is likely that such factors led to discrepancies in the location and/or spread of stimulation and may explain the inconsistent response rates. Ventral Capsule and Subthalamic Nucleus DBS for OCD whether stimulation of capsular white matter and/or nearby gray matter provides the optimal response (7,8). underwent 3T diffusion-weighted MRI for tractography anal- ysis (Magnetom Tim Trio; Siemens, Berlin, Germany). Following surgery, stimulation remained off for at least 4 weeks. At the beginning of each phase, participants were admitted to a neuropsychiatry ward for 2 weeks for DBS adjustment. Participants remained on optimized settings for 12 weeks with a contingency for readjustment if clinically indi- cated (Supplemental Figure S1). The anteromedial subthlamic nucleus (amSTN) is an alter- native DBS target for OCD following a randomized controlled trial by Mallet et al. (10), who found signiﬁcant symptomatic reduction. Which of the two sites is better for ameliorating severe OCD has yet to be established. In addition, preliminary evidence suggests that the mechanisms of action at the two sites may differ. VC/VS DBS studies have reported a pro- nounced improvement in mood (5,7,8) not evident following the amSTN study (10), leading Denys et al. (7) to suggest that different neural circuits may mediate OCD, with one stimulated by VC/VS DBS primarily improving mood and another affected by amSTN DBS primarily modulating compulsive behavior. ( pp g ) The initial two phases were double blind, randomized, and counterbalanced; each phase lasted 12 weeks. Participants received stimulation of either the amSTN (n = 3) or the VC/VS (n = 3) followed by the alternate condition. A 12-week open phase followed, during which electrodes at both sites were active (combined stimulation [COMB] phase). There were two additional 12-week open phases when optimized stimulation settings were delivered using data from previous phases (OPT phase), followed by the participants’ receiving CBT/exposure and response prevention in an inpatient unit (OPT plus adjunctive CBT phase) while continuing with the OPT DBS settings. Clinical and cognitive assessments were performed before surgery (baseline) and after each phase. In this study, using a randomized, double-blind, counter- balanced design, we investigated the effects of VC/VS and amSTN DBS in the same patients to 1) establish whether one site is more efﬁcacious than the other in improving OCD, 2) deter- mine the precise neuroanatomical locations for optimal effects by calculating volumes of tissue activation (VTAs) at each site, and 3) test the hypothesis of Denys et al. Stimulus Adjustments Optimal DBS parameters were derived in an iterative fashion over 2 weeks. Initially, each contact was screened with volt- ages up to 4 V (amSTN) or 8 V (VC/VS) using monopolar stimulation (pulse width 60 ms and frequency 130 Hz). Imme- diate clinical effects from stimulation delivered through each contact in turn and the threshold associated with positive and Participants Six patients were recruited from OCD specialized services. Eligibility required ICD-10 OCD, age .20 years, illness duration .10 years, unremitting symptoms for 2 years, Yale-Brown Obsessive Compulsive Scale (Y-BOCS) score $32, and DSM- IV General Assessment of Function Scale score #50. Patients were ineligible if they were pregnant or had ICD-10 substance misuse, organic brain syndrome, adult personality disorder (except obsessive-compulsive type), pervasive developmental disorder, schizophrenia or bipolar disorder, or contraindications to neurosurgery. Treatment resistance was deﬁned as no sus- tained beneﬁt from 1) at least two serotonin reuptake inhibitors for a minimum of 12 weeks at optimal doses; 2) augmentation of serotonin reuptake inhibitor treatment with antipsychotics or extension of the serotonin reuptake inhibitor dose beyond rec- ommended limits; and 3) two trials of CBT, one as inpatient, lasting 10 hours minimum. All patients provided written informed consent. The clinical trial was registered with the UK Clinical Research Network (No. 13158) and the ISRCTN registry (No. 18430630) and received ethical approval (clinical study: National Health Service Health Research Authority No. 12/LO/ 1087; MRI: National Health Service West London Research Ethics Committee No. 10/H0706/68). Neurosurgery Surgery was performed under general anesthesia. Patients underwent stereotactic 1.5T MRI (Magnetom Avanto; Siemens) for planning of amSTN and VC/VS coordinates and trajectories (12). Through 14-mm frontal bilateral burr holes, 1.5-mm- diameter radiofrequency electrodes were introduced to each target under dynamic impedance monitoring. These were replaced with DBS leads through the same trajectory to the target. Separate corticotomies, within the same burr hole, were used to implant the two DBS leads. Quadripolar DBS leads were used with 0.5-mm separation between contacts for amSTN leads (3389; Medtronic, Minneapolis, MN) and 1.5-mm sepa- ration between contacts for VC/VS leads (3387; Medtronic). The VC/VS lead was planned to locate one contact within the nu- cleus accumbens core, one contact within its shell, and the upper two contacts in the most ventral aspect of the anterior limb of the internal capsule. An immediate stereotactic MRI veriﬁed targeting accuracy (13). Two neurostimulators (Activa PC; Medtronic) were placed subcutaneously below the collar- bone, one on each side, and each was connected to bilateral leads from one of the electrodes via subcutaneous cables. The potential of synergistic beneﬁt of DBS at both sites and adjunctive CBT was additionally explored in open phases when both DBS sites were stimulated. Ventral Capsule and Subthalamic Nucleus DBS for OCD (7) by ﬁrst employing ratings of mood and a test of cognitive ﬂexibility previously shown to measure an endophenotype of OCD (11) and then investigating the neural circuitry associated with the DBS at each site with magnetic resonance imaging (MRI) tractography. Randomization and Blinding Computer-generated pairwise randomization was used so that equal numbers were recruited to receive amSTN or VC/VS stimulation ﬁrst, in a counterbalanced order. Two unblinded clinicians (TF, PL) held the randomization list and adjusted DBS parameters. All other team members, ward staff, and partici- pants were blinded to allocation. Biological Psychiatry 2 Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal ABSTRACT For example, within the area of the VC/VS, it is not clear (ventral capsule/ventral striatum [VC/VS]). Earlier, small studies found that relatively few patients responded to VC/VS DBS (5,6), but more recent, larger studies found level 1 evidence in favor of DBS (7,8). Neurosurgical targeting in these studies changed over time (9), and there were also differences in the choice of DBS electrodes, all of which had four contacts at their tip but variable distances between them. It is likely that such factors led to discrepancies in the location and/or spread of stimulation and may explain the inconsistent response rates. For example, within the area of the VC/VS, it is not clear Obsessive-compulsive disorder (OCD) has a lifetime prevalence of 1% to 2%. Effective treatments include cognitive behavioral therapy (CBT) and serotonin reuptake inhibitor medication (1), but there remains a severely impaired, treatment-refractory subgroup (2) for whom anterior cingulotomy or capsulotomy is an option (3). Obsessive-compulsive disorder (OCD) has a lifetime prevalence of 1% to 2%. Effective treatments include cognitive behavioral therapy (CBT) and serotonin reuptake inhibitor medication (1), but there remains a severely impaired, treatment-refractory subgroup (2) for whom anterior cingulotomy or capsulotomy is an option (3). Deep brain stimulation (DBS) for OCD was introduced as an alternative to neurosurgical ablation because it is reversible and adjustable (4). Four studies have included a randomized controlled trial of DBS at the site of the anterior capsulotomy ª 2019 Society of Biological Psychiatry. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1 Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal ISSN: 0006-3223 RESULTS Volumes corresponding to the active electrodes for each target were merged using Fslmerge (FSL v5.0) into a 4-dimensional data ﬁle for each hemisphere. Fslmaths (FSL v5.0) was then used to generate group average volumes of tissue activation. Six participants were recruited from NHS England Specialised OCD Services by HT, NF, LMD, and KM. Five were men; the age range was 38 to 62 years and the duration of illness was 20 to 30 years (Table 1). The study took place between January 10, 2013, and October 25, 2016. During the trial, patient 2 required further DBS adjustment during the amSTN, VC/VS, and COMB phases and required additional medication for Ventral Capsule and Subthalamic Nucleus DBS for OCD ﬁbers were estimated in each brain voxel using model 2 and graphics processing unit parallelization (17,18). Probtrackx was used on these estimates to obtain global connectivity (i.e., the probability of the existence of a path through the diffusion ﬁeld between any two distant points, a surrogate measure of anatomical connectivity) (19). Probabilistic trac- tography was generated in ProbtrackX2 graphics processing unit version (FSL v5.0) (number of samples = 5000, curvature threshold = 0.2, step length = 0.5 mm, subsidiary ﬁber volume fraction threshold = 0.01) (19). The process repetitively sam- ples from the distributions of voxelwise principal diffusion directions generated in BedpostX, each time computing a streamline through these local samples to generate a prob- abilistic streamline or a sample from the distribution on the location of the true streamline, building up a spatial connec- tivity distribution. Streamlines truly represent paths of minimal hindrance to diffusion of water in the brain, but they are reasonable indirect estimates of long-range white matter connections (20). negative effects were noted. Anticipated adverse effects of stimulation included hypomania and anxiety (9,10) and were documented with a visual analog scale. Stimulation parame- ters were reﬁned daily according to patient feedback, visual analog scale, and clinical assessment, including the use of stimulation through multiple monopolar electrode contacts per lead or using a bipolar conﬁguration. Volumes of Tissue Activation SureTune, Version 2 (Medtronic), a DBS therapy planning plat- form, was used to model activation volumes around individual contacts. This applies neuron models coupled to ﬁnite element simulations to generate DBS therapy activation volumes (15). The preprocessed postoperative magnetization prepared rapid acquisition gradient echo scans were manually aligned with the preimplantation magnetization prepared rapid acquisition gradient echo scans (see Supplement). Automatic coregistra- tion was carried out with a restricted volume of fusion cantered on the mesencephalon, diencephalon, and VS to minimize registration error resulting from brain shift incurred during sur- gery despite minimal brain shift with our surgical technique (16). Registration accuracy was inspected and the process iterated if necessary. All volumes were realigned with a plane parallel to the anterior commissure-posterior commissure line. Clinical and Cognitive Assessments The Y-BOCS was the primary outcome measure to test VC/VS and amSTN DBS effects on OCD symptoms. To test the mechanistic hypothesis, secondary measures were the Montgomery–Åsberg Depression Rating Scale (MADRS) to evaluate mood and the Cambridge Neuropsychological Test Automated Battery Intra-Extra Dimensional Set-Shift (IED) task to evaluate cognitive ﬂexibility. In the IED task, participants progress through nine stages assessing the ability to learn and reverse rules governing correct responses using computer feedback (14). In stages 1 to 7, responses to a speciﬁc stim- ulus dimension are correct. The ability to shift attention away from the previously correct stimulus dimension to a different dimension (i.e., cognitive ﬂexibility) is tested in extradimen- sional set-shifting task stage 8 (EDS). Probabilistic tractography streamlines were generated for each patient in native space. Individual patient DBS VTAs were used as seed masks resulting in four tracts per patient from the amSTN and the VC/VS bilaterally. The corpus callosum was used as an exclusion mask. Cerebrospinal ﬂuid termination masks were used to exclude false positive streamlines. Using the obtained transformations to and from standard space, resulting streamlines were transformed to Montreal Neurolog- ical Institute space, and group averages were generated. The Human Central Nervous System: A Synopsis and Atlas (21) was used to corroborate the relevant anatomical structures. Ventral Capsule and Subthalamic Nucleus DBS for OCD Statistical Analysis Friedman’s test was used to test for DBS effects during the double-blind crossover phases comparing baseline, amSTN, and VC/VS. Signiﬁcance levels were adjusted for multiple comparisons using the false discovery rate (FDR) method of Benjamini and Hochberg (22). Post hoc pairwise Conover tests for signiﬁcant effects were used (23), with FDR corrections also applied. For each variable, the effect of time, stimulation at both sites, and adjunctive CBT was assessed with Friedman’s test across all six time points independent of stimulation type. To test the effect of DBS of amSTN and VC/VS together, Wilcoxon’s tests were used to compare phases when one site was stimulated (time 2 1 time 3) and when both sites were stimulated (time 4 1 time 5) and the effect of CBT, by comparing time 5 and time 6. FDR corrections of signiﬁcance values were applied throughout. To substantiate our effects, we repeated these analyses using more powerful parametric statistics, again correcting for multiple comparisons (see Supplement). The postimplantation magnetization prepared rapidacquisition gradient-echo scan was used to ﬁt the DBS lead model within the MRI artifact produced by the leads. For each patient, DBS pa- rameters during the 12-week phase when each set of electrodes was active and optimized were used to generate activation vol- umes around active DBS contacts in the amSTN and VC/VS with corresponding voltages. Binary image ﬁles of activation volumes with corresponding transformation matrices were exported and processed in MATLAB, version R2016A (The MathWorks, Inc., Natick, MA)using an in-housesoftware to generate Neuroimaging Informatics Technology Initiative volumes for further analysis. Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal Study Design Eligible participants were offered stereotactic ablation or entry to the DBS trial. Prescribed medications were kept constant unless clinically indicated. Before surgery, all patients 2 Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal Connectivity Analysis Preprocessed data (see Supplement) were fed into BedpostX (FSL v5.0) to estimate ﬁber orientations. Up to three crossing Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 3 3 Ventral Capsule and Subthalamic Nucleus DBS for OCD Biological Psychiatry Ventral Capsule and Subthalamic Nucleus DBS for OCD l y Biological Psychiatry Ventral Capsule and Subthalamic Nucleus DBS for OCD Ventral Capsule and Subthalamic Nucleus DBS for OCD Table 1. Patient Details Table 1. Patient Details Patient No./Sex Age at Onset, Years Age at Surgery, Years Illness Duration, Years Disability at Study Entry Medication 1/Female 16 38 22 Living in inpatient unit; failed supportive accommodation Escitalopram 20 mg; risperidone 0.5 mg; trazodone 150 mg; clomipramine 200 mg 2/Male 16 38 22 Extreme avoidance; impaired social function Tramadol 200 mg; memantine 35 mg; pregabalin 300 mg; citalopram 120 mg; quetiapine 25 mg; clonazepam 0.75 mg 3/Male 32 62 30 Largely housebound; requiring help with ADLs Aspirin 75 mg; omeprazole 20 mg; sitagliptin 100 mg; nitrazepam 10 mg as needed 4/Male 17 37 20 Largely housebound; severely impaired in ADLs Escitalopram 40 mg; aripiprazole 10 mg 5/Male 32 55 23 Largely housebound; unable to live independently Aripiprazole 20 mg; chlorpromazine 400 mg; pregabalin 300 mg; propranolol 40 mg; sertraline 400 mg; zopiclone 7.5 mg 6/Male 15 43 28 Extreme avoidance; impaired social function Pregabalin 600 mg; aripiprazole 20 mg; sertraline 200 mg ADLs, activities of daily living. mood instability. Patient 6 required DBS adjustment during the OPT phase because of worsening of OCD. Patient 1’s OCD symptoms improved during VC/VS DBS but worsened when switched to amSTN. Stimulation adjustment over 2 weeks made no improvement. Because of patient distress, stimula- tion reverted to the VC/VS site. The scores entered for the amSTN stage for this participant were at the point of switching from amSTN back to VC/VS, akin to last entry carried forward. This participant continued with subsequent trial phases per protocol. VC/VS: p , .001; amSTN vs. VC/VS: p = .001). Changes in EDS errors were signiﬁcant for amSTN but not VC/VS DBS (baseline vs. amSTN: p = .003; baseline vs. VC/VS: p = .157; amSTN vs. VC/VS: p = .018). Parametric analyses substantiated these effects other than the post hoc comparison of baseline and amSTN DBS on the MADRS, which was not signiﬁcant (see Supplement). Previous studies have deﬁned a DBS response as $35% reduction in baseline Y-BOCS (5–8). Connectivity Analysis The proportion of patients achieving responder status at the end of each phase was the following: amSTN phase, 3 of 6; VC/VS phase, 5 of 6; COMB phase, 5 of 6; OPT phase, 6 of 6; OPT plus adjunctive CBT phase, 6 of 6. Comparison of VC/VS and amSTN DBS All participants completed the IED task, and the only signiﬁ- cant DBS effect was an improvement in EDS errors. There were signiﬁcant improvements in Y-BOCS score (Table 2), MADRS score, and EDS errors following DBS (Figure 1). All Friedman tests were signiﬁcant, controlling for FDR: Y-BOCS score (c2 2 = 9.0, p = .017), MADRS score (c2 2 = 10.33; p = .017), EDS errors (c2 2 = 7.00, p = .03). Y-BOCS scores signiﬁcantly improved following both amSTN and VC/VS DBS (baseline vs. amSTN: p , .001; baseline vs. VC/VS: p , .001; amSTN vs. VC/VS: p = 1.00). Changes in MADRS scores were signiﬁcantly different from baseline for both amSTN and VC/VS DBS. The magnitude of the amSTN effect was signiﬁcantly greater than VC/VS (baseline vs. amSTN: p = .023; baseline vs. Table 2. Y-BOCS Scores at Baseline and During Five Stimulation Phases Patient Baseline amSTN VC/VS COMB OPT AdCBT 1 38 32 (16)a 22 (42) 17 (55) 18 (53) 13 (66) 2 34 26 (23) 29 (15) 23 (32) 20 (41) 21 (38) 3 37 17 (55) 18 (51) 12 (68) 10 (73) 2 (95) 4 38 20 (47) 13 (66) 16 (58) 10 (74) 7 (82) 5 34 23 (32) 17 (50) 17 (50) 15 (56) 13 (62) 6 36 1 (97) 3 (92) 0 (100) 13 (64) 0 (100) Mean 6 SEM 36.17 6 0.75 19.83 6 4.32 17.00 6 3.57 14.17 6 3.18 14.33 6 1.69 9.33 6 3.21 Values in parentheses indicate % reduction from baseline. amSTN deep brain stimulation was the initial condition for patients 4, 5, and 6. AdCBT, optimal combined settings plus adjunctive cognitive behavioral therapy; amSTN, anteromedial subthalamic nucleus; COMB, combined phase; OPT, optimal combined settings; VC/VS, ventral capsule/ventral striatum; Y-BOCS, Yale-Brown Obsessive Compulsive Scale. aThis score is last entry carried forward. Y-BOCS scores range from 1 to 40 and are categorized according to severity as follows: 0–7 = subclinical; 8–15 = mild; 16–23 = moderate; 24–31 = severe; 32–40 = extreme. Effects of Time, Combined DBS, and Adjunctive CBT Following FDR corrections across all three Friedman’s tests, there was a signiﬁcant improvement in Y-BOCS and MADRS scores over time, and EDS errors did not change: Y-BOCS score (c2 5 = 24.11, p , .001), MADRS score (c2 5 = 16.49, p = .006), EDS errors (c2 5 = 6.59, p = .253). Following FDR corrections of post hoc comparisons, there were no statisti- cally signiﬁcant changes when combined DBS of both sites was compared with single-site stimulation (Y-BOCS: p = .116; MADRS: p = .146) or when DBS at both sites was compared Table 2. Y-BOCS Scores at Baseline and During Five Stimulation Phases Table 2. Y-BOCS Scores at Baseline and During Five Stimulation Phases Patient Baseline amSTN VC/VS COMB OPT AdCBT 1 38 32 (16)a 22 (42) 17 (55) 18 (53) 13 (66) 2 34 26 (23) 29 (15) 23 (32) 20 (41) 21 (38) 3 37 17 (55) 18 (51) 12 (68) 10 (73) 2 (95) 4 38 20 (47) 13 (66) 16 (58) 10 (74) 7 (82) 5 34 23 (32) 17 (50) 17 (50) 15 (56) 13 (62) 6 36 1 (97) 3 (92) 0 (100) 13 (64) 0 (100) Mean 6 SEM 36.17 6 0.75 19.83 6 4.32 17.00 6 3.57 14.17 6 3.18 14.33 6 1.69 9.33 6 3.21 Values in parentheses indicate % reduction from baseline. amSTN deep brain stimulation was the initial condition for patients 4, 5, and 6. AdCBT, optimal combined settings plus adjunctive cognitive behavioral therapy; amSTN, anteromedial subthalamic nucleus; COMB, combined phase; OPT, optimal combined settings; VC/VS, ventral capsule/ventral striatum; Y-BOCS, Yale-Brown Obsessive Compulsive Scale. aThis score is last entry carried forward. Y-BOCS scores range from 1 to 40 and are categorized according to severity as follows: 0–7 = subclinical; 8–15 = mild; 16–23 = moderate; 24–31 = severe; 32–40 = extreme. phase; OPT, optimal combined settings; VC/VS, ventral capsule/ventral striatum; Y-BOCS, Yale-Brown Obsessive Compulsive Scale. aThis score is last entry carried forward. Y-BOCS scores range from 1 to 40 and are categorized according to severity as follows: 0–7 = subclinical; 8–15 = mild; 16–23 = moderate; 24–31 = severe; 32–40 = extreme. Effects of Time, Combined DBS, and Adjunctive CBT 4 Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 4 Biological Psychiatry - -, 2019; -:-–- w Ventral Capsule and Subthalamic Nucleus DBS for OCD Biological Psychiatry Ventral Capsule and Subthalamic Nucleus DBS for OCD OCD Symptoms (Y-BOCS) Depressed Mood (MADRS) Cognitive Inﬂexibility (EDS Log Errors) NS NS Baseline amSTN VC/VS Both Optimal Post CBT 0 20 10 30 40 0 20 40 60 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Figure 1. Mean Yale-Brown Obsessive Compulsive Scale (Y-BOCS) score, Montgomery–Åsberg Depression Rating Scale (MADRS) score, and extra- dimensional set-shifting (EDS) log errors at baseline and following deep brain stimulation phases: anteromedial subthalamic nucleus (amSTN), ventral capsule/ ventral striatum (VC/VS); combined amSTN and VC/VS DBS (Both); optimal combined settings (Optimal); optimal combined settings plus cognitive behavioral therapy (Post CBT). The amSTN and VC/VS phases followed a randomized counterbalanced design. Both the Optimal and Post-CBT phases were open and sequential. Y-BOCS scores range from 1 to 40: 0–7 = subclinical; 8–15 = mild; 16–23 = moderate; 24–31 = severe; 32–40 = extreme. MADRS scores range from 1 to 60: 0–6 = normal; 7–19 = mild; 20–34 = moderate; 35–60 = severe. p , .05; p , .01; *p # .001. NS, not signiﬁcant; OCD, obsessive-compulsive disorder. Depressed Mood (MADRS) 20 40 Figure 1. Mean Yale-Brown Obsessive Compulsive Scale (Y-BOCS) score, Montgomery–Åsberg Depression Rating Scale (MADRS) score, and extra- dimensional set-shifting (EDS) log errors at baseline and following deep brain stimulation phases: anteromedial subthalamic nucleus (amSTN), ventral capsule/ ventral striatum (VC/VS); combined amSTN and VC/VS DBS (Both); optimal combined settings (Optimal); optimal combined settings plus cognitive behavioral therapy (Post CBT). The amSTN and VC/VS phases followed a randomized counterbalanced design. Both the Optimal and Post-CBT phases were open and sequential. Y-BOCS scores range from 1 to 40: 0–7 = subclinical; 8–15 = mild; 16–23 = moderate; 24–31 = severe; 32–40 = extreme. MADRS scores range from 1 to 60: 0–6 = normal; 7–19 = mild; 20–34 = moderate; 35–60 = severe. p , .05; p , .01; *p # .001. NS, not signiﬁcant; OCD, obsessive-compulsive disorder. DISCUSSION This is the ﬁrst study to compare, in the same patients, DBS of two brain sites for severe OCD. In a within-subjects, random- ized, double-blind, counterbalanced comparison of amSTN and VC/VS stimulation in 6 patients, we were able to address important questions regarding the efﬁcacy and mechanism of DBS at each site. All patients had been ill for at least 20 years and failed to respond to high doses of medication plus inten- sive CBT. DBS signiﬁcantly reduced OCD symptoms during stimulation of the VC/VS and amSTN, and the magnitude of the reduction at each site, measured with the Y-BOCS, did not differ. In contrast, there were different effects of DBS on mood and cognition. DBS of the amSTN, but not the VC/VS, signif- icantly improved cognitive ﬂexibility (EDS errors). DBS of the VC/VS elicited a striking improvement in mood, measured by the MADRS, which was to a greater degree than amSTN DBS. Tractography from optimally activated electrode contacts at each site suggests that these dissociated effects reﬂected DBS modulation of distinct brain networks. Adverse Events with adjunctive CBT (Y-BOCS: p = .092; MADRS p = .223). Parametric analyses substantiated these ﬁndings (see Supplement). with adjunctive CBT (Y-BOCS: p = .092; MADRS p = .223). Parametric analyses substantiated these ﬁndings (see Supplement). There were no adverse events associated with surgery. The most common adverse event during the DBS trial was hypo- mania (elevated mood or irritability, racing thoughts, disinhi- bition), occurring within hours after stimulation adjustment and remedied by further adjustment. This was witnessed twice each during amSTN and VC/VS DBS and three times during the COMB phase. One patient reported hypomania-like symptoms during the OPT phase on a more sustained basis (racing thoughts and urges to steal) and required frequent admissions for adjustment of DBS for unstable mood. The VC/VS battery became depleted and was replaced once in 3 patients and twice in 1 patient during open phases of the study, without surgical complications (Supplemental Table S3). Amplitude of Active Contacts VTAs Postoperative stereotactic MRI veriﬁed that amSTN electrodes had at least two contacts within the amSTN and that VC/VS electrodes had two or three contacts within the VC and one or two contacts in the nucleus accumbens. For VC/VS, the most dorsal contacts produced the optimal clinical response for all patients, and the mean stimulation amplitude was 5.85 6 1.2 V (Supplemental Tables S1 and S2). For the amSTN, the deepest contacts were most effective for all patients, and the mean stimulation amplitude was 1.56 6 0.82 V (Supplemental Tables S1 and S2). The average VC/VS VTA was centered on the white matter in the ventral portion of the anterior limb of the internal capsule and encroached slightly on adjacent portions of the nucleus accumbens, head of caudate, globus pallidus, and putamen (Figure 2). The average amSTN VTA was centered on the anterior-inferior medial border of the STN spreading into the ventral tegmental area (Figure 2). Exploratory multiple re- gressions showed that changes in Y-BOCS score, MADRS score, or EDS errors did not predict STN and VC volumes of tissue activation following STN and VC DBS, respectively (range of B values: 20.750 to 0.625; range of t values: 21.988 to 1.254). Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 5 Tractography The average streamlines generated from individual amSTN VTAs were connected to the lateral orbitofrontal cortex (OFC), dorsal anterior cingulate cortex (DACC), dorsolateral prefrontal cortex (DLPFC), and medial forebrain bundle. The average streamlines generated from individual VC VTAs were connected to the medial OFC, the mediodorsal thalamus, the amygdala via the amygdalofugal pathway, the hypothalamus, and the habe- nula via the habenulointerpeduncular tract (Figure 3A, B). Indi- vidual patient streamlines are shown in Supplemental Figure S2. Many studies suggest that orbitofrontal (OFC) cortico- striato-thalamic circuitry is dysfunctional in OCD. It is now recognized that the medial and lateral aspects occupy Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 5 Biological Psychiatry Ventral Capsule and Subthalamic Nucleus DBS for OCD Figure 2. Average deep brain stimulation volume of tissue activation (VTA) in the ventral capsule (VC-VTA) and anteromedial subthalamic nucleus (amSTN-VTA). Figure 2. Average deep brain stimulation volume of tissue activation (VTA) in the ventral capsule (VC-VTA) and anteromedial subthalamic nucleus (amSTN-VTA). Figure 2. Average deep brain stimulation volume of tissue activation (VTA) in the ventral capsule (VC-VTA) and anteromedial subthalamic nucleus ( STN VTA) brain stimulation volume of tissue activation (VTA) in the ventral capsule (VC-VTA) and anteromedial subthalamic nucleu Figure 2. Average deep brain stimulation volume of tissue activation (VTA) in the ventral capsule (VC-VTA) and ante (amSTN-VTA) form the limbic and cognitive components of the hyperdirect pathway (28). The STN is integral to basal ganglia circuitry, and input from the hyperdirect pathway provides a mechanism whereby cortical inﬂuences can rapidly suppress the mani- festation of behaviors that are already being programmed (29). Taking all ﬁndings together, amSTN DBS may regulate aber- rant information processing in the hyperdirect pathway from the lateral OFC, DLPFC, and DACC (28) and enable patients to interrupt their compulsive cycle of repetitive acts and thoughts through improving cognitive ﬂexibility and goal-directed planning. separate trajectories within the OFC cortico-striato-thalamic circuitry and are functionally different (24). Abnormal func- tional connectivity between lateral OFC (Brodmann areas 10, 11, 47) and caudate nucleus is associated with EDS errors in OCD (25). Compatible with this, amSTN DBS improves glucose metabolism in OFC (Brodmann areas 10, 11) in association with better Y-BOCS scores (26). 6 Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal Tractography A recent functional MRI study of OCD found speciﬁc, abnormal connectivity between the basolateral amygdala and medial OFC in OCD, which was predictive of successful treatment with CBT (35). The habenulointerpeduncular tract has been implicated in the development of depression via in- hibition of brainstem serotonergic raphe nuclei (36). In OCD, selective serotonin reuptake inhibitors increase serotonin neurotransmission and are the mainstay of pharmacological symptoms (37). Thus, involving the medial pathway, and the habe the striking improveme We anticipated that enable patients to utiliz (38), but this was not fo Y-BOCS changes befo were already at a mild making it difﬁcult for Figu anter activa lines tion corte intern medi amygdalofugal pathway is an output tract from the basolateral nucleus of the amygdala to the mediodorsal thalamus and OFC. A recent functional MRI study of OCD found speciﬁc, abnormal connectivity between the basolateral amygdala and medial OFC in OCD, which was predictive of successful treatment with CBT (35). The habenulointerpeduncular tract has been implicated in the development of depression via in- hibition of brainstem serotonergic raphe nuclei (36). In OCD, selective serotonin reuptake inhibitors increase serotonin neurotransmission and are the mainstay of pharmacological symptoms (37). Thus, modulation by VC DBS of circuitry involving the medial OFC, the associated amygdalofugal pathway, and the habenulointerpeduncular tract may mediate the striking improvement in mood as well as OCD symptoms. We anticipated that a positive response to DBS would enable patients to utilize CBT and boost the OCD effect further (38), but this was not found. However, inspection of individual Y-BOCS changes before CBT showed that 4 of 6 patients were already at a mild level of OCD symptom severity, thus making it difﬁcult for further measurable gains to be made Figure 3. (A) Group average streamlines from anteromedial subthalamic nucleus volumes of tissue activation (amSTN-VTA). (B) Group average stream- lines from ventral capsule volumes of tissue activa- tion (VC-VTA). DACC, dorsal anterior cingulate cortex; DLPFC, dorsolateral prefrontal cortex; IC, internal capsule; LAT, lateral; MED, medial; MFB, medial forebrain bundle; OFC, orbitofrontal cortex. Figure 3. (A) Group average streamlines from anteromedial subthalamic nucleus volumes of tissue activation (amSTN-VTA). (B) Group average stream- lines from ventral capsule volumes of tissue activa- tion (VC-VTA). DACC, dorsal anterior cingulate cortex; DLPFC, dorsolateral prefrontal cortex; IC, internal capsule; LAT, lateral; MED, medial; MFB, medial forebrain bundle; OFC, orbitofrontal cortex. symptoms (37). Tractography The EDS tests the ability to adapt to changing circumstances by shifting the focus of attention and acquiring new responses (i.e., cognitive ﬂexi- bility); this ability is a putative endophenotype of OCD, being also impaired in ﬁrst-degree unaffected relatives (11). These ﬁndings are compatible with the observations of this study that the amSTN DBS site was associated with improved EDS performance and that tractography streamlines from activated contacts connected to the lateral OFC. For all patients, the VC/VS dorsal-most electrode contacts were the most effective, suggesting that stimulation of the ventral anterior limb of the internal capsule (VC), rather than the VS/nucleus accumbens, mediated the clinical effect of improved OCD and mood. This was conﬁrmed by the volume of tissue activation, which was centered on the VC with only minor encroachment on the surrounding gray matter. Other studies support this conclusion (8,30). Connectivity analysis also showed streamlines linking the DLPFC and DACC with the amSTN. In addition to the OFC, the function of these two cortical areas has been linked to the severity of OCD symptoms measured by the Y-BOCS. For example, in a functional MRI study, reduced functional con- nectivity between the DLPFC and putamen was associated with both impaired goal-directed planning and OCD symptom severity measured by the Y-BOCS (25). In addition, ablation of the DACC, performed during cingulotomy, is an established neurosurgical procedure shown to alleviate symptoms in pa- tients with medically refractory OCD (27). Nonhuman primate tracing studies, shown to substantiate human tractography (31), suggest that VC DBS captures ﬁbers from the medial OFC to the thalamus and more posterior areas (32,33). This is in keeping with the tractography ﬁnding here of a streamline extending anteriorly to the medial OFC and pos- teriorly to the dorsomedial thalamus, as well as streamlines involving the amygdalofugal pathway and habenulointerpe- duncular tract. The medial OFC is hyperactive during the early processing of threat-related stimuli in OCD (34). The In nonhuman primates, the amSTN receives uninterrupted projections from the OFC, DLPFC, and DACC, considered to 6 Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 6 Ventral Capsule and Subthalamic Nucleus DBS for OCD Biological Psychiatry Ventral Capsule and Subthalamic Nucleus DBS for OCD Biological Psychiatry Ventral Capsule and Subthalamic Nucleus DBS for OCD Ventral Capsule and Subthalamic Nucleus DBS for OCD amygdalofugal pathway is an output tract from the basolateral nucleus of the amygdala to the mediodorsal thalamus and OFC. Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 7 ACKNOWLEDGMENTS AND DISCLOSURES This work was supported by Medical Research Council Grant No. MR/ J012009/1, National Institute for Health Research University College London Hospitals Biomedical Research Centre (to EMJ), the Monument Trust and the Parkinson’s Appeal UK (to MH, LZ, TF, PL), the Brain Research Trust (to HA), Wellcome Trust Award Grant No. WT 104631/Z/14/Z (to TWR), and the National Institute for Health Research Cambridge Biomedical Research Centre mental health theme (to TWR, BJS, AMA-S). 2. Boschen MJ, Drummond LM (2012): Community treatment of se- vere, refractory obsessive-compulsive disorder. Behav Res Ther 50:203–209. 3. Matthews K, Eljamel MS (2003): Status of neurosurgery for mental disorder in Scotland. Selective literature review and overview of cur- rent clinical activity. Br J Psychiatry 182:404–411. We thank the patients who participated in the study, Lesley Morton and staff on Hughlings Jackson ward, and the Queen Square DBS nursing team. The full trial protocol is available at https://www.ucl.ac.uk/ ion/research/departments/motor-neuroscience-and-movement-disorders/ research/professor-eileen-joyce. 4. Nuttin B, Cosyns P, Demeulemeester H, Gybels J, Meyerson B (1999): Electrical stimulation in anterior limbs of internal capsules in patients with obsessive-compulsive disorder. Lancet 354:1526. 5. Abelson JL, Curtis GC, Sagher O, Albucher RC, Harrigan M, Taylor SF, et al. (2005): Deep brain stimulation for refractory obsessive-compulsive disorder. Biol Psychiatry 57:510–516. TF has received honoraria for speaking at meetings sponsored by Bial, Proﬁle Pharma, and Britannia Pharmaceuticals and serving on advisory boards for Bial and Oxford Biomedica. NAF has received honoraria for speaking at meetings from Abbott, Wiley, and the British Association for Psychopharmacology; ﬁnancial support to attend scientiﬁc meetings from the International Society for Affective Disorders, International Forum of Mood and Anxiety Disorders, European College of Neuro- psychopharmacology, British Association for Psychopharmacology, and the Royal College of Psychiatrists; ﬁnancial royalties for publications from Oxford University Press; and payment for editorial duties from Taylor and Francis. KM has received honoraria for chairing advisory boards sponsored by Medtronic; received research project funding from Merck Serono, Lundbeck, Reckitt Benckiser, St. Jude Medical (Abbott), and Indivior; and received travel and accommodation support from Medtronic and St Jude Medical (Abbott) to attend scientiﬁc meetings. TWR has received royalties from Cambridge Cognition; consulted for Cambridge Cognition, Lundbeck, Mundipharma, and Unilever; and received research grants from Lundbeck and Shionogi. BJS has received honoraria as a consultant to Cambridge Cognition, PEAK, and Mundipharma. MH, PL, and LC have has received honoraria and travel expenses from Medtronic, St. ARTICLE INFORMATION From the Department of Clinical and Movement Neurosciences (HT, HA, TF, PL, MJ, LZ, MH, EMJ), University College London Queen Square Institute of Neurology, London; The National Hospital for Neurology and Neurosurgery (HT, HA, TF, PL, LZ, MH, EMJ), London; Highly Specialised Service for OCD and BDD (England) (LMD), SW London and St George’s NHS Trust, London; Departments of Psychology (AMA-S, TWR) and Psychiatry (BJS), Behav- ioural and Clinical Neuroscience Institute, University of Cambridge, Cam- bridge; Highly Specialised Service for OCD and BDD (England) (NAF), Hertfordshire Partnership University NHS Foundation Trust, Welwyn Garden City; Centre for Clinical & Health Research Services (NAF), School of Life and Medical Sciences, University of Hertfordshire, Hatﬁeld; Division of Neuroscience (KM), School of Medicine, University of Dundee, Dundee; and Advanced Interventions Service (KM), NHS Tayside, Ninewells Hospital and Medical School, Dundee, United Kingdom. In summary, DBS of the VC and amSTN signiﬁcantly alle- viated OCD symptoms, and the magnitude of effect did not differ between these sites, suggesting that both targets are equally efﬁcacious. The ﬁnding that amSTN but not VC DBS improved cognitive ﬂexibility and that the effect of DBS on mood was signiﬁcantly greater for VC DBS implicates the involvement of different neural circuitries associated with distinct symptoms in OCD. Tractography ﬁndings revealed that VC and amSTN DBS modulate distinct brain networks impli- cated in OCD and are compatible with these clinical and cognitive observations. Address correspondence to Eileen M. Joyce, FRCPsych, Ph.D., Box 19, National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, United Kingdom; E-mail: e.joyce@ucl.ac.uk. Received Mar 29, 2018; revised Dec 13, 2018; accepted Jan 3, 2019. Supplementary material cited in this article is available online at https:// doi.org/10.1016/j.biopsych.2019.01.017. Ventral Capsule and Subthalamic Nucleus DBS for OCD other authors report no biomedical ﬁnancial interests or potential conﬂicts of interest. Adverse effects were mainly stimulation-induced hypoma- nia, also reported in other studies. These were not more common during stimulation of either site or combined stimulation. ISRCTN: Deep Brain Stimulation for Severe Obsessive Compulsive Disorder; http://www.isrctn.com/ISRCTN18430630; ISRCTN18430630. There are several limitations to the study, the main one being the small sample size. However, when comparing the efﬁcacy of the two DBS sites, patients served as their own controls in an innovative design, and the conclusions were robust to adjustment for multiple comparisons and parametric and nonparametric analyses. Nevertheless, it would be important to test our hypothesis in a larger group of patients when the mechanistic actions of STN and VC DBS on recovery from OCD can be evaluated in more detail. Another limitation is the possible confounding effect of combined stimulation and CBT with time. Future studies could compare the effect of additional CBT at an earlier stage. REFERENCES 1. Skapinakis P, Caldwell DM, Hollingworth W, Bryden P, Fineberg NA, Salkovskis P, et al. (2016): Pharmacological and psychotherapeutic interventions for management of obsessive-compulsive disorder in adults: A systematic review and network meta-analysis. Lancet Psy- chiatry 3:730–739. Tractography Thus, modulation by VC DBS of circuitry involving the medial OFC, the associated amygdalofugal pathway, and the habenulointerpeduncular tract may mediate the striking improvement in mood as well as OCD symptoms. We anticipated that a positive response to DBS would enable patients to utilize CBT and boost the OCD effect further (38), but this was not found. However, inspection of individual Y-BOCS changes before CBT showed that 4 of 6 patients were already at a mild level of OCD symptom severity, thus making it difﬁcult for further measurable gains to be made during CBT. amygdalofugal pathway is an output tract from the basolateral nucleus of the amygdala to the mediodorsal thalamus and OFC. A recent functional MRI study of OCD found speciﬁc, abnormal connectivity between the basolateral amygdala and medial OFC in OCD, which was predictive of successful treatment with CBT (35). The habenulointerpeduncular tract has been implicated in the development of depression via in- hibition of brainstem serotonergic raphe nuclei (36). In OCD, selective serotonin reuptake inhibitors increase serotonin neurotransmission and are the mainstay of pharmacological treatment because they improve both mood and core OCD amygdalofugal pathway is an output tract from the basolateral nucleus of the amygdala to the mediodorsal thalamus and OFC. A recent functional MRI study of OCD found speciﬁc, abnormal connectivity between the basolateral amygdala and medial OFC in OCD, which was predictive of successful treatment with CBT (35). The habenulointerpeduncular tract has been implicated in the development of depression via in- hibition of brainstem serotonergic raphe nuclei (36). In OCD, selective serotonin reuptake inhibitors increase serotonin neurotransmission and are the mainstay of pharmacological treatment because they improve both mood and core OCD Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 7 7 Biological Psychiatry Biological Psychiatry 8 Biological Psychiatry - -, 2019; -:-–- w Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal Ventral Capsule and Subthalamic Nucleus DBS for OCD Ventral Capsule and Subthalamic Nucleus DBS for OCD 12. Holl EM, Petersen EA, Foltynie T, Martinez-Torres I, Limousin P, Hariz MI, et al. (2010): Improving targeting in image-guided frame- based deep brain stimulation. Neurosurgery 67:437–447. stimulation in obsessive-compulsive disorder: A positron emission tomography study. Biol Psychiatry 68:1016–1022. stimulation in obsessive-compulsive disorder: A positron emission tomography study. Biol Psychiatry 68:1016–1022. 27. Dougherty DD, Baer L, Cosgrove GR, Cassem EH, Price BH, Nierenberg AA, et al. (2002): Prospective long-term follow-up of 44 patients who received cingulotomy for treatment-refractory obsessive- compulsive disorder. Am J Psychiatry 159:269–275. 13. Zrinzo L, Yoshida F, Hariz MI, Thornton J, Foltynie T, Yousry TA, et al. (2011): Clinical safety of brain magnetic resonance imaging with implanted deep brain stimulation hardware:Large case seriesandreview of the literature. World Neurosurg 76:164–172; discussion 169-173. 28. Haynes WI, Haber SN (2013): The organization of prefrontal- subthalamic inputs in primates provides an anatomical substrate for both functional speciﬁcity and integration: Implications for Basal Ganglia models and deep brain stimulation. J Neurosci 33:4804– 4814. 14. Sahakian BJ, Owen AM (1992): Computerized assessment in neuro- psychiatry using CANTAB: Discussion paper. J R Soc Med 85:399–402. 15. Astrom M, Zrinzo LU, Tisch S, Tripoliti E, Hariz MI, Wardell K (2009): Method for patient-speciﬁc ﬁnite element modeling and simulation of deep brain stimulation. Med Biol Eng Comput 47:21–28. 29. Aron AR, Herz DM, Brown P, Forstmann BU, Zaghloul K (2016): Frontosubthalamic circuits for control of action and cognition. J Neurosci 36:11489–11495. 16. Petersen EA, Holl EM, Martinez-Torres I, Foltynie T, Limousin P, Hariz MI, et al. (2010): Minimizing brain shift in stereotactic functional neurosurgery. Neurosurgery 67:ons213–ons221; discussion ons221. 30. van den Munckhof P, Bosch DA, Mantione MH, Figee M, Denys DA, Schuurman PR (2013): Active stimulation site of nucleus accumbens deep brain stimulation in obsessive-compulsive disorder is localized in the ventral internal capsule. Acta Neurochir Suppl 117:53–59. 17. Jbabdi S, Sotiropoulos SN, Savio AM, Grana M, Behrens TE (2012): Model-based analysis of multishell diffusion MR data for tractography: How to get over ﬁtting problems. Magn Reson Med 68:1846–1855. 31. Safadi Z, Grisot G, Jbabdi S, Behrens TE, Heilbronner SR, McLaughlin NCR, et al. (2018): Functional segmentation of the anterior limb of the internal capsule: Linking white matter abnormalities to speciﬁc connections. J Neurosci 38:2106–2117. 18. Hernandez M, Guerrero GD, Cecilia JM, Garcia JM, Inuggi A, Jbabdi S, et al. Ventral Capsule and Subthalamic Nucleus DBS for OCD (2013): Accelerating ﬁbre orientation estimation from diffusion weighted magnetic resonance imaging using GPUs. PLoS One 8:e61892. 32. Haber SN, Knutson B (2010): The reward circuit: Linking primate anatomy and human imaging. Neuropsychopharmacology 35:4–26. 19. Behrens TE, Berg HJ, Jbabdi S, Rushworth MF, Woolrich MW (2007): Probabilistic diffusion tractography with multiple ﬁbre orientations: What can we gain? Neuroimage 34:144–155. 33. Lehman JF, Greenberg BD, McIntyre CC, Rasmussen SA, Haber SN (2011): Rules ventral prefrontal cortical axons use to reach their targets: Implications for diffusion tensor imaging tractography and deep brain stimulation for psychiatric illness. J Neurosci 31:10392– 10402. 20. Jbabdi S, Johansen-Berg H (2011): Tractography: Where do we go from here? Brain Connect 1:169–183. 21. Nieuwenhuys R, Voogd J, van Huijzen C (2008): The Human Central Nervous System: A Synopsis and Atlas, 4th ed. Berlin, Germany: Springer, 2008. 34. Apergis-Schoute AM, Gillan CM, Fineberg NA, Fernandez-Egea E, Sahakian BJ, Robbins TW (2017): Neural basis of impaired safety signaling in obsessive compulsive disorder. Proc Natl Acad Sci U S A 114:3216–3221. 22. Benjamini Y, Hochberg Y (1995): Controlling the false discovery rate: A practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 57:289–300. 23. Pohlert T (2014): The Pairwise Multiple Comparison of Mean Ranks Package (PMCMR). R Package. Available at: https://CRAN.R-project. org/package=PMCMR. Accessed August 7, 2018. 35. Fullana MA, Zhu X, Alonso P, Cardoner N, Real E, Lopez-Sola C, et al. (2017): Basolateral amygdala-ventromedial prefrontal cortex connec- tivity predicts cognitive behavioural therapy outcome in adults with obsessive-compulsive disorder. J Psychiatry Neurosci 42:378–385. 24. Fettes P, Schulze L, Downar J (2017): Cortico-Striatal-thalamic loop circuits of the orbitofrontal cortex: Promising therapeutic targets in psychiatric illness. Front Syst Neurosci 11:25. 36. Hikosaka O (2010): The habenula: From stress evasion to value-based decision-making. Nat Rev Neurosci 11:503–513. 25. Vaghi MM, Vertes PE, Kitzbichler MG, Apergis-Schoute AM, van der Flier FE, Fineberg NA, et al. (2017): Speciﬁc frontostriatal circuits for impaired cognitive ﬂexibility and goal-directed planning in obsessive- compulsive disorder: Evidence from resting-state functional connec- tivity. Biol Psychiatry 81:708–717. 37. Fineberg NA, Reghunandanan S, Simpson HB, Phillips KA, Richter MA, Matthews K, et al. (2015): Obsessive-compulsive disorder (OCD): Practical strategies for pharmacological and somatic treatment in adults. Psychiatry Res 227:114–125. 38. Mantione M, Nieman DH, Figee M, Denys D (2014): Cognitive- behavioural therapy augments the effects of deep brain stimulation in obsessive-compulsive disorder. Psychol Med 44:3515–3522. 26. ACKNOWLEDGMENTS AND DISCLOSURES Jude, and Boston Scientiﬁc for speaking at meetings. EMJ has received funding from Boston Scientiﬁc, the National Institutes of Health, the International College of Neuropharmacology, and the European College of Neuropsychopharmacology to attend meetings. The 6. Goodman WK, Foote KD, Greenberg BD, Ricciuti N, Bauer R, Ward H, et al. (2010): Deep brain stimulation for intractable obsessive compulsive disorder: Pilot study using a blinded, staggered-onset design. Biol Psychiatry 67:535–542. 7. Denys D, Mantione M, Figee M, van den Munckhof P, Koerselman F, Westenberg H, et al. (2010): Deep brain stimulation of the nucleus accumbens for treatment-refractory obsessive-compulsive disorder. Arch Gen Psychiatry 67:1061–1068. 8. Luyten L, Hendrickx S, Raymaekers S, Gabriels L, Nuttin B (2016): Electrical stimulation in the bed nucleus of the stria terminalis alleviates severe obsessive-compulsive disorder. Mol Psychiatry 21:1272–1280. 9. Greenberg BD, Gabriels LA, Malone DA Jr, Rezai AR, Friehs GM, Okun MS, et al. (2010): Deep brain stimulation of the ventral internal capsule/ventral striatum for obsessive-compulsive disorder: World- wide experience. Mol Psychiatry 15:64–79. 10. Mallet L, Polosan M, Jaafari N, Baup N, Welter ML, Fontaine D, et al. (2008): Subthalamic nucleus stimulation in severe obsessive- compulsive disorder. N Engl J Med 359:2121–2134. 11. Chamberlain SR, Fineberg NA, Menzies LA, Blackwell AD, Bullmore ET, Robbins TW, et al. (2007): Impaired cognitive ﬂexibility and motor inhibition in unaffected ﬁrst-degree relatives of patients with obsessive-compulsive disorder. Am J Psychiatry 164:335–338. 11. Chamberlain SR, Fineberg NA, Menzies LA, Blackwell AD, Bullmore ET, Robbins TW, et al. (2007): Impaired cognitive ﬂexibility and motor inhibition in unaffected ﬁrst-degree relatives of patients with obsessive-compulsive disorder. Am J Psychiatry 164:335–338. 8 Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 8 Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 9 Ventral Capsule and Subthalamic Nucleus DBS for OCD Le Jeune F, Verin M, N’Diaye K, Drapier D, Leray E, Du Montcel ST, et al. (2010): Decrease of prefrontal metabolism after subthalamic Biological Psychiatry - -, 2019; -:-–- www.sobp.org/journal 9 Biological Psychiatry - -, 2019; -:-–- w
https://openalex.org/W4221023792	https://dspace.lib.cranfield.ac.uk/bitstream/1826/17644/1/Non-intrusive_flow_diagnostics-2022.pdf	English	null	Non-intrusive flow diagnostics for unsteady inlet flow distortion measurements in novel aircraft architectures	Progress in aerospace sciences/Progress in Aerospace Sciences	2,022	cc-by	25,340	Non-intrusive flow diagnostics for unsteady inlet flow distortion measurements in novel aircraft architectures Non-intrusive flow diagnostics for unsteady inlet flow distortion measurements in novel aircraft architectures Ulrich Doll a,∗, Matteo Migliorini b, Joni Baikie b, Pavlos K. Zachos b, Ingo Röhle c, Sergey Melnikov c, Jonas Steinbock d, Michael Dues d, Ralf Kapulla a, David G. MacManus b, Nicholas J. Lawson e Experimental Thermal-Hydraulics Group, Paul Scherrer Institute, Forschungstrasse 111, Villigen PSI, 5232, Switzerland P l i E i i C C fi ld U i i MK43 0AL C fi ld U i d Ki d a Experimental Thermal-Hydraulics Group, Paul Scherrer Institute, Forschungstrasse 111, Villigen PSI, 5232, Switzerland b Propulsion Engineering Centre, Cranfield University, MK43 0AL, Cranfield, United Kingdom c Berliner Hochschule für Technik, Luxemburger Str. 10, Berlin, 13353, Germany d ILA R&D GmbH, Rudolf-Schulten-Straße 3, Jülich, 52428, Germany e School of Aerospace, Mechanical & Mechatronic Engineering, The University of Sydney, Rm N304, J11 Level 3, Sydney, NS a Experimental Thermal-Hydraulics Group, Paul Scherrer Institute, Forschungstrasse 111, Villigen PSI, 5232, Switzerlan b Propulsion Engineering Centre, Cranfield University, MK43 0AL, Cranfield, United Kingdom e School of Aerospace, Mechanical & Mechatronic Engineering, The University of Sydney, Rm N304, J11 Level 3, Sydney, NSW 2006, Aust A B S T R A C T Inlet flow distortion is expected to play a major role in future aircraft architectures where complex air induction systems are required to couple the engine with the airframe. The highly unsteady distortions generated by such intake systems can be detrimental to engine performance and were previously linked with loss of engine stability and potentially catastrophic consequences. During aircraft design, inlet flow distortion is typically evaluated at the aerodynamic interface plane, which is defined as a cross-flow plane located at a specific upstream distance from the engine fan. Industrial testing currently puts more emphasis on steady state distortions despite the fact that, historically, unsteady distortions were acknowledged as equally important. This was partially due to the limitations of intrusive measurement methods to deliver unsteady data of high spatial resolution in combination with their high cost and complexity. However, as the development of aircraft with fuselage-integrated engine concepts progresses, the combination of different types of flow distortions is expected to have a strong impact on the engine’s stability margin. Therefore, the need for novel measurement methods able to meet the anticipated demand for more comprehensive flow information is now more critical than ever. In reviewing the capabilities of various non-intrusive methods for inlet distortion measurements, Filtered Rayleigh Scattering (FRS) is found to have the highest potential for synchronously characterising multiple types of inlet flow distortions, since the method has the proven ability to simultaneously measure velocity, static pressure and temperature fields in challenging experimental environments. The attributes of the FRS method are further analysed aiming to deliver a roadmap for its application on ground-based and in-flight measurement environments. Keywords: Air induction systems Convoluted diffusers Inlet flow distortion Unsteady flow Propulsion integration Non-intrusive flow diagnostics Seeding-free measurements Laser based flow measurements Filtered Rayleigh Scattering (FRS) associated engine stability margins are relatively mastered on current aircraft designs with under-wing engine configurations. However, fu- ture commercial aircraft concepts are moving towards novel, closely coupled airframe-engine architectures with convoluted intakes to sup- ply the propulsion system with the required mass flow. This presents a growing need to better understand the influence of unsteady inlet distortions on the stability of the engine across all phases of flight. Unsteady distortions generated by the intake were previously found to be detrimental to engine performance, but may also trigger rotor blade vibrations that can result in reduced component lifetimes or, in extreme cases, cause compressor surge or catastrophic damage to Progress in Aerospace Sciences 130 (2022) 100810 Progress in Aerospace Sciences 130 (2022) 100810 ∗Corresponding author. E-mail address: ulrich.doll@psi.ch (U. Doll). Available online 9 March 2022 0376-0421/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ∗Corresponding author. E-mail address: ulrich.doll@psi.ch (U. Doll). https://doi.org/10.1016/j.paerosci.2022.100810 Received 8 October 2021; Received in revised form 10 February 2022; Accepted 19 February 2022 1. Introduction Novel propulsion systems and their integration with the airframe are expected to play a key role in achieving aviation’s long-term sus- tainability targets. International aviation strategic reports from EASA, NASA and ICAO call for a dramatic reduction in CO2 and NOx emis- sions [1–3]. Reaching these goals will require substantial developments to the airframe and the propulsion system, resulting in unconventional architectures such as boundary layer ingestion (BLI) and distributed propulsion. In these future designs, the engine face will be confronted with highly unsteady and distorted inflow characteristics. Steady-state inlet flow distortion (i.e. variations in air pressure, temperature and angularity at the inlet plane of an aero-engine) and Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. Roman symbols 𝛥𝑃 Total pressure variation 𝑐 Concentration 𝑃 Total pressure 𝑝 Static pressure 𝑃avg Average total pressure over full AIP 𝑇 Temperature 𝑣 Velocity Subscripts c Circumferential r Radial Nomenclature Acronyms 3C Three-component AIM Advanced In-flight Measurement techniques AIP Aerodynamic Interface Plane AIR Aerospace Information Report ARP Aerospace Recommended Practice BLI Boundary Layer Ingestion CRBS Coherent Rayleigh–Brillouin Scattering CRM Common Research Model CW Continuous Wave DGV Doppler Global Velocimetry FLEET Femtosecond Laser Electronic Excitation Tagging FRS Filtered Rayleigh Scattering FWHM Full Width at Half Maximum IR Infrared IRS Interferometric Rayleigh Scattering L2F Laser-2-Focus LDA Laser Doppler Anemometry LIDAR Light Detection And Ranging LIF Laser Induced Fluorescence MPR Multiple Per Revolution MTV Molecular Tagging Velocimetry NGV Nozzle Guide Vane NTF National Transonic Facility OPO Optical Parametric Oscillator PDV Planar Doppler Velocimetry RELIEF Raman Excitation and Laser-Induced Elec- tronic Fluorescence S-PIV Stereoscopic Particle Image Velocimetry SAE Society of Automotive Engineers SD Swirl Directivity SI Swirl Intensity SNR Signal-to-Noise-Ratio SP Swirl Pairs SS Sector Swirl UV Ultra violet VTOL Vertical Take-Off and Landing Greek symbols 𝜌 Density 𝜃 Critical sector angle Circumferential Radial instantaneous data to aid the understanding of the flow distortions that lead to engine stall events remains a key requirement. This becomes even more critical as industry is moving towards an era of strongly close-coupled engine-aircraft configurations. Given the known limita- tions of conventional methods (low spatial and temporal resolution, and intrusive instruments that interact with the flow), existing technologies are inadequate to sufficiently reduce the risk on the development of future systems. 2. Inlet flow distortion in novel propulsion systems engine components. These assessments will be required throughout the aircraft’s development phases as the design is matured, initially through numerical means, then ideally in ground test facilities followed by in- flight testing. To unlock the full potential of these novel concepts, a close integration of airframe, intake and engine design is imperative. 1. Introduction To support this ongoing transformation process, the purpose of this review article is to provide a comprehensive overview on the topic of inlet flow distortion in the context of future airframe-integrated propul- sion systems. After presenting a few examples of these novel concepts, the article provides an introduction to the phenomenology of inlet flow distortion, discusses the effects that inlet flow distortion can have on a propulsion system, how inlet flow distortion is described math- ematically and current measurement practices of the pertinent flow quantities. The second part of the review initially describes the state-of- the-art in non-intrusive distortion measurement, points to drawbacks of current methods and formulates requirements that future measurement technology should be able to meet. This motivates a review of seeding- free laser-optical measurement techniques from the viewpoint of their potential for characterising complex technical flows, specifically with respect to the future ability to use these methods along the entire engine development cycle in ground and flight tests, which ultimately requires the use of optical probes. From the short list of available methods, the filtered Rayleigh scattering (FRS) technique is found to clearly have the highest potential to characterise inlet flow distortions in convoluted intake architectures. The article then continues with a description of current technological hurdles and concludes with an outlook on future FRS instrumentation as an enabler for advanced, seeding-free, optical flow measurements pertinent to in-situ ground and in-flight test scenarios. 2.1. Overview of novel aero-engine architectures This is implemented using a boundary layer ingestion (BLI) fan at the rear of the fuselage. The study compared this to an equivalent reference aircraft entering into service in 2035, where the reference aircraft incorporates 2035 technology but uses a conventional engine arrangement, without the fuselage fan. The propulsive fuselage concept was predicted to reduce CO2 emissions by 4.6% [8]. The ingestion of the boundary layer by the propulsor at the tail results in highly distorted inlet airflow for the fan, which was evaluated computation- ally and experimentally as part of the CENTRELINE project. Using simulations of the fluid flow around the fuselage and wind tunnel experiments on a sub-scale model, the inlet airflow profile was pre- dicted. This inlet profile was then reproduced in a low-speed BLI fan test rig using a distortion screen and the fan performance was mea- sured. A second fan, designed with features to mitigate the detrimental effect of the distortion, demonstrated improved efficiency and stabil- ity [7]. Other concepts of a similar design include the NASA STARC- ABL [9], Boeing SUGAR FREEZE (https://www.boeing.com/features/ innovation-quarterly/aug2017/feature-technical-sugar.page), and Dis- PURSAL (http://www.dispursal.eu/). DisPURSAL is another European design which was introduced before the CENTRELINE concept where the aft propulsor was powered by a gas turbine in the tail, fed by an s-duct, whereas the others are all turboelectric designs. S-ducts also generate high levels of inlet distortion [10,11] which is then ingested by the engine. Fig. 3. NASA N3-X Turboelectric Distributed Propulsion blended wing-body aircraft concept. Source: Courtesy of NASA https://www.nasa.gov/content/hybrid-wing-body-goes- hybrid. boundary layer ingestion reduces the wasted kinetic energy normally in the jet exhaust by ‘‘filling-in’’ the momentum deficit in the fuselage wake. However, the boundary layer growth along the fuselage results in a non-uniform inflow, which is ingested by the intake duct and presented to the engine. This requires consideration as the benefit of BLI may be partially offset by the effect of the distorted propulsor inflow on fan performance. The ONERA Nova is an aircraft concept with a similar fuselage configuration, which also incorporates boundary layer ingesting engines mounted at the rear of the fuselage [15]. y g g g g The N3-X is a 300 passenger, 7500 nmi range civil transport aircraft concept designed to meet the NASA Subsonic Fixed Wing project ‘N+3 generation’ fuel burn reduction goal. 2.1. Overview of novel aero-engine architectures Novel concepts for civil transport aircraft incorporate unconven- tional aero-engine propulsion system architectures (Figs. 1–3). Al- though the designs have overall benefits in terms of efficiency and noise [4], these architectures present more significant engine-airframe interaction effects compared to the conventional arrangement of en- gines on pylons under the wing [5,6]. At cruise conditions, conven- tional under-wing podded engines ingest relatively ‘‘clean’’, undis- turbed airflow, while the unconventional designs embed the engine intakes in the fuselage, which leads to the inlet airflow ingested by the engines being highly distorted by the upstream airframe geometry. This has consequences for engine stability and efficiency [6,7]. Current practice for aero-engine testing and safety certification re- lies on only a few intrusive measurements of pressure and temperature (at a low temporal and spatial resolution) to quantify the flow distortion levels at the engine face. Although unsteady flow distortion has been identified as a key detrimental factor to aero-engine stability, the low maturity of advanced flow measurement methods in combination with the high risk of integrating complex measurement systems into engine test facilities have prevented the development of design metrics for distortion tolerant aero-engines. Consequently, the necessity for richer 2 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 2. Render of the D8 ‘‘Double Bubble’’ aircraft concept. Source: Courtesy of NASA https://www.nasa.gov/content/ the-double-bubble-d8-0. Fig. 1. Artist’s Impression of the CENTRELINE turbo-electric propulsive fuselage aircraft design. Source: https://www.centreline.eu/. Fig. 2. Render of the D8 ‘‘Double Bubble’’ aircraft concept. Source: Courtesy of NASA https://www.nasa.gov/content/ the-double-bubble-d8-0. Fig. 1. Artist’s Impression of the CENTRELINE turbo-electric propulsive fuselage aircraft design. Source: https://www.centreline.eu/. Source: https://www.centreline.eu/. Fig. 3. NASA N3-X Turboelectric Distributed Propulsion blended wing-body aircraft concept. Source: Courtesy of NASA https://www.nasa.gov/content/hybrid-wing-body-goes- hybrid. For example, the CENTRELINE project was a recent concept valida- tion study for a wake-filling propulsive fuselage aircraft design [8]. This studied a 340 passenger, 6500 nmi range wide-body aircraft (shown in Fig. 1) with a specified entry into service date of 2035. The aircraft aims to reduce CO2 emissions through the use of a ‘‘wake-filling’’ propulsion design, whereby the momentum lost in the fuselage boundary layer is recovered, or ‘‘filled’’, by using this low-momentum air as the intake air for the propulsion system. This lowers the jet exhaust velocity for the same net thrust and so results in a lower kinetic energy expenditure. 2.2. Inlet flow distortion phenomenology and characterisation Inlet flow distortion can arise from a range of aerodynamic as- pects such as boundary layer ingestion, lip separation, shock induced separation, vortex ingestion and secondary internal flows [19]. As a consequence, perturbations of total pressure, temperature and velocity are presented to the engine system. This constitutes an element of risk for the development of propulsion systems for future aircraft designs presented in the previous section. Often, these are of concern not only for the aerodynamic efficiency and stability of the propulsion, but also for the effect on the aeromechanical compatibility between the intake and the engine. Inlet distortion is generally classified into three cate- gories: total pressure distortion [20], total temperature distortion [21] and swirl distortion [22]. This section will focus on the sources of the inlet distortion, the phenomenology and the expected impact onto the propulsion system. gear, canards and aerodynamic surfaces which may interact with the intake flow and produce flow separations [20,24]. For novel aircraft configurations in which engines are rear mounted and semi-embedded in the fuselage [25,26], the thick inlet boundary layer will generate non-uniform total pressure distributions at the entry of the air induction system (Fig. 5). Due to the shock interactions at the inlet, supersonic aircrafts can also be exposed to total pressure distortions, especially during manoeuvres with large angles of side slip [19]. Devices or excrescences at the intake inlet such as screens to avoid debris ingestion may also cause non-uniform pressure distributions [19]. A number of distortion descriptors were previously proposed to characterise inlet total pressure distortion patterns in industrial intake- engine integration test campaigns. The philosophy behind these formu- lations was to bring together the pressure data collected by an intrusive pressure rake installed at the interface between intake and engine, or Aerodynamic Interface Plane (AIP), as defined by the S-16 committee in [20,27]. As such, different descriptor formulations were introduced to quantify total pressure variations in the radial and circumferential directions across the AIP (𝛥𝑃r∕𝑃avg and 𝛥𝑃c∕𝑃avg respectively, see [27]), whereas aero-engine manufacturers developed additional formulations such as the descriptors based on a critical sector angle 𝜃, as defined by Reid in [28]. This was developed based on the observation that upon increasing the angular extent of the ‘‘spoiled’’ low-pressure region, the effect on the compressor in terms of surge delivery static pressure was dramatic up to a certain value beyond which it stabilised at some minimum value. 2.1. Overview of novel aero-engine architectures The airframe is a blended wing- body design where the fuselage forms part of the lifting surface and blends continuously into the wing, as shown in Fig. 3. Two gas tur- bines with superconducting generators power an array of 14 electric propulsors near the trailing edge of the fuselage. The gas turbine power-generating engines are wingtip mounted to minimise losses and stability issues from inlet distortion. The Turboelectric Distributed propulsion architecture used in this design allows the decoupling of the locations of the power-generating gas turbines from the thrust- generating fans. By incorporating many fans, this generates a very high effective bypass ratio to improve propulsive efficiency and employs boundary layer ingestion while avoiding the ingestion of high levels of distortion into the engine core. Using a Boeing 777-200LR as a reference aircraft, the design was predicted to have a 70% reduction The MIT-Aurora developed D8 ‘‘Double Bubble’’ is a 180 passenger, 3000 nmi transcontinental range concept civil transport aircraft [12], shown in Fig. 2. It is designed for the Boeing 737/Airbus A320 category of operation and incorporates technologies associated with a 2035 year of entry into service. The ‘‘Double Bubble’’ name comes from the two intersecting cylindrical pressure vessels used to form the fuselage into a shape which allows for the inclusion of a lifting nose [13]. The unconventional location for the aircraft’s engines, flush mounted in the top surface of the fuselage, between the two vertical stabilisers, means they ingest roughly 40% of the fuselage boundary layer [14]. Boundary Layer Ingestion is an enabling technology for achieving the fuel efficiency levels of the D8 which is estimated to have the potential to reduce fuel burn by 36% compared to a conventional tube-and- wing aircraft [14]. In the same way as with the CENTRELINE concept, 3 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 4. Inlet total pressure distortion due to intake lip separation at high angle of attack flight condition. Source: Republished with permission of American Institute of Aeronautics and As- tronautics, from [26]; permission conveyed through Copyright Clearance Center, Inc. in mission fuel consumption [16]. Other blended wing-body aircraft designs exist such as the MIT-Cambridge University Silent Aircraft Initiative SAX-40 and the Boeing N2B [5,17]. These use a different engine architecture with pods of embedded ‘‘Tri-fans’’. 2.1. Overview of novel aero-engine architectures This is another method by which the effective bypass ratio is improved, where a single gas turbine is connected via shafts to three fans transmitting power mechanically rather than electrically. In an analysis of the SAX-40 propulsion system, de la Rosa Blanco et al. [17] remark that embedding the engines into the fuselage can reduce the installation drag, increase noise attenuation and enable boundary layer ingestion. However, they emphasise that total pressure losses have a significant impact on per- formance, and there is a large uncertain risk to the design from the effects of inlet flow distortion. Similarly, in the case of the N2B design, flow simulations by Kim and Liou [5] also present the significant effect of inlet flow distortion in the design. In all the above mentioned novel propulsion concepts, the swirl distortion is likely to increase along with the ingestion of a distorted total pressure field. Firstly, the approaching fuselage flow will have some 3-d aspects and therefore will not contain as uniform a swirl distribution as compared to a podded under-wing configuration. Secondly, when an air induction system ingests total pressure distortion, such as a fuselage boundary layer, this augments the swirl distortions generated in the intake duct train [18]. Fig. 4. Inlet total pressure distortion due to intake lip separation at high angle of attack flight condition. Source: Republished with permission of American Institute of Aeronautics and As- tronautics, from [26]; permission conveyed through Copyright Clearance Center, Inc. Fig. 5. Boundary Layer Ingestion (BLI) concept NOVA by ONERA [15]. Source: Courtesy of ONERA https://www.onera.fr/en/news/nova- nextgen-onera-versatile-aircraft. This overview of novel aero-engine architectures demonstrates both the variety and commonality among a range of proposed future aircraft designs. Common features arise among the concepts, such as boundary layer ingestion, lifting-body fuselages, turboelectric propulsion, and the augmentation of fan pressure ratio through electric or mechanical power transfer. An important consideration in the design of the air- frames and propulsion systems is the highly distorted and unsteady nature of the inlet airflow. The impact of this flow distortion on the propulsion system stability and performance is further discussed in the following sections. Fig. 5. Boundary Layer Ingestion (BLI) concept NOVA by ONERA [15]. Source: Courtesy of ONERA https://www.onera.fr/en/news/nova- nextgen-onera-versatile-aircraft. 2.2. Inlet flow distortion phenomenology and characterisation That point is the critical angle, and the pair of descriptors used with this method are defined by Campbell in [29]. A number of further total pressure distortion descriptor formulations is introduced in the same reference. 2.2.1. Total pressure distortion Total pressure distortion is the variation in stagnation pressure ingested by the engine caused by irregular flow features upstream of the engine through their effect on the pressure field. The S-16 committee, formed as a working group of SAE International, recognises this distortion type as the predominant destabilising element and often the most common among the different applications [23]. There are multiple sources of total pressure distortion. For example, the shape of the inlet determines boundary layer growth along the inlet walls, possible separation zones, and streamwise vorticity, all of which influence the total pressure of the flow reaching the engine. Moreover, civil aircraft at high angles of attack or cross-wind conditions can develop separation from the lip of the nacelle and a subsequent total pressure decrease in the separated region of the fan face (Fig. 4). Other aircraft configurations with highly integrated propulsive systems are also exposed to wakes and vortices generated by the forebody, nose 4 U. Doll et al. U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 6. Tightly-wound ground vortex during taxiing. Source: The U.S. National Archives. Inlet total pressure distortion was previously found to influence the propulsion system efficiency. Results from a turbofan engine test conducted at a ground testing facility with a circumferential inlet distortion pattern showed loss in gross thrust of 2% and specific fuel consumption increased by 6% [27]. More importantly, inlet distortion can also influence the stability limits of the engine. Total pressure losses in the inlet airflow lead to variations in pressure recovery across the engine face which affects the pressure ratio delivered by the com- pressor behind the distorted regions. The flow physics of how exactly distortion behaves in turbomachinery is an area of active research. However, it is inferred that when total pressure distortion is present, there are regions of high and low total pressure over the rotor face. This means a blade passes into and out of the ‘‘spoiled regions’’ and the blade incidence and, consequently, the blade loading changes as it does so [19]. Depending on the magnitude of the change and on the radial and circumferential extent of the spoiled region, this may cause the blades to stall and may trigger compressor instabilities such as mechanical excitation and, ultimately, compressor surge [30]. 2.2.1. Total pressure distortion This is highly detrimental to efficient compressor operation and very often catastrophic damage can be caused to the hardware as shown by Song et al. [31]. Fig. 6. Tightly-wound ground vortex during taxiing. Source: The U.S. National Archives. More investigations are needed to address the total temperature dis- tortion problem and define methodologies for stability assessments. Computational and experimental investigations on this distortion type are limited and this is acknowledged in SAE standard AIR5867 [21]. From the published data, common parameters to characterise temper- ature distortions quantify the rate of change of temperature variations and also the spatial distribution of the AIP distorted sectors as described in [21]. More investigations are needed to address the total temperature dis- tortion problem and define methodologies for stability assessments. Computational and experimental investigations on this distortion type are limited and this is acknowledged in SAE standard AIR5867 [21]. From the published data, common parameters to characterise temper- ature distortions quantify the rate of change of temperature variations and also the spatial distribution of the AIP distorted sectors as described in [21]. Investigations on the impact of inlet total pressure distortion re- vealed that both the magnitude and the distribution of the spoiled regions at the compressor fan face play a role in the reduction of the compressor stability margin. Since Reid’s experiments on a 6-stage com- pressor exposed to radial and circumferential distortion patterns, it was inferred that the impact on the compressor surge margin is a function of the area of spoiling [28]. However, the extensive investigations of Melick [32] revealed that the response of the engine to the distortion depends on the length-scale of distortion and compressor chord. This was later associated with the concept of rotor reduced frequency, which was applied to typical transonic compressor fans by Cousins [33]: If the time required for the distortion to travel from the blade leading edge to the blade throat is less than the time for the blade to travel across the distorted sector, the blade response is maximised. This theory is supported by Longley et al. [34]. They observed a severe stability margin degradation when the distortion frequency matched a charac- teristic speed of the compressor flow field such that the blade exposure time to the distortion became similar to the through-flow time. 2.2.1. Total pressure distortion The reduction in stability margin could be as high as 90% compared to the margin associated with a steady distortion pattern [34]. Therefore, it is concluded that the impact of the total pressure distortion must be evaluated on a case-by-case basis because it is strongly dependent on the response time of the compressor blades. Key references on the matter are collected in Table 1. Additional considerations on unsteady total pressure distortion are discussed in Section 2.2.4. 2.2.3. Swirl distortion Swirl distortion can be described as the variation of the circumfer- ential portion of the flow velocity vector at the AIP. The development of methodologies to assess the swirl distortion is very recent, since it became clear that the assessment of only total pressure distortion is not sufficient to define the engine’s surge margin loss, in cases where swirl distortion is also present [19]. In engine configurations with inlet guide vanes (IGVs) part of the inlet swirl is reduced from the flow approaching the first rotor, which mitigates some of the stability risk linked with inlet swirl. In engines with no IGVs in place, swirl distortion issues may still arise and become detrimental to the engine’s stability and performance as described by Cousins in [19]. The source of inlet swirl distortion can be internal or external to the intake. In embedded air induction systems, complex (S-shaped) subsonic diffusers are very frequently used to drive the required amount of flow into the propulsion system. Such diffuser ducts are known to generate large levels of swirl distortion due to the secondary flows which develop because of the curved geometries [49]. Additionally, there have been cases whereby swirl generation originates externally to the intake from various aerodynamic surfaces of the aircraft as described in the relevant SAE standard [22] . In novel aircraft configurations, where the engine is closely coupled and rear mounted, there is an increased likelihood of vortex ingestion from the strakes, leading edge extensions, fuselage and aerodynamic surfaces [22]. For an embedded engine with a convoluted air induction system, the swirl distortion generated within the ducts depends on the total pressure distribution at the inlet such that, for example, the ingestion of a fuselage boundary layer can cause swirl distortions at the AIP [50]. Tightly wound vortices can also be generated by the interaction of the inlet flow with the ground under no wind, head-wind, cross-wind or tail-wind conditions [51], and also during thrust reverser operation (Fig. 6). Moreover, swirl distortion is a concern also in gas turbine intakes for helicopters, in which the inlet flow is subject to the rotor downwash [19]. In turboprop engines and in future hybrid electric distributed propulsion systems, the inlet flow is expected to cause peak swirl fluctuations due to the wake of the propeller blade passing in front of the intake [52]. 2.2.2. Total temperature distortion p Total temperature distortions can arise from thrust reverse op- eration, ingestion of hot gases from firing of armaments or during take-off queues [21]. Total temperature distortion is less investigated than total pressure distortion but nevertheless it can be detrimental to the engine, causing performance degradation, power loss due to engine flame out or compressor instability. This is expected to become more relevant for future formation flight on civil aircraft. Preliminary feasibility studies reported a fuel burn saving of up to 16% for a mid- range passenger aircraft comparable to a A320-200 [45]. Formation flight was recently trialed by Airbus, reporting fuel burn savings of more than 5% on long-haul flights with A350 aircraft [46]. Cases of inlet total temperature distortion were also recorded in helicopter and V/STOL (Vertical and/or Short Take-Off and Landing) aircraft operation when hovering close to the ground [47]. A significant effect on engine stability was observed, especially in cases in which a single distorted region with a considerably large circumferential extent was presented to the engine, rather than multiple distortion sectors [48]. Similarly to the other distortion types, a number of swirl dis- tortion descriptors were introduced to characterise engine face swirl 5 5 Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. Table 1 Experimental (E) and computational (C) studies on engine response to inlet total pressure distortion based on blade resident time theory and total pressure fluctuations. Distortion type E/C Model Distortion generation method Key findings Ref. Year Total pressure E 6-stage axial compressor Mesh screens for radial, circumferential distortion sectors Minimum area of spoiling which induces maximum loss in surge pressure ratio [28] 1969 Total pressure E GE, PW, RR rigs and compressors Mesh screens for circumferential distortion sectors Stall margin as analytical function of the distortion pattern, distortion level and compressor rotor reduced frequency [32] 1973 Total pressure E Square inlet duct Random frequency generator with mechanical actuators Engine responds to unsteady distortion patterns [35] 1974 Total pressure C Parallel compressor model Steady and dynamic disturbances simulated with analogue computer Trade-off pulse amplitude vs duration for instability. Table 1 ground vortex ingestion or S-shaped intakes swirl distortion, respec- tively. Mitchell [58] demonstrated that the impact of discrete wing-tip vortex ingestion depends on many parameters including the vortex ingestion position relative to the intake inlet, the vortex strength, the swirling direction and the trajectory from inlet to fan face. The highest loss in surge margin was for counter-swirling vortices near the hub, which caused a loss in surge margin up to 6% for relatively high rotor speed [58]. In a more recent computational work, Mehdi [59] demonstrated correlations between the swirl angle and the loss in compressor surge margin for bulk, single and multiple vortex ingestion. Based on his CFD simulations, the reduction of surge margin caused by a single vortex was relatively greater than the one caused by multiple or bulk vortices [59]. Key references and findings on the response of aero-engines to swirl distortion are collected in Table 2. patterns. A first set of formulations was initially shown by Sheoran and Bouldin [53], based on swirl-angle data at the AIP using the engine rotation direction as the positive direction. These formulations include expressions to characterise Sector Swirl (SS), Swirl Intensity (SI), Swirl Directivity (SD), and Swirl Pairs (SP) at an AIP. The mathematical definitions of the four swirl distortion descriptors are given in She- oran and Bouldin [53] along with examples of descriptor values for a variety of swirl patterns. The SAE Aerospace information Report AIR5686 [22] gives more in-depth examples with example calculations for representative cases. Swirl distortion has not previously attracted as much attention as total pressure distortion, and the impact on the aero-engine is still relatively poorly understood. Initial studies used velocity triangle based assessments to quantify the influence of swirl patterns on axial com- pressors [22]. Some studies showed that the vibratory response due to unsteady swirl can be equally severe as total pressure distortion and can excite the blade resonant frequencies [54–57]. Vortex flows can also set up harmonic effects in the flow field that drive high- cycle fatigue leading to reduced operational lifespan of the engine [19]. More recent studies assessed the swirl distortion in the form of tightly- wound discrete vortex and paired swirl, for example in the case of 2.2.2. Total temperature distortion Steady and dynamic distortion combine linearly for reduction of surge margin [36] 1977 Total pressure E GE low-speed 4-stage axial compressor rig Rotor mismatch, large tip clearance and mesh screen for circumferential distortion sector Evidence that compressors have a two natural frequencies associated with modal stall and spike stall [37] 1995 Total pressure E Large scale low speed compressor Mesh screens for circumferential distortion sector Severe stability margin degradation if distortion frequency matches a characteristic speed of the compressor flow field [34] 1996 Total pressure C Low-speed compressor Mesh screens for circumferential distortion sectors If distortion transport time is less than blade resident time, the blade response is maximised [33] 1998 Total pressure E Ground testing of large scale turbofan engine Unsteady distortion induced by transient wind gusts Rotating stall triggered by delay between the response of the inlet and of the fan to incoming fluctuations of low frequency [38] 1999 Total pressure E/C Review of previous work Experimental and parallel compressor models Engine response depends on compressor reduced frequency and blade resident time for each configuration [19] 2004 Total pressure E/C Review of previous work Experimental and parallel compressor models Very long-length scale disturbances affect rotor in quasi-steady manner while very short ones may not affect the rotor at all [39] 2010 Total pressure and swirl E/C Review of previous work Unsteady spike-type distortion Modal or spike-type stall inception. Spike-type stall inception triggered by peak fluctuation of pressure, vorticity, velocity [40] 2010 Total pressure C 1/10 RR transonic axial fan stage Step, 1 pulse, continuous pulses of total pressure Trade-off pulse amplitude vs. duration for instability. Fan less tolerant to continuous stream of pulses rather than single one [41] 2015 Total pressure E Subsonic axial flow fan stage Mesh screens for radial distortion sectors Stall inception occurs mainly through long-scale disturbances with embedded spikes. Inflow distortion amplifies spikes [42] 2017 Total pressure E NASA GRC 8 × 6’ wind tunnel for BLI tests Thick boundary layers BLI-type inlet distortion can excite natural frequencies of the fan rotor [43] 2019 Total pressure C Transonic axial-flow fan stage NASA R67 Circumferential distortion sector Stall inception triggered by periodic tip leakage flow of the blade encountering distortion. Longer exit ducts worsened engine stability [44] 2019 2.2.4. Unsteady distortion However, it was soon realised that engine surge events could neither be reproduced nor predicted via a time-averaged, steady-state picture of inlet distortion, which supported the hypothesis that time-dependent distortions may have large effects on the engine’s stability limit [63]. In fact, the unsteady part of the distortions was found to add as much as 30% range to the steady-state measurements [19]. These peak fluctuations were considered large enough to impact engine performance and stability. total pressure distortion and that severe inlet distortion amplifies the likelihood of spikes [42]. Similar conclusions are also drawn by Zhang et al. [44] who reported that stall inception is triggered by the periodic leakage flow of the blade encountering the distortion. More recent computational and experimental studies for a modern low pressure ratio transonic fan have also identified the impact of unsteady total pressure distortions on the rotating stall cell inception, rotation speed and extent [64]. A certain level of effort was recently made to include unsteady total pressure distortions into engine industrial testing and apply extreme value statistics to reduce testing and computational requirements for quantifying its effect [65]. The SAE S-16 turbine engine inlet flow dis- tortion committee recently acknowledged the importance of unsteady pressure distortion and classified them under the definition of planar waves [66]. These are one-dimensional, unsteady pressure fluctuations at the compressor face. Planar waves can be generated internally within the inlet in the case of flow separations, shock-boundary layer inter- actions, supersonic flight buzz, un-start or interactions with adjacent engines. These can be also linked to instabilities developed during subsonic flight at low mass flow ratio or the response to inlet control system inputs during supersonic flight. Additional external causes may trigger these fluctuations, such as vortex ingestion, ingestion of wakes from nose gear and bomb bay doors, atmospheric gusts and explosions, and armament firings. The occurrence of unsteady distortions is not restricted to extreme manoeuvres, but instead, may appear also in the normal flight envelope [66], and thus this is an element of risk for the engine stability during normal flight conditions. Although the SAE standard AIR5866 [66] summarises a number of methodologies to assess planar waves, there is no evidence yet related to the sensitivity of the engine to these unsteady oscillations. 2.2.4. Unsteady distortion Often, this distortion is addressed through several iterations between engine manufacturers and airframe designers where design modifications are made to avoid or suppress the generation of such planar waves as described in [23]. g p g p y Although the engine response to unsteady inlet distortion is not fully understood, many researchers demonstrated that unsteady flow fluctuations can trigger stall inception mechanisms in aero-engine com- pressors. The experiments of Longley et al. [34] were among the first to identify two mechanisms which are associated with the stability margin degradation of the engine: modal stall and spike stall inception. The first is associated with long length-scale distortions with a signature frequency at about 30% of the rotor speed [34]. The second is associ- ated with short and localised disturbances at about 70% of the rotor speed [34]. While modal stall usually occurred because of the whole compressor system instabilities, the spike-stall is much more common especially in modern, highly loaded compressors because it can be triggered by localised peak fluctuations of pressure, vorticity, and ve- locity [40]. In the context of spike-type disturbances, some researchers investigated the impact of unsteady fluctuations of total pressure. One of the first studies by Wenzel and Blaha [36] involved the generation of pulses with an analogue computer which fed steady and dynamic dis- turbances to a parallel compressor model. It revealed that there was a trade-off between pulse amplitude and pulse duration which generated instabilities [36]. Interestingly, this study also demonstrated that steady and dynamic distortion combined linearly for the reduction of surge margin [36]. These conclusions were verified by a recent CFD study by Shaw [41] of a transonic axial fan stage which was exposed to step-like, single and multiple total pressure pulses. The trade-off between pulse amplitude and duration was verified, and it demonstrated that a range of pulse duration between 20% and a full rotor revolution can stall the fan depending on the operating condition of the compressor. Moreover, the fan was less tolerant to a continuous stream of pulses rather than a single one [41]. Latest studies on the spike-type distortion showed that spikes are also embedded in long-scale disturbances due to inlet Overall, it is concluded that there is no general rule to predict the engine response for different unsteady total pressure distortion patterns, amplitudes and frequencies. 2.2.4. Unsteady distortion For many years, unsteady variations of total pressure, swirl or temperature distortion were considered of low importance despite some earlier suggestions made in the 70s to include the time-variant element of inlet total pressure distortion in the characterisation of an engine’s 6 Progress in Aerospace Sciences 130 (2022) 100810 Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. Table 2 Experimental (E) and computational (C) studies on engine response to inlet swirl distortion. Distortion type E/C Model Distortion generation method Key findings Ref. Year Swirl E J-85 engine Wing tip vortex ingested in S-duct intake Vortex impact is function of ingestion position, vortex strength, swirling direction and trajectory [58] 1975 Swirl E Review of previous work Canards wing tip vortex, swirl induced by S-duct intakes Total pressure distortion not adequate to define stability limit if swirl distortion is present [54] 1982 Swirl and total pressure E/C Review of previous work Experimental and CFD models Vibratory response to swirl can be more severe than total pressure distortion [55] 1997 Swirl C Transonic axial fan stage NASA R67, high-pressure compressor stage NASA R37 Bulk and tightly wound vortices Correlation between swirl descriptors and loss in surge margin. Greatest surge margin loss for the ingestion of co-rotating vortex near the hub [59] 2014 Swirl and total pressure E Transonic axial fan stage NASA R67 Tip-radial and hub-radial total pressure loss. Co- and counter-bulk swirl Superposition of radial total pressure and bulk swirl distortion is not valid to predict compressor stability [60] 2016 Swirl and total pressure E/C Review of previous work Bulk, paired and multiple swirl in parallel compressor models and experiments Impact of combined paired swirl and total pressure distortion is not predictable with superposition of effects [22] 2017 Swirl C Transonic high-pressure compressor stage NASA R37 Bulk swirl and paired swirl with IGVs Bulk swirl more predictable than twin swirl. Need to consider unsteady flow to predict stall margin [61] 2018 stability degradation [62]. Hence, limited effort went into understand- ing their nature and their influence on an engine’s performance and stability penalties [63]. As a result, large engine development cam- paigns focused only on evaluating the impact of steady-state distortion patterns on the propulsion system. In order to reproduce such patterns at engine test-beds, various methods were applied, mostly based on the imposition of distortion profiles, generated by appropriate screens installed forward to the AIP [63]. 2.2.5. Combined distortion In most configurations, the presence of one type of inlet distortion is not exclusive and more than one distortion type can co-exist at the fan face, which can further deteriorate the engine’s stability margin as described in [22]. For example, wakes from bodies upstream the intake can generate a wide range of combined swirl and total pressure distortions [67]. Moreover, previous observations suggest that new engine developments may require the characterisation of combined pressure and temperature distortions [68]. Accountability of combined total pressure distortion and planar waves should be also in place for engine stability assessments [66]. The SAE guidelines recommend that when considering combined distortions, it should be verified that the superposition of effects is appropriate for modelling the combined effect of distortion types on the engine stability, and this should be evaluated on a case-by-case basis [66]. For example, it is believed that total pressure and total temperature do not interact with each other and thus the superposition of effects is granted [21]. However, Naseri et al. [60] demonstrated that the superposition of total pressure and swirl distortion can only be linked to compressor performance and less to compressor stability. The complexity around combined distortions is also acknowledged by other researchers who highlighted the need to include both sources of steady and unsteady distortion for the understanding of stability limits and mechanical forced excita- tion on the compressor blades [54–57]. During the engine operability considerations a key concern related to unsteady distortions is the non-synchronous blade forcing that these can cause. However, no solid guidelines exist regarding testing and simulation with combined distortion effects primarily due to the lack of understanding of such flow mechanisms and hence the inability to link these with engine performance and stability penalties. Fig. 7. Pressure rake used for total pressure distortion characterisations. Source: Reproduced from [69]. © 2018 Elsevier Masson SAS. All rights reserved. indicated in [7]. In such cases, the acquired information is no more synchronous in time across the plane and, hence, only time-averaged distortion measurements can be obtained without acknowledging the unsteady part of it. This was often found to provide misleading in- formation about the flow non-uniformities, whose mean values were significantly lower than instantaneous peak events that could trigger engine instabilities [70,71]. 2.2.5. Combined distortion Although the standards prescribing the total pressure distortion measurement methods have existed for many decades [20], recom- mended practices about swirl measurements have only recently been introduced [22]. However, it is easily realised that, although unsteady total pressure and swirl distortion was historically identified as a key detrimental factor to an engine’s stability, the relatively low maturity of advanced flow measurement methods in combination with the high complexity of more sophisticated measurement systems, has limited the understanding of the complex, unsteady flows linked with advanced air induction systems. Numerous historic examples reinforcing this statement were previously reported and comprehensively summarised in [72]. A key message from this work focuses on the fact that the poor characterisation of the unsteady part of the various types of distortions along with the inability of existing measurement techniques to synchronously measure more than one type of dynamic distortion, yields poor interpretation of the influence of such complex flows on engine stability and the loss of surge margin. This was acknowledged as a major pitfall in some previous engine development programmes such as the YF-12, the F-15, F-18 and F-22 among others [72]. Some further emphasis was given to the currently insufficient capability to capture unsteady total temperature distortions at a sufficiently high temporal frequency pertinent to engine response [21]. As such, the necessity for richer data primarily in space but also in time, to aid the understanding of these complex distortions that lead to engine failure events remains a key requirement. The timeliness of such developments is now more critical than ever as an era of closely coupled aircraft-engine systems emerges in both the civil and defence markets, whose development and engine integration risks cannot be easily mitigated by current practices. Imaging based laser-optical flow measurement techniques have the potential to address this limitation by providing flow data of particularly high resolution in space and time. Recent work suggested that optical velocimetry techniques such as Particle Image Velocimetry (PIV) deliver a spatial resolution of cross-flow velocity measurements by at least one order of magnitude greater than conventional intru- sive distortion measurement methods [73]. As such, a currently open 2.2.4. Unsteady distortion Previous work suggests a typical range between 1-per-rev and blade passing frequency for disturbances that could contribute to the instability of a compression system. Alter- natively, for axial compressors, a circumferential spoiled sector of 4–5 blades can also generate instability. However, this must be evaluated on a case-by-case basis as it can be affected by a range of parameters even low frequency ambient wind perturbations of less than 5 Hz [38]. Since instabilities such as spikes are localised in the blade spacing, many flow features such as boundary layers, tip leakage and blade wakes can play a role in compressor instability. Furthermore, the nature of these 7 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 7. Pressure rake used for total pressure distortion characterisations. Source: Reproduced from [69]. © 2018 Elsevier Masson SAS. All rights reserved. instabilities can be pressure, vorticity, and velocity fluctuations [40] and overall the phenomenon is still not well understood. Nevertheless, the importance of unsteady distortions is now widely acknowledged within the relevant community along with the necessity of more ad- vanced experimental and data analysis methods, to be able to provide richer flow field data in space and time. Such advancements will play a critical role in improving the current understanding of complex, highly unsteady distorted flows and will eventually support the development of design rules for closely integrated aircraft and engine systems. 2.3. Current industrial practices in distortion measurements and knowledge gaps Current practice for inlet distortion measurements in industry in- cludes the use of a number of intrusive pressure rakes at prescribed positions at an engine’s AIP as shown in [27]. An example of such a distortion rake installed inside an intake duct is shown in Fig. 7. These pressure measurements are most commonly of low temporal resolution, although time-dependent rakes were also shown previously in selected cases where time-resolved data was required [69]. The ARP1420 aerospace standard [27] prescribes pressure probe locations across an AIP, to ensure representative data acquisition, without miss- ing significant flow details and without causing too much blockage. Alternative intrusive methods include the use of miniature high-speed pressure transducers embedded in rake arrays to minimise blockage effects. This allows unsteady pressure measurements across an engine face plane as well as in turbomachinery passages [33]. Although pressure probes have the disadvantage of being intrusive to the flow, these systems can capture data at very high temporal rates in the order of >10 kHz or also provide swirl characteristics [69]. These systems include multi-hole probe arrays able to capture flow angularities as well as total pressure variations across the plane of interest. Very often, only a small number of multi-hole probes is available during a certain test campaign hence the rake on which they are mounted has to be traversed across the measurement plane as 8 8 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 8. Example visualisation of the mean secondary flow behind a customised distortion screen measured with S-PIV inside Virginia Tech’s full-scale engine research rig. Colours represent secondary flow magnitudes (red large, blue small) and streamlines the local flow direction. Source: Republished with permission of Emerald Publishing Limited, from [74]; permission conveyed through Copyright Clearance Center, Inc. Fig. 8. Example visualisation of the mean secondary flow behind a customised distortion screen measured with S-PIV inside Virginia Tech’s full-scale engine research rig. Colours represent secondary flow magnitudes (red large, blue small) and streamlines the local flow direction. Source: Republished with permission of Emerald Publishing Limited, from [74]; permission conveyed through Copyright Clearance Center, Inc. y g py g , Fig. 9. S-PIV measurements of time-averaged (⟨⟩) and unsteady (standard deviation, std()) streamwise velocity component (w) and swirl angle (𝛼) downstream of an S-shaped diffuser [18]. 3.1. State-of-the-art in non-intrusive distortion measurements 3.1. State-of-the-art in non-intrusive distortion measurements To capture the full range of the various types of inlet flow distortion and determine their impact on the stability and performance of a propulsion system, measurements of static pressure, temperature and three-component (3C) velocity are required. Given the known limita- tions of currently used intrusive distortion measurement methods (low spatial and temporal resolution, intrusive instruments that interact with the flow), existing technologies are inadequate to sufficiently reduce the risk on the development of future closely coupled engine-airframe configurations. In the context of swirl distortion measurements, planar laser-optical techniques such as Particle Image Velocimetry (PIV) and Doppler Global Velocimetry (DGV) have demonstrated their ability to spatially resolve complex flow patterns and capture the associated unsteady characteristics. Fig. 9. S-PIV measurements of time-averaged (⟨⟩) and unsteady (standard deviation, std()) streamwise velocity component (w) and swirl angle (𝛼) downstream of an S-shaped diffuser [18]. The PIV technique enables flow velocity measurements in a two- dimensional plane by illuminating small tracer particles added to the flow and capturing two images in quick succession, subsequently us- ing computational methods to determine the displacement of particle patterns between the two images. Stereoscopic PIV (S-PIV) extends this method by using multiple cameras to capture flow velocity in three dimensions, across a given flow plane [75]. The application of S-PIV methods for cross-flow swirl distortion measurements in civil intake configurations was previously demonstrated by Hoopes [76] and Guimaraes-Bucalo et al. [77], who also developed a method to prescribe swirl distortion patterns at the inlet of a propulsion system via tailored made distortion screens. The resulting distorted inlet flow fields were investigated in several studies at a small scale rig and a full scale engine test facility [77–81]. Example flow fields measured with S-PIV at different axial stations behind the distortion screen are presented in Fig. 8. Previous work at Cranfield University focused on S-PIV measurements in convoluted subsonic diffusers [70,73]. Therein, the superior spatial and temporal resolution of the resulting PIV velocity fields was exploited to highlight flow unsteadiness associated with the swirl angle, whose fluctuations (highlighted by the standard deviation, std(𝛼)) could double the time-averaged distortion levels (Fig. 9). This work was further expanded by high-speed S-PIV velocity measurements at repetition rates of up to 8 kHz. 2.3. Current industrial practices in distortion measurements and knowledge gaps research question is now emerging about the development and ap- plication of non-intrusive, optical methods for the characterisation of unsteady distortions in novel air induction systems aiming to further educate the development of stall tolerant, closely coupled propulsion systems. An additional question is related with the applicability of such non-intrusive methods at in-flight tests which were previously found of critical importance in certain development programmes, due to the inability of ground- or wind-tunnel test campaigns to capture extreme, instantaneous distortion events [72]. Overall, it is anticipated that the application of non-intrusive, laser based flow diagnostics for unsteady inlet flow distortions at ground but also in in-flight test campaigns, is one of the most promising routes to address the design challenges associated with the development of the next generation, closely coupled, aircraft architectures. 3.1. State-of-the-art in non-intrusive distortion measurements A half-model of the aircraft was attached to a six-degree of freedom positioning system inside the wind tunnel’s open jet test section and an aspiration system was used to model compressor suction, enabling the simulation of a large set of test conditions in terms of mass flux, angle of attack and yawn angle, as shown in Fig. 11. From the diagnostics point of view, the main technological hurdle was the anticipated usage of DGV to measure flow velocities inside the air- craft models convoluted intake duct. The resulting optical accessibility constraints were addressed by replacing a segment of the intake duct with an optical module. Thereby, the AIP was consecutively illuminated with laser light from three directions using miniaturised light sheet optics, which were supplied with laser light via 15 meter long optical wave guides. Observation occurred through an optical probe located three tube diameters downstream from the measurement plane and an imaging fibre was used to transfer the imaged scene to the DGV camera system. The contour plot in Fig. 11 shows a sample flow field obtained with the described system, exhibiting a characteristic asymmetry of axial velocity and a dominating eccentric vortical structure rotating in counterclockwise direction. Although DGV has demonstrated the ability to achieve fully probe- based measurements of the mean three-dimensional velocity field in complex experimental scenarios, the aforementioned approach lacks the required time resolution to handle the unsteady flow regime at the AIP. To date, only few DGV approaches to measure flow velocity at high acquisition rates of several kHz have been presented [90,91]. These methods rely on avalanche photodiode or photomultiplier tube arrays for signal detection and, thereby, trade the capability to resolve large coherent flow structures for time resolution. Recently, time-resolved DGV measurements of a single velocity component in a supersonic heated jet were achieved at 50 kHz repetition rate by combining high power continuous wave (CW) laser illumination with high-speed camera technology [92]. gy Even though recent progress was accomplished, the characteristics of continuous wave laser light sources are the main limiting factor for time-resolved and large-field measurements with DGV. For these lasers, the scattered intensity per resolution element is small so that camera exposure times are typically on the order of 10 to 20 ms [84], which is too long to capture flow dynamics in the Mach number range relevant to intake flows. 3.1. State-of-the-art in non-intrusive distortion measurements To perform whole-field measurements with DGV resolv- ing the relevant time and spatial scales, pulsed laser sources would have to be used. Several studies applied pulsed frequency tunable injection- seeded Nd:YAG lasers to acquire instantaneous velocity information with DGV [93–95]. A major concern with these systems is a spatial variation of laser frequency within the light sheet, which is varying from pulse to pulse and can reach values of up to 100 MHz, potentially resulting in velocity errors of tens of meters per second [93,96,97]. To achieve the necessary temporal resolution to capture the unsteady aerodynamics at the AIP, novel pulsed laser concepts with enhanced spectral quality are required. Even though notable progress in the diagnostic capability to char- acterise inlet flow distortion has been accomplished, laser-optical mea- surement techniques such DGV or PIV require the use of tracer parti- cles, which comes with a number of caveats, including the fouling of the optical access as well as the requirement of a uniform seeding distribu- tion across the measurement plane and the installation of seeding rakes within the intake sub-system, posing particular challenges for airborne measurements. In addition, DGV is prone to measurement bias caused by multiple scattering in densely seeded flows [85,86]. In summary, the state-of-the-art in swirl distortion measurement has been significantly expanded by laser-optical PIV and DGV methods, from a few intrusive probe measurements to whole-field unsteady flow characterisation. While PIV has clear advantages in terms of capturing the unsteady component of swirl distortion, DGV shows great potential for experimental scenarios with aggravated optical accessibility. How- ever, in light of the above limitations concerning flow seeding, optical accessibility for PIV and time resolution for DGV, it is questionable whether the capabilities of both methods can be made available for the entire engine development cycle from ground testing to in-flight experimentation and certification. Moreover, both PIV and DGV lack the ability to capture scalar flow quantities and, thus, total pressure and temperature distortion is left out of the picture. For further detailed information on the previously discussed measurement techniques, in- cluding error characteristics and limitations, we refer to work outlined in [75,98]. Aside from seeding related issues, the application of the PIV tech- nique requires uninterrupted optical accessibility to the test section, which might not be available in ground test scenarios with more complex intake geometries and will certainly not be feasible for in- flight application. 1 German–Dutch Wind Tunnels, Niedergeschwindigkeits-Windkanal Braun- schweig. 3.1. State-of-the-art in non-intrusive distortion measurements A half-model of the aircraft was attached to a six-degree of freedom positioning system inside the wind tunnel’s open jet test section and an aspiration system was used to model compressor suction, enabling the simulation of a large set of test conditions in terms of mass flux, angle of attack and yawn angle, as shown in Fig. 11. From the diagnostics point of view, the main technological hurdle was the anticipated usage of DGV to measure flow velocities inside the air- craft models convoluted intake duct. The resulting optical accessibility constraints were addressed by replacing a segment of the intake duct with an optical module. Thereby, the AIP was consecutively illuminated with laser light from three directions using miniaturised light sheet optics, which were supplied with laser light via 15 meter long optical wave guides. Observation occurred through an optical probe located three tube diameters downstream from the measurement plane and an imaging fibre was used to transfer the imaged scene to the DGV camera system. The contour plot in Fig. 11 shows a sample flow field obtained with the described system, exhibiting a characteristic asymmetry of axial velocity and a dominating eccentric vortical structure rotating in counterclockwise direction. advantage for probe-based applications since the technique’s underly- ing principle does not require high-quality imaging of small objects such as 𝜇m-sized particles, but instead analyses the Doppler-shifted light scattered from an ensemble of particles detected at the respective resolution element [86]. In addition, DGV potentially has access to all three velocity components with just a single camera view, whereas PIV requires a stereoscopic camera arrangement to capture both the two in-plane and the out-of-plane velocities. and illuminated with a thin laser sheet. However, instead of analysing the particle pattern displacement between two consecutive frames us- ing correlation techniques, in DGV, the Doppler frequency shift of the light scattered from particles moving through the measurement plane is transformed into a detectable intensity change by means of molecular absorption. In the context of inlet flow distortion, the DGV method was successfully used to derive the time-averaged swirl distortion inside the intake duct of a fighter aircraft model [84]. These measurements were conducted at the DNW NWB1 wind tunnel facility in Brunswick, Germany. 3.1. State-of-the-art in non-intrusive distortion measurements Proper orthogonal decomposition (POD) analysis was used to identify the most energetic unsteady struc- tures and the frequency content of the unsteady distortion descriptors was analysed with a spectral analysis [18,71] (Fig. 10). The high temporal and spatial resolution of high-speed S-PIV enabled also more advanced analysis methods for tracking unsteady localised distortion events presented to the engine [50]. fields was exploited to highlight flow unsteadiness associated with the swirl angle, whose fluctuations (highlighted by the standard deviation, std(𝛼)) could double the time-averaged distortion levels (Fig. 9). This work was further expanded by high-speed S-PIV velocity measurements at repetition rates of up to 8 kHz. Proper orthogonal decomposition (POD) analysis was used to identify the most energetic unsteady struc- tures and the frequency content of the unsteady distortion descriptors was analysed with a spectral analysis [18,71] (Fig. 10). The high temporal and spatial resolution of high-speed S-PIV enabled also more advanced analysis methods for tracking unsteady localised distortion events presented to the engine [50]. Doppler Global Velocimetry (DGV or Planar Doppler Velocimetry — PDV) methods were introduced in the early 90’s by Meyers and Komine [82,83]. Similar to PIV, the flow is seeded with small particles 9 Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. Fig. 10. Spectral signature of the streamwise velocity downstream of an S-shaped diffuser [18]. U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 10. Spectral signature of the streamwise velocity downstream of an S-shaped diffuser [18 ature of the streamwise velocity downstream of an S-shaped diffuser Fig. 10. Spectral signature of the streamwise velocity downstream and illuminated with a thin laser sheet. However, instead of analysing the particle pattern displacement between two consecutive frames us- ing correlation techniques, in DGV, the Doppler frequency shift of the light scattered from particles moving through the measurement plane is transformed into a detectable intensity change by means of molecular absorption. In the context of inlet flow distortion, the DGV method was successfully used to derive the time-averaged swirl distortion inside the intake duct of a fighter aircraft model [84]. These measurements were conducted at the DNW NWB1 wind tunnel facility in Brunswick, Germany. 3.1. State-of-the-art in non-intrusive distortion measurements Instead, flexible probe-based measurement solutions will be required. Some examples of probe-based PIV, relying on obser- vation with rigid borescopes exist in the literature [87–89]. But these devices lack flexibility to be adapted to complex geometries and the optical resolving power of instruments relying on flexible imaging fibre technology, is insufficient to resolve discrete particles as required for PIV [86,88]. Therefore, the intensity based DGV method has a clear 10 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. Fig. 11. The photograph (upper right) shows the investigated fighter aircraft half-model mounted on a six-degree of freedom positioning mechanism. The inlet duct is connected to the aspiration system (yellow segment and hose on the right). DGV camera system and associated electronics are mounted behind the aircraft model (metallic grey boxes). Optical wave guides and an imaging fibre (grey bundle on top of yellow bend) are used to deliver laser light to and transfer the measured signal from the measurement plane. A schematic overview of the optical module is provided below and a measured time-averaged velocity field is shown on the left [84]. Fig. 11. The photograph (upper right) shows the investigated fighter aircraft half-model mounted on a six-degree of freedom positioning mechanism. The inlet duct is connected to the aspiration system (yellow segment and hose on the right). DGV camera system and associated electronics are mounted behind the aircraft model (metallic grey boxes). Optical wave guides and an imaging fibre (grey bundle on top of yellow bend) are used to deliver laser light to and transfer the measured signal from the measurement plane. A schematic overview of the optical module is provided below and a measured time-averaged velocity field is shown on the left [84]. 3.2. Non-intrusive seeding-free measurement techniques for complex inter- nal flows from 200 to 450 nm with about 70% of the power between 220 and 280 nm [100]. Since excitation as well as detection occur in the UV, this sets aggravated requirements to the quality of the optical access. Likewise, camera sensors are typically insensitive in the UV spectral range, so that image intensifiers have to be used for imaging applications. 3.1. State-of-the-art in non-intrusive distortion measurements The successful application of optical flow diagnostics to measure velocity, static pressure or temperature in air flows, by adding neither particles scattering light in the Mie regime (diameters between 1 and 100 μm), nor seeding tracer molecules such as acetone, toluene or nitrogen oxide, is a challenging task. Since the scattered light intensity scales with the scattering cross section, which is several orders of mag- nitude higher for particle scattering compared to molecular scattering or fluorescence, powerful lasers and sensitive detectors are required to apply these molecular techniques. Owing to the different photo- physical mechanisms involved, the following overview of non-intrusive seeding-free measurement techniques is further subdivided into three families: Laser Induced Fluorescence (LIF) methods, methods utilising molecular tagging and methods relying on molecular scattering. The section concludes with a final comparison and evaluation of the pre- sented techniques, with respect to their qualification of characterising inlet flow distortion. Under AIP relevant flow conditions, populated ground state transi- tions are excited to the oxygen B-state, which lies above the dissociation energy of O2, leading to a very short upper state lifetime of a few picoseconds only [100]. The consequences of the short upper state lifetime are summarised by Miles and Lempert [102]: Due to the rapid depopulation of the upper state the emitted fluorescence is almost independent of collisional (pressure) quenching. The fluorescence yield is proportional to the number of excited molecules and, thus, density measurements at constant temperature or vice versa are feasible in principle. However, since the major part of the absorbed laser energy is lost to predissociation and only 0.004% of the excited molecules fluoresce, resulting LIF intensities are very weak. Finally, the short lifetime leads to an increased natural broadening of individual absorp- tion lines (FWHM > 100 GHz), so that a Doppler shift caused by flow velocity of several tens to hundreds of MHz cannot be distinguished from the fluorescence emission spectrum. To date, the potential of O2 LIF to be applied to aerodynamic flows as a point measurement technique was only explored in laboratory scale studies [103–105]. As a remark, since at higher temperatures additional vibrational states emitting stronger fluorescence signals are thermally populated [102], so called hot oxygen LIF was introduced as a planar technique for flame studies [106,107]. Molecular tagging velocimetry The term Molecular Tagging Velocimetry (MTV) describes a group of laser-optical techniques based on the excitation of a long-lasting fluorescence signal along a laser beam from molecules naturally present in or introduced into the flow of interest. The velocity of the molecules is determined by detecting the fluorescence of the tagged line at two consecutive instants within the radiation lifetime of the tracer and by measuring the shift of the fluorescence pattern between the images [98, 112]. The method can be extended from a one-dimensional single- component velocity measurement to the full 3C velocity vector field by writing a crossed pattern of fluorescence lines and by stereoscopic imaging of the pattern displacement [113]. However, most MTV meth- ods require the addition of molecular tracers to supply the required luminescent properties and to date, there are only two techniques which have the capability to be applied to unseeded air flows. The so-called RELIEF (Raman Excitation and Laser-Induced Electronic Fluo- rescence) technique involves the usage of three laser wavelengths [114] and, thus, is too complex to be applied to flows of practical interest. Consequently, only the FLEET (Femtosecond Laser Electronic Excitation Tagging) method will be further outlined. g A different approach with the potential to derive concentration, temperature, pressure as well as velocity information from a CO2 LIF signal was introduced in [111]. The concept is based on wavelength scanning the output of a CW quantum cascade laser emitting IR radia- tion at 4.3 μm across an appropriate absorption line. The integrated LIF signal below the resulting lineshape scan is a measure of CO2 concentration. Furthermore, the ratio of the integrated signals of two neighbouring absorption lines can be used to derive temperature. Also, the gas pressure is calculated from the collisional width obtained from fitting a lineshape model to the scanned absorption line and flow velocity can be inferred from a bulk Doppler shift of the absorption line. Since fluorescence emission and absorption occur at the same wavelength range, the CO2 LIF signal is prone to fluorescence trapping and the optical path through the absorbing medium has to be kept short to minimise its influence. To prove the concept, planar pressure and concentration fields were measured in a CO2-Ar jet in Ar co-flow by expanding the laser into a small sheet of 5 mm height. LIF methods In Laser Induced Fluorescence (LIF) methods, the emission wave- length of a laser has to be aligned to an occupied electronic state of a single atomic or molecular species, such that a higher energy level is ex- cited by absorbing part of the incident radiation. After a specific upper state lifetime, the excited molecule relaxes back to its ground energetic state, with a certain probability of emitting a fluorescence photon. The LIF signal can in principle be used to measure number density, tem- perature, species concentration, the velocity component along the laser propagation direction and pressure [99]. Without the addition of tracer molecules, the usage of resonance techniques such as LIF methods must rely on the natural constituents of air. In this context, only O2 (oxygen) and CO2 (carbon dioxide) possess absorption lines that can potentially be probed with available laser light sources to derive the relevant flow quantities in applications of practical relevance. Co2LIF. Laser induced fluorescence of carbon dioxide based on IR (infrared) radiation is a promising approach to measure pressure, tem- perature or flow velocity in flows with CO2 content. The 532 nm output of a Nd:YAG nanosecond-pulse laser was used to pump a wavelength tunable OPO (Optical Parametric Oscillator) to excite CO2 vibrational transitions near 2.0 μm in [108]. Since the resulting fluorescence emission occurs in the infrared at 4.3 μm, a liquid nitrogen cooled IR sensitive InSb (Indium Antimonide) camera was used for detection. A single-pulse example CO2 LIF image is shown in Fig. 12. A strong quenching effect of N2 and H2O on the CO2 fluorescence was identi- fied, so that linear excitation was deemed appropriate for qualitative O2 LIF. In oxygen LIF, transitions in the Schumann-Runge absorption system near 193 nm [100] or 248 nm [101] are excited with ArF (argon fluoride) or KrF (krypton fluoride) excimer lasers. The resulting fluorescence emission spans a wavelength range in the UV (ultraviolet) 11 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 12. Example CO2 LIF visualisation of a 6-mm-diameter forced CO2 jet at room- temperature mixing with ambient air. The fluorescence signal is excited with a 8 mJ pulse at 2.0 μm and an InSb camera collects fluorescence at 4.3 μm. Source: Reprinted with permission from [108]. © 2002 The Optical Society. Fig. 13. LIF methods Single-shot images of displaced FLEET lines at different downstream locations corresponding to heights above the jet exit of 1, 5, 10, and 15 mm. Fluorescence is collected 2 μs after the tagging laser, and the camera gate width is 1 μs. The unperturbed flow position is indicated by the fluorescent lines outside of the free jet. Source: Reprinted with permission from [115]. © 2011 The Optical Society. Fig. 12. Example CO2 LIF visualisation of a 6-mm-diameter forced CO2 jet at room- temperature mixing with ambient air. The fluorescence signal is excited with a 8 mJ pulse at 2.0 μm and an InSb camera collects fluorescence at 4.3 μm. Source: Reprinted with permission from [108]. © 2002 The Optical Society. Fig. 13. Single-shot images of displaced FLEET lines at different downstream locations corresponding to heights above the jet exit of 1, 5, 10, and 15 mm. Fluorescence is collected 2 μs after the tagging laser, and the camera gate width is 1 μs. The unperturbed flow position is indicated by the fluorescent lines outside of the free jet. Source: Reprinted with permission from [115]. © 2011 The Optical Society. visualisation only. The same authors used an identical camera setup, combined with a CO2 laser generating laser pulses at 10.6 μm of 5 J pulse energy and 2 μs length, to realise a saturated excitation scheme [109]. The method was successfully applied to visualise a transverse CO2 jet in a laminar co-flow as well as the CO2 content of a CO/H2 diffusion flame with a large field of view of 11 x 11 cm2. The collected LIF signal was found to be proportional to CO2 mole fraction and a minimum detection limit of about 1000 ppm could be achieved. In a first effort to provide quantitative results by means of the CO2 LIF technique, the saturated approach was applied to measure time-averaged temperature and pressure fields in an under-expanded jet consisting of 30% CO2 and 70% N2 issuing into a 300 mbar nitrogen ambient atmosphere [110]. Similar to [108], an optimised Nd:YAG pump laser OPO combination with an output energy of 75 mJ was used to excite the saturated CO2 LIF signal. A small sheet of 12 mm height was formed and the laser wavelength was consecutively tuned to two absorption lines near 2 μm. From the ratio of the respective LIF signals, time-averaged temperature and pressure fields were derived. LIF methods configuration, which is potentially sufficient for applications in dry standard air (∼400 ppm). Molecular tagging velocimetry Due to superior SNR (signal-to-noise-ratio) compared to previous approaches, the CO2 detection limit was lowered to 200 ppm in the planar measurement FLEET. The FLEET technique is a non-intrusive laser diagnostic method to measure flow velocity in nitrogen containing gas flows introduced by Michael et al. in 2011 [115]. In this work, a femtosecond laser with a pulse width of 120 fs and 1.2 mJ pulse energy, emitting laser light at 810 nm with 10 Hz repetition rate is directed onto the examination area above a high velocity air jet. The very short pulse results in high irradiance at the laser focus, so that nitrogen molecules dissociate into atomic nitrogen. When the nitrogen atoms recombine to N2, a long-lived fluorescence signal in the spectral range between 560 to 660 nm is emitted, tagging the flow along a line of ∼1 cm length. A single component of flow velocity is finally obtained by imaging 12 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 g p ( ) Fig. 14. FLEET measurements in the wake downstream of the CRM wing. A motorised mirror was used to scan the laser beam to eight different spanwise measurement locations. Since the optical setup did not allow vertical translation of the laser beam, the CRM was rolled by ±3◦to obtain velocity profiles above and below the wing at each spanwise position. The 24 measured profiles were combined to a two-dimensional representation of the mean streamwise velocity (freestream velocity 285.9 m/s) (a). The location of the area covered by the FLEET measurements in relation to the aircraft body is shown in (b). Source: Reprinted with permission from Springer Nature, [116]. © 2021. Fig. 14. FLEET measurements in the wake downstream of the CRM wing. A motorised mirror was used to scan the laser beam to eight different spanwise measurement locations. Since the optical setup did not allow vertical translation of the laser beam, the CRM was rolled by ±3◦to obtain velocity profiles above and below the wing at each spanwise position. The 24 measured profiles were combined to a two-dimensional representation of the mean streamwise velocity (freestream velocity 285.9 m/s) (a). The location of the area covered by the FLEET measurements in relation to the aircraft body is shown in (b). Source: Reprinted with permission from Springer Nature, [116]. © 2021. Molecular tagging velocimetry Due to the technique’s capability to measure the concentration of major gas species as well as temperature, laser based spontaneous Raman scattering has extensively been used for pointwise or one- dimensional flame studies [130]. Static pressure or velocity measure- ments are in principle possible, but have not yet been carried out. Raman scattering is about three orders of magnitude weaker compared to (already weak) Rayleigh scattering, it has no demonstrated ability to measure the pertinent flow quantities and is susceptible to strong laser stray light from walls or windows. Therefore, the technique is not discussed further in the current framework of inlet flow distortion measurement. For a single-component gas (air can be treated as such), the laser-induced Rayleigh scattering radiating from a probed volume is proportional to the gas density. Temperature or pressure can then be deduced by applying the ideal gas law, provided, that the one or the other is known. Due to the relative strength of the signal, the laser induced Rayleigh scattering can be used for planar measurements in combination with powerful lasers and sensitive detectors. However, the main difficulty to apply the technique to technical flows is posed by Mie scattering from large particles, which are commonly present in such environments, and laser stray light from surfaces like viewing windows or flow channel walls. Both occur at a similar wavelength and are typically many orders of magnitude stronger compared to molecular Rayleigh scattering. Additional flow information can be extracted from the Rayleigh signal when it is spectrally resolved: The amplitude of the spectrum is related to density, width and shape are associated with pressure and temperature and flow velocity is propor- tional to a Doppler shift of the entire spectrum to higher or lower frequencies [112,131]. Some point-wise measurement approaches such as CRBS (Coherent Rayleigh–Brillouin Scattering) [132–134] or IRS (Interferometric Rayleigh Scattering) [135–138] exist, which exploit the spectral properties of the scattered signal to measure multiple flow quantities simultaneously, but all suffer from the aforementioned om- nipresent laser stray light in enclosed flow environments. In addition, these point-wise techniques lack sufficient spatial detail to analyse and understand complex flow topologies. Due to its stray light suppression capabilities the FRS (Filtered Rayleigh Scattering) technique is the only Rayleigh scattering approach that has the potential to compete under the demanding conditions of inlet flow distortion measurement. Molecular tagging velocimetry the displacement of the tagged region at a later instant in time with an intensified camera. Sample single-pulse FLEET images at different heights above the jet are depicted in Fig. 13. The same authors provided evidence that the FLEET signal in oxygen containing flows is almost one order of magnitude lower compared to pure nitrogen flows in [117]. Subsequently, temperature measurements were performed in heated nitrogen and air jets by spectrally resolving the FLEET emission, indi- cating the potential of the method for combined temperature/velocity measurements [118]. By using stereoscopic observation and operating the intensified cameras in burst mode, the measurement of 3C flow velocity, three-dimensional flow traces as well as acceleration was achieved [119]. Further development efforts of the method targeted the complex dependencies of the fluorescence signal with respect to thermodynamic conditions as well as gas composition [120–124]. In investigating the FLEET technique’s uncertainty limits under thermo- dynamic conditions relevant to cryogenic wind tunnel testing, accuracy of the measured velocities was found to be of the order of 1% with a precision of 0.4 m/s [123]. In this study, the femtosecond laser was operated at 1 kHz repetition rate and the FLEET signal was captured at 200 kHz with a lens-coupled image intensifier/high-speed camera combination. To reach beyond the limitations imposed by the Ti:Sapphire femtosecond laser technology (low pulse energy and repetition rate limited to 10 kHz) used for FLEET tagging, the available measurement rate and pulse energy were further increased by intro- ducing either picosecond [125–127] or femtosecond [128] burst-mode laser technology, to write the tag lines. Recently, the FLEET technique was applied at the cryogenic National Transonic Facility (NTF), NASA Langley Research Center, where it was used to identify a velocity deficit region in the wake of a Common Research Model (CRM) wing (Fig. 14) [116]. A recently published review article by Danehy et al. provides a conclusive summary on the fundamentals, past applications and future lines of development of the FLEET technique [129]. Molecular scattering techniques In contrast to resonant LIF techniques, where the excitation fre- quency of the laser has to coincide with a populated electronic energy level of the probed molecule, the non-resonant scattering of light from molecules is a process that can occur at any wavelength. The incident photon excites the molecule into a virtual state, which immediately relaxes by emitting a photon, so that no lifetime can be associated with this interaction. The molecular scattering of light is further classified into two subcategories. If the wavelength of the excitation and the scattered photon coincide, the ‘‘elastic’’ scattering process is termed Rayleigh scattering. If there is a net energy transfer from the excitation photon to the molecule, ‘‘inelastic’’ spontaneous Raman scattering oc- curs, producing a red- (Stokes) and blue-shifted (anti-Stokes) scattering response [98,112]. FRS. The FRS method relies on the spectral discrimination between i) weak elastic laser light scattering from gas molecules inside a probe volume, which carries information on density, pressure, temperature and flow velocity (the latter through the optical Doppler frequency- shift) and ii) intense laser stray light from surfaces or large particles (Mie scattering) by means of molecular absorption. The basic principle of the method is indicated in Fig. 15. The scattered light from surfaces or particles has the same narrow bandwidth as the illuminating light 13 Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. U. Doll et al. Fig. 15. Narrow bandwidth laser light scattering (Mie/geometric) is attenuated by the molecular filter (dashed–dotted), while portions of the spectrally broadened Rayleigh scattering pass through and form the FRS signal. temperature and velocity fields in gaseous flows with absolute uncer- tainties as low as 15 hPa, 2.5 K and 2.7 m/s, respectively [164]. The modified system hardware, as well as the novel theoretical considera- tions, enabled the characterisation of an internal flow phenomenon by means of the FRS technique, for the first time in the literature [165]. Therein, the modified technique was used to measure pressure, tem- perature and flow velocity inside a circular duct using endoscopic image acquisition. By adopting the idea of observing the measure- ment plane from multiple views to acquire three-component velocities originally introduced for DGV [166], an FRS velocimetry system was developed [131]. Molecular scattering techniques In utilising multiple-branch imaging fibre technology and thus observing the measurement plane from three different sides, the simultaneous acquisition of planar pressure and temperature dis- tributions, as well as three-component velocity fields in the near-field and far-field of a turbulent air jet was achieved (Fig. 16, for far field results see original paper). The CW laser approach was subsequently adapted by Boyda and Lowe, who extended the evaluation procedure by a cross-correlation method [167]. Fig. 15. Narrow bandwidth laser light scattering (Mie/geometric) is attenuated by the molecular filter (dashed–dotted), while portions of the spectrally broadened Rayleigh scattering pass through and form the FRS signal. Apart from the FRS method’s further development in the fields of theory and measurement accuracy, the novel FRS implementation was used for fundamental flow investigations in canonical laboratory flows as well as at large scale experimental facilities related to turbomachin- ery component testing. Regarding the former, the FRS technique was employed to gather detailed temporally averaged planar information on the temperature and velocity fields inside a Ranque–Hilsch vor- tex tube [169]. Observed flow structures suggested a strong coupling between acoustic phenomena and the time-averaged flow field. In addition, FRS was applied to investigate the aero-thermal flow behind the nozzle guide vane (NGV) cascade of a three-sector gas turbine combustor simulator (Fig. 17) [168]. A direct comparison of non- intrusive FRS results with data measured by a conventional five-hole probe revealed a significant bias to velocities measured by the probe, which can be attributed to strong pressure gradients present in the NGV airfoils’ wake areas. Finally, the FRS technique was used to characterise the aero-thermal properties at the outlet of a high-pressure single sector combustor, operated under engine representative conditions [170,171]. In this work, a fully probe-based FRS implementation was presented, which relied on the transport of high-power laser light through optical fibres and observation with a flexible imaging fibre borescope. Results were found to be in good agreement with complementary velocity measurements by PIV and OH-LIF thermometry. Whereas OH-LIF is restricted to temperatures above 1300 K [172], FRS is capable of resolving the whole temperature range from low temperatures near the air-cooled liner walls to high temperatures at the combustor’s centreline. source, while light scattered from gas molecules is spectrally broad- ened. The broadening is mainly caused by the stochastic molecular (Brownian) motion. Molecular scattering techniques In utilising a molecular absorption line as a notch filter, narrow bandwidth laser stray light can be strongly attenuated, while portions of the Rayleigh scattered light pass through the filter and constitute the FRS signal. By adopting the general principle of molecular filtering originally developed for LIDAR2 applications [139], Miles and Lempert intro- duced the FRS technique for aerodynamic diagnostics in 1990 [140]. Miles research group and others have used the FRS technique for ap- plications ranging from planar flow visualisation in high speed aerody- namic flows [141–145], to temperature field measurements in combus- tion flows [146–152]. Other work involves the combined application of PIV and FRS to measure flow and temperature field simultane- ously [153,154]. Furthermore, a frequency scanning FRS method was introduced, which allows the simultaneous derivation of time-averaged pressure, temperature and velocity information [96,155–157]. How- ever, application of the FRS technique has generally been limited to unconfined flows, i.e. to laboratory experiments, where the probed volume of interest was not inside some apparatus and therefore the laser light illumination and scattered light collection did not occur through windows. The main reason for this limitation was constituted by the intense laser light scattered from windows and walls being insufficiently attenuated by the molecular filter. This insufficient at- tenuation resulted from laser-line broadening effects and frequency impurities, both of which are associated with the type of pulsed laser light sources used in the past (typically injection seeded Nd: YAG lasers) [149,151,155,158–163]. 2 Light Detection And Ranging. Ability for seeding-free distortion measurement The previous sections provide an overview on the few laser-optical techniques with the potential to execute non-intrusive seeding-free air flow measurements. In the context of inlet flow distortion measurement in a complex experimental facility, O2 LIF has to be considered inappro- priate. The method is the least mature, relies on complex hardware and the signal-to-noise ratio is very low even under controlled laboratory conditions. CO2 LIF based on IR laser radiation might offer an interest- ing alternative, since time-resolved measurements of static pressure are feasible and lineshape scanning via CW laser excitation can be used for time-averaged multi-parameter measurements. However, the technique is still in an early stage of development and further fundamental research is needed to assess the capabilities of the method. Moreover, the measurement of flow velocity using LIF is only possible along the direction of incidence of the laser, which requires two laser sheets with different directions of propagation to resolve the two in-plane velocity components. The out-of-plane component remains inaccessible to the method [96]. The development of the FLEET technique has kept progressing over the past decade, resulting in an accurate instrument for measuring flow velocity in challenging environments at acquisition Motivated by the fact that the deficient spectral purity of the formerly used pulsed laser light sources constitutes the major obstacle in exploiting the FRS method’s full potential, the Engine Measurement Systems group at DLR (German Aerospace Center) Cologne developed an alternative approach to the FRS technique: In focusing on time- averaged measurements rather than instantaneous results, the system was established around a continuous wave laser. In combining a com- pact and robust design with superior spectral characteristics, this step forward formed the basis of opening the technique to a wide field of applications, ranging from precision laboratory experiments to turbo- machinery component testing under realistic operating conditions. In adopting the aforementioned frequency scanning approach, the system has the capability to simultaneously measure time-averaged pressure, 14 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 16. (Left) Near-field characterisation of a turbulent jet: Light scattered from the interrogation area is collected from three directions by means of a multiple-branch image fibre bundle and transmitted through the detection unit, which is composed of two lenses in retro-arrangement (grey), the molecular iodine filter (pink) as well as a bandpass filter (blue). Quadrants of the resulting image represent the differing camera views. Ability for seeding-free distortion measurement (Right) Near-field jet topology measured by FRS: Temperature and velocity fields both exhibit constant values inside the potential core near the jet axis, enveloped by strong gradients in the shear layer and ambient conditions in the surroundings. Source: Reprinted and adapted with permission from [131]. © 2017 The Optical Society. Fig. 16. (Left) Near-field characterisation of a turbulent jet: Light scattered from the interrogation area is collected from three directions by means of a multiple-branch image fibre bundle and transmitted through the detection unit, which is composed of two lenses in retro-arrangement (grey), the molecular iodine filter (pink) as well as a bandpass filter (blue). Quadrants of the resulting image represent the differing camera views. (Right) Near-field jet topology measured by FRS: Temperature and velocity fields both exhibit constant values inside the potential core near the jet axis, enveloped by strong gradients in the shear layer and ambient conditions in the surroundings. Source: Reprinted and adapted with permission from [131]. © 2017 The Optical Society. Fig. 17. A sectional view of the test rig’s CAD model is shown on the left. Pre-heated air enters the three-sector lean-burn combustor simulator through the swirler elements from the left and as a result relevant temperature distortions together with a high degree of swirl (±50◦) and turbulence intensity (up to ∼30%) approach the NGV cascade. The investigations focus on the central sector which is coloured in orange. Measurements behind the NGV cascade were performed using a conventional five-hole probe with mounted temperature sensor and FRS. The data analysis revealed pronounced differences between the velocity results obtained by both methods, which, when combined with a CFD solution, could be attributed to erroneous probe readings due to strong pressure gradients in the wake zones of the NGV airfoils. Source: Reprinted with permission from Springer Nature, [168]. © 2018. Fig. 17. A sectional view of the test rig’s CAD model is shown on the left. Pre-heated air enters the three-sector lean-burn combustor simulator through the swirler elements from the left and as a result relevant temperature distortions together with a high degree of swirl (±50◦) and turbulence intensity (up to ∼30%) approach the NGV cascade. The investigations focus on the central sector which is coloured in orange. Measurements behind the NGV cascade were performed using a conventional five-hole probe with mounted temperature sensor and FRS. Ability for seeding-free distortion measurement By using a CW laser with excellent spectral characteristics, the FRS method can be used to simultaneously measure time-averaged pressure, one velocity component and temperature fields in complex internal flows. The si- multaneous measurement of time-averaged thermodynamic quantities and 3C velocity field was achieved in a free jet experiment. Even though the time-resolved measurement of multiple flow variables still needs to be demonstrated, the FRS method clearly shows the highest potential to characterise inlet flow distortions in a complex experimental installa- tion. Unlike seeding based PIV or DGV velocimetry methods used so far, the FRS technique is not limited to swirl distortion measurement but has the unique capability to simultaneously capture velocity and scalar fields and, thus, to resolve total pressure and temperature distortions. The characteristics of the different methods and their development potential for inlet flow distortion measurement are summarised in Table 3. light sources to capture unsteady inlet flow distortions. In this context, the aforementioned deficient spectral purity of the pulsed injection- seeded Nd:YAG laser systems used to date significantly limits the applicability of the FRS method for unsteady measurements in confined flow environments. The lack of spectral quality originates from the injection-seeded slave oscillator and is inherent for this type of laser systems [158,162]. To this end, the long pulse laser concept presented in Fig. 183 might prove a viable alternative. In this laser architecture, the only oscillator in the system is found inside the seed laser. Since the realisation of the required spectral properties is much simpler to achieve in these CW solid state lasers [173] and no properties of a second larger cavity have to be taken into account, the spectral purity of the system is only determined by the respective seed pulse entering the amplification stages. Thus, a significantly improved suppression of laser induced stray light is expected, which would enable the FRS technique to measure instantaneous flow data in the direct vicinity of highly luminous surfaces. The general aim of any FRS acquisition and evaluation method is finding a solution to the multiple parameter dependency of the measured signal. One measured intensity value stands against up to five unknown flow quantities4 (three velocity components, tempera- ture, pressure). In addition, a minimum of three linear independent observation directions are required to unambiguously derive the three components of the velocity vector. Ability for seeding-free distortion measurement The data analysis revealed pronounced differences between the velocity results obtained by both methods, which, when combined with a CFD solution, could be attributed to erroneous probe readings due to strong pressure gradients in the wake zones of the NGV airfoils. Source: Reprinted with permission from Springer Nature, [168]. © 2018. rates of up to 1 MHz. However, the high fluence required to dissociate nitrogen molecules at the measurement location, can only be achieved 0-D, 1-D along a line of ∼1 cm length or quasi-2-D by using multiple lines. To spatially resolve the 3C velocity distribution at the AIP would 0-D, 1-D along a line of ∼1 cm length or quasi-2-D by using multiple lines. To spatially resolve the 3C velocity distribution at the AIP would 15 Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. Table 3 Summary of seeding-free optical techniques for inlet flow distortion measurement. Specified quantities are static pressure (𝑝), flow velocity (𝑣), static temperature (𝑇), density (𝜌), concentration (𝑐). Simultaneous measurement of two or more quantities is denoted by ‘‘/’’ (e.g. 𝑝/𝑣/𝑇), time-averaged by TA, time-resolved by TR. 3 Amplitude Technologies (Continuum) Agilite laser family, https: //amplitude-laser.com/products/nanosecond-lasers/nanosecond-advanced- lasers/agilite/. 4 An equation of state is typically used to couple density and pres- sure/temperature. Ability for seeding-free distortion measurement Technique Measured quantities Current state of maturity Potential for inlet flow distortion measurement O2 LIF To date: Visualisation Potential: 𝑇(TR), 𝜌(TR) • Single-shot • Simple laboratory flows, mostly flames • No quantitative measurements to date • No spatial resolution/uncertainty data available NO • Complex hardware (deep UV laser, intensified cameras) • Very weak signal CO2 LIF To date: 𝑝(TA), 𝑇(TA), 𝑝/𝑐(TA) Potential: 𝑝(TR), 𝑇(TR), 𝑝/𝑇/𝑣/𝑐 (TR) Simple laboratory flows • Pulsed: TA 𝑝or 𝑇, large interrogation area • CW: multi-parameter with lineshape scan at 330 fps (0.5 Hz) • No spatial resolution/uncertainty data available Pulsed: YES • Detection limit too high for natural air • Quantitative TR not yet shown CW: YES • Further fundamental investigations needed • Laser-sheet too small FLEET To date: 3C-𝑣(TR), 𝑇(TR) Potential: 3C-𝑣/𝑇(TR) • 0-D, 1-D (1 cm), quasi 2D with multiple taglines • Spatial resolution: 1 mm transverse, 2–3.3 mm streamwise [123] • 𝑣with high accuracy (1%) and precision (0.4 m/s) [123] • Up to 1 MHz repetition rate • Applied to complex flows (cryogenic wind tunnel) YES • Interrogation area too small for TR flow topology • Signal quenching by oxygen FRS To date: 𝑣(TR), 𝑇(TR), 𝑝/3C-𝑣/𝑇 (TA) Potential: 𝑝(TR), 3C-𝑣(TR), 𝑝/3C-𝑣/𝑇(TR) • Planar technique, typical light sheet height 20–150 mm • Spatial resolution 0.2 mm [154] • TR measurements in simple flows only (accuracy 3–10% for single shot 𝑇) • TA 𝑝/𝑣/𝑇in complex flows (accuracy: 50–90 hPa/2-3 K/2–3 m/s [168]), TA 𝑝/3C-𝑣/𝑇in simple flow (accuracy: 5 hPa/1.2 K/1.4 m/s [131]) YES • p accuracy in complex flow to be improved • TA 𝑝/3C-𝑣/𝑇in complex flow not yet demonstrated • Multi-parameter TR not yet demonstrated Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. YES • p accuracy in complex flow to be improved • TA 𝑝/3C-𝑣/𝑇in complex flow not yet demonstrated • Multi-parameter TR not yet demonstrated require a stereo camera setup as well as traversing of the laser through the plane of interest, which is very time consuming and technically demanding. Furthermore, due to the small interrogation area, FLEET cannot be used to resolve the instantaneous flow topology. The FRS technique has shown its potential to measure time-resolved planar distributions of a single flow quantity (predominantly temperature) in simple flow configurations such as free flames or jets. Ability for seeding-free distortion measurement The aforementioned frequency scan- ning method has successfully been used to measure multiple flow quantities simultaneously. By modulating the laser’s output frequency along the molecular filter’s transmission curve in discrete frequency steps, this results in intensity spectra at each camera sensor element. The number of discrete scanning frequencies is chosen to be much larger than the actual number of unknown flow quantities, which turns the evaluation into an over-determined mathematical problem. However, since an image has to be taken at each frequency step, the acquisition procedure is time consuming and cannot be applied to acquire instantaneous flow field data. To overcome these limitations, Table 3. 4 An equation of state is typically used to couple density and pres- sure/temperature. 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing In its time-averaged variant using CW laser illumination, the seeding-free FRS technique has an immediate high potential to be used for the characterisation of intake flow distortion in complex experimental scenarios. Since the FRS method is closely related to DGV in terms of hardware requirements, the DGV approach presented in Fig. 11 serves as an excellent blueprint for applying FRS in simi- lar configurations. To unlock the unsteady measurement potential of FRS to simultaneously measure instantaneous velocity field and scalar flow quantities, however, progress in instrumentation and novel data acquisition and evaluation strategies will be required. Compared to DGV signals from particle scattering, the available scattered intensity by FRS is several orders of magnitude weaker. To achieve the necessary time resolution and signal-to-noise-ratio, FRS measurements at the AIP will require the usage of powerful pulsed laser 16 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 same. Likewise, the detected Doppler frequency shift varies with the scattering geometry. The second approach (Fig. 19, right) uses two acquisition channels as indicated by the two cameras, each equipped with an absorption filter of differing vapour concentration (variants of this concept have been published in [174,175]). Such a configuration would require a minimum of three observation directions, which results in six independent intensity values to derive the five unknown flow quantities. In combination with pulsed laser illumination, both concepts have the capability for synchronous instantaneous 3C velocity, pressure and temperature field measurement. Fig. 18. Long pulse laser design: The single-longitudinal mode CW seed laser (red) is sliced and shaped into an appropriate seed pulse by an electro-optical modulator (yellow). These seed pulses are then amplified in four amplification stages (blue) and finally converted into narrow bandwidth high energy green 532 nm radiation in the second harmonic generator (green). The conceptual approach outlined above describes a possible path- way towards a comprehensive experimental description of both the steady and unsteady components of inlet flow distortion, using the FRS method. Further technological hurdles are introduced by an intended deployment of FRS instrumentation during flight tests. The challenges compared to ground-based measurements arise from the harsh envi- ronment (e.g. vibration, noise, altitude and relative movement of the equipment) and limitations by the aircraft platform (e.g. limited space, quality of optical access, power supply, airworthiness/certification). 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing Furthermore, electronic equipment requiring active cooling such as lasers or scientific cameras must be placed in ambient controlled zones of the aircraft or the aircraft’s operating envelope must be revised (e.g. by reduced flight altitudes) to ensure safe and reliable opera- tion. More information on equipment specific requirements as well as certification and safety aspects in the context of advanced optical diag- nostics can be drawn from the AIM (Advanced In-flight Measurement techniques) and AIM2 project documentation and respective publica- tions [176–178]. Previous examples of using laser-optical diagnostics in-flight are applications of point-wise LDA (Laser Doppler Anemome- try) and L2F (Laser-2-Focus) techniques [179], LIDAR (Light Detection And Ranging) [180,181] and PIV [182,183] for characterising flight speeds close to the fuselage, wake vortices or overwing flow structures and turbulence. Apart from general complexities associated with the in- flight application of laser-optical flow measuring instruments regarding dimensions, weight, energy consumption and optical alignment, the reliable supply of tracer particles under flight conditions is the main challenge for techniques such as LDA, L2F or PIV [74]. Typically, the measurements have to rely on natural occurring seeding like wa- ter droplets inside clouds or particles originating from air pollution. The size distribution and concentration of these particles is highly dependent on the actual weather conditions and has significant impact on data quality and validity [179,182,183]. In view of the envisaged application scenario of measuring the flow inside extremely compact and highly integrated engine inlets, further difficulties arise from the fact that optical accessibility will be reduced to a minimum, which makes the usage of probe-based technology unavoidable. Point-wise LDA or L2F are well suited to be integrated into compact and robust probe solutions. However, these techniques lack the necessary spatial information to assess the expected complex flow patterns. In the light of these drawbacks of current in-flight measuring approaches, the seeding-free FRS method has an immediate high potential for airborne velocimetry and, in addition, has the capability to simultaneously provide static pressure and temperature [74]. In the context of opti- cal access limitations, a fully probe-based implementation of the FRS technique with endoscopic image acquisition and high-power laser light transfer through optical fibres has already been realised [170,171]. This indicates the capabilities of the method to achieve in-situ measurements in challenging flow environments with aggravated optical accessibility requirements. Fig. 18. 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing (2) Since significantly reduced laser energy needs to be introduced into the test section, this notably increases the chance of transmitting the laser light by means of optical waveguides for pulsed laser operation. Earlier work on fibre optic beam delivery for PIV measurements showed that pulse energies of 30 mJ can be transmitted [185]. (3) No installation of beam forming optics is required at the test section. This not only eliminates a potential source of error, but also significantly reduces the optical access requirements for laser illumination to a small bore instead of a large window. controlled zones of the aircraft. Furthermore, optical light sheet and imaging probes could be used to fully accommodate the restricted optical accessibility. In contrast, the application of pulsed laser tech- nology to measure unsteady inlet flow distortions has certainly to be considered less mature. Apart from the significantly larger dimensions, higher weight, power and cooling requirements of such a laser system, it is questionable whether the transfer of the laser light to the test object can occur through optical fibres or whether the necessary high pulse energy (>1 J) would require an actively stabilised laser beam guidance with mirrors. A schematic of a possible FRS in-flight implementation is provided in Fig. 20. Sensitive equipment such as the laser as well as detection unit containing the camera system and temperature stabilised iodine filter are placed inside the ambient controlled part of the aircraft. The laser beam is coupled into the light delivery system, which will preferably be based on fibre technology for both CW or pulsed laser operation. In addition to a high degree of flexibility, no optical interface between ambient controlled aircraft cabin and the non-regulated zone will be required when using fibres. If active beam guidance with mirrors is to be used for pulsed laser operation, an optical port must be designed and the controller has to account for changes in the beam path, caused by deformation of the aircraft body through thermal, pressure and g-load effects. Hollow elements of the aircraft structure can be used to deliver the laser light to the test section. Optical fibres can be installed as simple as electrical cables, while the mirror solution would require mounting brackets with remotely adjustable mirrors on the aircraft structure and additional cables for the control signal. 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing An enlarged schematic view of such an optical probe is shown in Fig. 21. The front end of the probe consists of a collecting lens, which images the plane of interest onto the entry face of an image fibre bundle. The oblique observation results in object areas outside the front lens’ specified depth of field, so that the collecting lens and image fibre bundle surface are aligned at an angle to satisfy the Scheimpflug criterion for sharp imaging of the light sheet throughout the visible domain. Since the perspective arrangement may result in areas that are obscured from observation, this has to be compensated by the additional probes. Similar to the optical fibres used for laser light transport, flexible image fibres can be guided through hollow structural elements to the ambient controlled aircraft cabin containing the detection unit. Fig. 20. Concept of an in-flight FRS measurements system installed to a closely coupled engine configuration. The integrated engine design is inspired by the Bauhaus Luftfahrt Propulsive Fuselage concept [184]. g A possible solution to relax the requirements for unsteady measure- ments using pulsed laser radiation, is posed by the multi-pass concept shown in Fig. 22. Instead of spanning a light sheet to illuminate the complete cross section of the inlet duct, the unconditioned laser beam enters the test section from below. After the first pass, the beam is deflected by two mirrors fixed inside a hollow shroud and directed back into the flow. This is repeated several times, so that a regular pattern of crossing laser beams fills the cross section (the arrangement shown is only to illustrate the concept, several more passes could be implemented). Except for the red transparent region, where the laser direction is undefined and detector saturation can be expected, 3C velocity, static pressure and temperature can be inferred at each resolution element along the individual beam traces. If a sufficient angular resolution is realised from a large number of passes, two- dimensional maps can be generated by interpolation. Such a setup would be beneficial in a number of ways: (1) By illuminating the cross section with a laser beam pattern instead of spanning the full width with a light sheet, more than two orders of magnitude less laser energy is required to reach similar FRS signal levels. 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing Long pulse laser design: The single-longitudinal mode CW seed laser (red) is sliced and shaped into an appropriate seed pulse by an electro-optical modulator (yellow). These seed pulses are then amplified in four amplification stages (blue) and finally converted into narrow bandwidth high energy green 532 nm radiation in the second harmonic generator (green). two imaging concepts with the potential to simultaneously acquire multiple instantaneous flow variables are depicted in Fig. 19. Both are inspired by the FRS velocimetry concept [131] and rely on multiple- branch imaging fibre technology to observe the measurement plane from multiple directions. The first configuration (Fig. 19, left) requires a minimum of five observation branches to unambiguously derive the five flow quantities of interest. The approach takes advantage of the fact that the detected intensity per resolution element is not only a function of the wanted flow quantities, but also of the scattering angle, which is defined as the inverse cosine of the scalar product of the unit vector in the laser and observer direction. The influence of the scat- tering angle on the signal intensity is twofold: For multiple observers, the Rayleigh scattering’s spectral width and shape changes with the scattering angle, even though the thermodynamic conditions stay the From a technological point of view, there is a high chance of successfully implementing CW FRS technology on a flying test bed to perform time-averaged in-situ flow measurements inside closely inte- grated engine inlet configurations. Volume, weight and electrical power consumption of such an FRS system remain moderate. The usage of fibre technology for laser light transfer and image detection allows for flexible and convenient installation through unpressurised and ambient 17 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 Fig. 19. Two alternative versions of FRS-camera system setups for the instantaneous measurement of 3C-velocity, temperature and pressure. Two alternative versions of FRS-camera system setups for the instantaneous measurement of 3C-velocity, temperature and pressure. Fig. 20. Concept of an in-flight FRS measurements system installed to a closely coupled engine configuration. The integrated engine design is inspired by the Bauhaus Luftfahrt Propulsive Fuselage concept [184]. of flow velocity combined with static pressure and temperature, at least three camera views are required for CW and five for pulsed laser operation. Furthermore, a stronger inclination of the different views is potentially beneficial for measurement accuracy. 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing Multi-hole or total pressure probe rakes are typ- ically used for time-averaged measurement of the relevant quantities to derive total pressure and swirl distortion metrics. However, the steady state picture utterly neglects the dynamic component of inlet flow distortion, which can reach magnitudes of up to 30% of the time- averaged quantities. Furthermore, the combination of different types of distortions is an underdeveloped field and there is a great lack of understanding of the impact that these events can have on the stability budget of an engine. To capture the unsteady dimension of the problem, the state-of-the-art for measuring total pressure distortion relies on miniature high-speed pressure transducers mounted on rake arrays, rotor blades or in blade passages, at acquisition rates on the order of 10 kHz. However, since the method is intrusive, a balance has to be found between the unavoidable flow channel blockage and spatial resolution necessary to capture the relevant flow details. Efforts to characterise swirl distortion at the AIP employing non-intrusive laser- optical DGV and stereoscopic PIV, increased the number of available datapoints by orders of magnitude. But the provision of appropriate seeding poses a major difficulty of these methods, which motivated the review on seeding-free laser-optical measurement techniques. In this context, the FRS technique is found to have the highest potential for characterising inlet flow distortions, since the method has the proven ability to simultaneously measure velocity, static pressure and temperature fields in complex experimental installations. Moreover, a fully probe-based realisation of FRS has already been implemented, underpinning the unique ability to use FRS technology along the entire engine development cycle in ground and in-flight testing. Fig. 21. Possible layout of a fibre-optical imaging probe and optical arrangement. Fig. 22. Concept of a multi-pass arrangement: The laser beam enters from below and is deflected multiple times into the test section. The red area marks the intersection region, where evaluation will not be feasible due to beam overlap. Even though non-intrusive diagnostics has been successfully intro- duced in the framework of inlet flow distortion measurement, there is an urgent need to develop novel measuring and analytical methods to meet the anticipated demand for more comprehensive flow infor- mation, due to the effects of highly convoluted intake architectures. 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing The laser is then directed into the beam forming optics and the light sheet enters the test section through an optical access to illuminate the AIP. The collection of the FRS signal occurs through observation probes, which are flush-mounted at several positions (two are indicated in red in Fig. 20) around the flow channel. To measure all three components As far as the future prospects of such in-flight measuring systems are concerned, it can be anticipated that their development will signif- icantly improve the current state-of-the-art in several aspects. Firstly, in-flight measurements could be used for engine stability assessment in line with the established stability metrics to determine the sensi- tivity of the propulsion system to the intake distortion, especially for transient in-flight operating conditions, which are not possible to be reproduced in ground tests. Secondly, the assessment of the unsteady 18 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 U. Doll et al. Fig. 21. Possible layout of a fibre-optical imaging probe and optical arrangement. Fig. 22. Concept of a multi-pass arrangement: The laser beam enters from below and is deflected multiple times into the test section. The red area marks the intersection region, where evaluation will not be feasible due to beam overlap. swirl distortion could aid in the determination of the aeromechanical compatibility between intake and rotor blades. New methods have been recently developed to assess the flow distortion approaching rotating blades [50]. The evaluation of the unsteady incidence signature on the blades can give insights on the blade loading distribution. This can be linked with the formation of stall cells [186] and, thus, can b l d h h b l ll h h ll Fig. 21. Possible layout of a fibre-optical imaging probe and optical arrangement. Fig. 21. Possible layout of a fibre-optical imaging probe and optical arrangement. the distorted propulsor inflow on fan performance. To unlock the full potential of these novel concepts, a close integration of airframe and engine design and development is imperative. Historically, inlet distortion has been treated as a steady state event and several descriptors have been developed in academics and industry. Despite efforts to develop standardised metrics by the S-16 committee of SAE International, no unique set of distortion descriptors has been established to date. 3.3. FRS for in-situ laser-optical distortion measurement in ground and in-flight testing To this end, this article identifies FRS measurement technology as a promising candidate, but further development efforts in suitable laser light sources, acquisition and evaluation procedures is needed to open the technique to unsteady inlet flow distortion measurement. The modification of established steady-state distortion descriptors to incorporate previously unavailable unsteady flow data and the develop- ment of novel distortion metrics, is currently an area of active research and will continue to gain importance as future developments of air- craft with fuselage-integrated engine concepts progress. This alignment of capability is setting the stage for exciting research questions and provides opportunities for close collaboration of academia and in- dustry, to foster faster and more educated design choices, ultimately leading to shorter innovation cycles and reduced time-to-market of next-generation aircraft designs. Fig. 22. Concept of a multi-pass arrangement: The laser beam enters from below and is deflected multiple times into the test section. The red area marks the intersection region, where evaluation will not be feasible due to beam overlap. swirl distortion could aid in the determination of the aeromechanical compatibility between intake and rotor blades. New methods have been recently developed to assess the flow distortion approaching rotating blades [50]. The evaluation of the unsteady incidence signature on the blades can give insights on the blade loading distribution. This can be linked with the formation of stall cells [186] and, thus, can be correlated with the engine stability. Finally, the FRS technique will offer additional capabilities to simultaneously measure velocity and scalar fields, so that data required to derive all established metrics could be obtained with a single, seeding-free, non-intrusive instrument. Moreover, the potential to simultaneously measure unsteady velocity, pressure and temperature by FRS opens the path for the development of completely new distortion metrics based on scalar-velocity correlations. Declaration of competing interest The authors declare that they have no known competing finan- cial interests or personal relationships that could have appeared to influence the work reported in this paper. References rep, National Aeronautics and Space Administration (NASA), 2010, p. 183. [14] A. Uranga, M. Drela, E.M. Greitzer, D.K. Hall, N.A. Titchener, M.K. Lieu, N.M. Siu, C. Casses, A.C. Huang, G.M. Gatlin, J.A. Hannon, Boundary layer ingestion benefit of the D8 transport aircraft, AIAA J. 55 (11) (2017) 3693–3708, http://dx.doi.org/10.2514/1.J055755. [37] P.D. Silkowski, Measurements of Rotor Stalling in a Matched and a Mismatched Multistage Compressor, Tech. rep, GTL-221GTL, Gas Turbine Laboratory, Mas- sachusetts Institute of Technology, Cambridge, MA, 1995, URL http://hdl. handle.net/1721.1/1047644. [15] L. Wiart, O. Atinault, B. Paluch, D. Hue, R. Grenon, Development of NOVA air- craft configurations for large engine integration studies, in: 33rd AIAA Applied Aerodynamics Conference, American Institute of Aeronautics and Astronautics, Dallas, USA, 2015, http://dx.doi.org/10.2514/6.2015-2254. [38] C. Freeman, A.L. Rowe, Intake engine interactions of a modern large turbofan engine, in: ASME 1999 International Gas Turbine and Aeroengine Congress and Exhibition, American Society of Mechanical Engineers, Indianapolis, IN, USA, 1999, http://dx.doi.org/10.1115/99-GT-344. [16] H.D. Kim, J.L. Felder, M. Armstrong, Revolutionary aeropropulsion concept for sustainable aviation: Turboelectric distributed propulsion, in: XXI International Symposium on Air Breathing Engines, ISABE, Busan, Korea, 2013, URL https: //core.ac.uk/display/42733509. [39] T. Breuer, N.C. Bissinger, Basic principles - gas turbine compatibility - gas turbine aspects, in: Encyclopedia of Aerospace Engineering, John Wiley & Sons, Ltd, Chichester, UK, 2010, http://dx.doi.org/10.1002/9780470686652.eae573, arXiv:arXiv:1011.1669v3. [17] E. de la Rosa Blanco, C. Hall, D. Crichton, Challenges in the silent aircraft engine design, in: 45th AIAA Aerospace Sciences Meeting and Exhibit, American Institute of Aeronautics and Astronautics, Reno, USA, 2007, http://dx.doi.org/ 10.2514/6.2007-454. [40] C. Tan, I. Day, S. Morris, A. Wadia, Spike-type compressor stall inception, detection, and control, Annu. Rev. Fluid Mech. 42 (1) (2010) 275–300, http: //dx.doi.org/10.1146/annurev-fluid-121108-145603. [41] M. Shaw, An Assessment of CFD for Transonic Fan Stabilities Studies, (Ph.D. thesis), Queens’ College, University of Cambridge, 2015. [18] G. McLelland, D.G. MacManus, P.K. Zachos, D. Gil-Prieto, M. Migliorini, Influence of upstream total pressure profiles on S-duct intake flow distortion, J. Propul. Power 36 (3) (2020) 346–356, http://dx.doi.org/10.2514/1.B37554. [42] T. Dejene Toge, A.M. Pradeep, Experimental investigation of stall inception and its propagation in a contra rotating axial fan under radial inflow distortion, in: ASME Turbo Expo: Turbine Technical Conference and Exposition, American Society of Mechanical Engineers, Charlotte, North Carolina, USA, 2017, http: //dx.doi.org/10.1115/GT2017-63432. [19] W.T. References Greitzer, Review–axial compressor stall phenomena, J. Fluids Eng. 102 (2) (1980) 134–151, http://dx.doi.org/10.1115/1.3240634. [31] K.-S. Song, S.-G. Kim, Y.-H. Hwang, Failure of the J79 engine compressor blade due to stall, J. Fail. Anal. Prev. 7 (3) (2007) 212–217, http://dx.doi.org/10. 1007/s11668-007-9034-4. [8] A. Seitz, A.s.L. Habermann, F. Peter, F. Troeltsch, A. Castillo Pardo, B. Della Corte, M. van Sluis, Z. Goraj, M. Kowalski, X. Zhao, T. Grönstedt, J. Bijewitz, G. Wortmann, Proof of concept study for fuselage boundary layer ingesting propulsion, Aerospace 8 (1) (2021) http://dx.doi.org/10.3390/ aerospace8010016. [32] J. Melick, H. C., Analysis of inlet flow distortion and turbulence effects on compressor stability, Tech. rep, NASA CR-114577, LTV Aerospace Corp.; Vought Systems Div.;, Dallas, TX, US, 1973, p. 215, URL https://ntrs.nasa.gov/search. jsp?R=19730012966. [9] J. Welstead, J.L. Felder, Conceptual design of a single-aisle turboelec- tric commercial transport with fuselage boundary layer ingestion, in: 54th AIAA Aerospace Sciences Meeting, American Institute of Aeronautics and Astronautics, San Diego, USA, 2016, http://dx.doi.org/10.2514/6.2016-1027. [33] W. Cousins, A theory for the prediction of compressor blade aerodynamic response, in: 34th AIAA/ASME/SAE/ASEE Joint Propulsion Conference and Exhibit, in: Joint Propulsion Conferences, American Institute of Aeronautics and Astronautics, Cleveland,USA, 1998, http://dx.doi.org/10.2514/6.1998-3308. [10] R.P. Rademakers, R. Niehuis, S. Bindl, Influence of secondary flow within integrated engine inlets on the performance and stability of a jet engine, in: XXII International Symposium on Air Breathing Engines, ISABE, Phoenix, USA, 2015, URL http://hdl.handle.net/2374.UC/745645. [34] J.P. Longley, H.W. Shin, R.E. Plumley, P.D. Silkowski, I.J. Day, E.M. Greitzer, C.S. Tan, D.C. Wisler, Effects of rotating inlet distortion on multistage com- pressor stability, J. Turbomach. 118 (2) (1996) 181–188, http://dx.doi.org/10. 1115/1.2836624. [11] M.M. Wojewodka, C. White, K. Kontis, A review of flow control techniques and optimisation in S-shaped ducts, Int. J. Heat Fluid Flow (2017) 48, http: //dx.doi.org/10.1016/j.ijheatfluidflow.2018.06.016. [35] B. Brimelow, T. Collins, G. Pfefferkorn, Engine testing in a dynamic environ- ment, in: 10th Propulsion Conference, American Institute of Aeronautics and Astronautics, San Diego, CA, USA, 1974, http://dx.doi.org/10.2514/6.1974- 1198. g j j [12] M. Drela, Development of the D8 transport configuration, in: 29th AIAA Applied Aerodynamics Conference, American Institute of Aeronautics and Astronautics, Honolulu, Hawaii, 2011, http://dx.doi.org/10.2514/6.2011-3970. [36] R.J. Wenzel, L. M.; Blaha, Analysis of Dynamic Inlet Distortion Applied to a Parallel Compressor Model, Tech. rep, NASA-TM-X-3522,NASA Technical Memorandum TM-X-3522, NASA Lewis Research Center;, Cleveland, OH, US, 1977, p. 25, URL https://ntrs.nasa.gov/search.jsp?R=19770015161. [13] E.M. Greitzer, H.N. Slater, N+3 Aircraft Concept Designs and Trade Studies, Final Report, Tech. References References [23] S-16 Turbine Engine Inlet Flow Distortion Committee, Guidelines for Char- acterization of Gas Turbine Engine Total-Pressure, Planar-Wave, and Total- Temperature Inlet-Flow Distortion, ARP6420, Tech. rep, SAE International, 2021, p. 34, http://dx.doi.org/10.4271/ARP6420. [1] NASA Stragetic Implementation Plan - 2019 Update, Tech. rep, National Aeronautics and Space Administration (NASA), 2019, URL https://www.nasa. gov/sites/default/files/atoms/files/sip-2019-v7-web.pdf. [24] Sutrisno, T.A. Rohmat, S.B. Wibowo, S. Iswahyudi, Vortex dynamics study of the canard deflection angles’ influence on the Sukhoi Su-30-like model to improve stall delays at high AoA, Aerosp. 6 (2) (2019) 12, http://dx.doi.org/10.3390/ aerospace6020012. [2] Strategic Research and Innovation Agenda - 2017 Update, Tech. rep, Advisory Council for Aviation Research and Innovation in Europe (ACARE), 2017, URL https://www.acare4europe.org/sites/acare4europe.org/files/document/ACARE- Strategic-Research-Innovation-Volume-1.pdf. [25] A. Dowling, T. Hynes, Towards a silent aircraft, Aeronaut. J. 110 (1110) (2006) 487–494, http://dx.doi.org/10.1017/S000192400000138X. [3] European commission – directorate general for research and innovation & directorate general for mobility and transport, in: Flightpath 2050: Europe’s vision for aviation, The Publications Office of the European Union, Luxembourg, 2011, http://dx.doi.org/10.2777/50266. [26] A. Coschignano, H. Babinsky, Boundary-layer development downstream of normal shock in transonic intakes at incidence, AIAA J. 57 (12) (2019) 5241–5251, http://dx.doi.org/10.2514/1.J058508. [27] S-16 Turbine Engine Inlet Flow Distortion Committee, Gas Turbine Engine Inlet Flow Distortion Guidelines, ARP1420C, Tech. rep, SAE International, 2017, p. 25, http://dx.doi.org/10.4271/ARP1420C. [4] C.A. Hall, D. Crichton, Engine and installation configurations for a silent aircraft, in: XVII International Symposium on Air Breathing Engines, ISABE, American Institute of Aeronautics and Astronautics, Munich, Germany, 2005. [5] H. Kim, M.-S. Liou, Flow simulation of N2B hybrid wing body configuration, in: 50th AIAA Aerospace Sciences Meeting Including the New Horizons Forum and Aerospace Exposition, American Institute of Aeronautics and Astronautics, Nashville, USA, 2012, http://dx.doi.org/10.2514/6.2012-838. [28] C. Reid, The response of axial flow compressors to intake flow distortion, in: ASME 1969 Gas Turbine Conference and Products Show, American Society of Mechanical Engineers, Cleveland, USA, 1969, http://dx.doi.org/10.1115/69- GT-29. [6] J.R. Lucas, W.F. O’Brien, A.M. Ferrar, Effect of BLI–type inlet distortion on turbofan engine performance, in: ASME Turbo Expo: Turbine Technical Con- ference and Exposition, American Society of Mechanical Engineers, Düsseldorf, Germany, 2014, http://dx.doi.org/10.1115/GT2014-26666. [29] A.F. Campbell, An Investigation of Distortion Indices for Prediction of Stalling Behavior in Aircraft Gas Turbine Engines, (Ph.D. thesis), Virginia Polytechnic In- stitute and State University, 1981, URL https://vtechworks.lib.vt.edu/bitstream/ handle/10919/95571/LD5655.V855_1981.C343.pdf?sequence=1. [7] A. Castillo Pardo, C.A. Hall, Aerodynamics of boundary layer ingesting fuselage fans, J. Turbomach. 143 (4) (2021) 041007, http://dx.doi.org/10.1115/1. 4049918. [30] E.M. Acknowledgements The SINATRA project leading to this publication has received fund- ing from the Clean Sky 2 Joint Undertaking (JU) under grant agreement No 886521. The JU receives support from the European Union’s Hori- zon 2020 research and innovation programme and the Clean Sky 2 JU members other than the Union. Novel airframe designs, with integrated engine architectures, offer potential benefits in terms of an increased aircraft efficiency and noise reduction. However, a major risk arises from inlet flow distortion caused by BLI and convoluted engine intakes, such that the possible gain in aerodynamic efficiency might be partially offset by the effect of 19 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 References [57] M. Lecht, H. Weyer, Unsteady Rotor Blade Loading in an Axial Compressor with Steady-state Inlet Distortions, Tech. rep, AGARD CP 248-30, AGARD CP 248-30, Cleveland, OH, US, 1978. [81] T. Guimarães, K.T. Lowe, W.F. O’Brien, Particle image velocimetry for distorted turbofan engine inlet applications, in: 19th International Symposium on the Application of Laser and Imaging Techniques To Fluid Mechanics, Lisbon, Portugal, 2018. [58] G.A. Mitchell, Effect of Inlet Ingestion of a Wing Tip Vortex on Compressor Face Flow and Turbojet Stall Margin, Tech. rep, NASA-TM-X-3246, E-8213,NASA Technical Memorandum TM-X-3246, Washington D.C., US, 1975, p. 78, URL https://ntrs.nasa.gov/search.jsp?R=19750018896. [59] A. Mehdi, Effect of Swirl Distortion on Gas Turbine Operability, (Ph.D. thesis), Propulsion Engineering Centre, School of Aerospace, Transport and Manufacturing, Cranfield University, 2014. [82] J.F. Meyers, H. Komine, Doppler global velocimetry-a new way to look at velocity, in: ASME Fourth International Conference on Laser Anemometry, Cleveland, USA, 1991. [60] A. Naseri, M. Boroomand, S. Sammak, Numerical investigation of effect of inlet swirl and total-pressure distortion on performance and stability of an axial transonic compressor, J. Therm. Sci. 25 (6) (2016) 501–510, http://dx.doi.org/ 10.1007/s11630-016-0891-6. [83] H. Komine, System for measuring velocity field of fluid flow utilizing a laser- doppler spectral image converter, (US4919536A) 1990, URL https://patents. google.com/patent/US4919536/en. [84] I. Roehle, Laser-Doppler-Velocimetry auf der Basis frequenzselektiver Absorp- tion: Aufbau und Einsatz eines Doppler Global Velocimeters, (Ph.D. thesis), Ruhr Universität Bochum, Bochum, 1999. [61] B. Tu, L. Zhang, J. Hu, Effect of swirl on the performance and stability of transonic axial compressor, Proc. Inst. Mech. Eng., Part A 232 (6) (2018) 608–621, http://dx.doi.org/10.1177/0957650917742320. [85] I. Roehle, Three-dimensional Doppler global velocimetry in the flow of a fuel spray nozzle and in the wake region of a car, Flow Meas. Instrum. 7 (3) (1996) 287–294, http://dx.doi.org/10.1016/S0955-5986(97)00011-3. [62] J. Jacocks, Statistical analysis of distortion factors, in: AIAA 1972-1100, 8th Joint Propulsion Specialist Conference, November 1972, 1972, http://dx.doi. org/10.2514/6.1972-1100. [86] C. Willert, G. Stockhausen, J. Klinner, C. Lempereur, P. Barricau, P. Loiret, J.C. Raynal, Performance and accuracy investigations of two Doppler global velocimetry systems applied in parallel, Meas. Sci. Technol. 18 (8) (2007) 2504, http://dx.doi.org/10.1088/0957-0233/18/8/027, publisher: IOP Publishing. g [63] M. Davis, A. Hale, D. Beale, An argument for enhancement of the current inlet distortion ground test practice for aircraft gas turbine engines, J. Turbomach. 124 (2) (2002) 235–241, http://dx.doi.org/10.1115/1.1451087. [64] T. Williams, C. Hall, M. Wilson, Low pressure ratio transonic fan stall with radial distortion, J. References Cousins, History, philosophy, physics, and future directions of aircraft propulsion system/inlet integration, in: ASME Turbo Expo: Turbine Technical Conference nd Exposition, Vol.2, American Society of Mechanical Engineers, Vienna, Austria, 2004, http://dx.doi.org/10.1115/GT2004-54210. [43] A.J. Provenza, K.P. Duffy, M.A. Bakhle, Aeromechanical response of a distortion-tolerant boundary layer ingesting fan, J. Eng. Gas Turbines Power 141 (1) (2019) http://dx.doi.org/10.1115/1.4040739. [20] S-16 Turbine Engine Inlet Flow Distortion Committee, Inlet Total-Pressure- Distortion Considerations for Gas-Turbine Engines, AIR1419C, Tech. rep, SAE International, 2017, p. 226, http://dx.doi.org/10.4271/AIR1419C. [44] W. Zhang, M. Vahdati, A parametric study of the effects of inlet distortion on fan aerodynamic stability, J. Turbomach. 141 (1) (2019) http://dx.doi.org/10. 1115/1.4041376. [21] S-16 Turbine Engine Inlet Flow Distortion Committee, Assessment of the Inlet/Engine Total Temperature Distortion Problem, AIR5867, Tech. rep, SAE International, 2017, p. 57, http://dx.doi.org/10.4271/AIR5867. [45] Y. Liu, E. Stumpf, Estimation of vehicle-level fuel burn benefits of aircraft formation flight, J. Aircraft 55 (2) (2018) 853–861, http://dx.doi.org/10.2514/ 1.C034296. [22] S-16 Turbine Engine Inlet Flow Distortion Committee, A Methodology for Assessing Inlet Swirl Distortion, AIR5686, Tech. rep, SAE International, 2017, p. 110, http://dx.doi.org/10.4271/AIR5686. 20 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 [71] D. Gil-Prieto, P.K. Zachos, D.G. MacManus, G. McLelland, Unsteady characteris- tics of S-duct intake flow distortion, Aerosp. Sci. Technol. 84 (2019) 938–952, http://dx.doi.org/10.1016/j.ast.2018.10.020. [46] Airbus and its partners demonstrate how sharing the skies can save airlines fuel and reduce CO2 emissions, Tech. rep, Airbus Press Release, 2021. [47] G.A. Richardson, W.N. Dawes, A.M. Savill, An unsteady, moving mesh CFD simulation for Harrier hot-gas ingestion control analysis, Aeronaut. J. 111 (1117) (2007) 133–144, http://dx.doi.org/10.1017/S0001924000004395. [72] S-16 Turbine Engine Inlet Flow Distortion Committee, Inlet/Engine Compat- ibility - From Model to Full Scale Development, AIR5687A, Tech. rep, SAE International, 2016, http://dx.doi.org/10.4271/air5687a. [48] T.J. Biesiadny, W.M. Braithwaite, R.H. Soeder, M. Abdelwahab, Summary of Investigations of Engine Response to Distorted Inlet Conditions, Tech. rep, NASA TM-87317, Lewis Research Center, Cliveland, Ohio, 1986, p. 215, URL https://ntrs.nasa.gov/citations/19860016864. [73] P.K. Zachos, D.G. MacManus, D.G. Prieto, N. Chiereghin, Flow distortion measurements in convoluted aeroengine intakes, AIAA J. 54 (9) (2016) 2819– 2832, http://dx.doi.org/10.2514/1.J054904, Publisher: American Institute of Aeronautics and Astronautics. p g [49] S.R. Wellborn, B.A. Reichert, T.H. Okiishi, Study of the compressible flow in a diffusing S-duct, J. Propuls. Power 10 (5) (1994) 668–675, http://dx.doi.org/ 10.2514/3.23778. [74] K.T. Lowe, Laser velocimetry for turbofan inlet distortion applications, Aircr. Eng. Aerosp. Tec. References 92 (1) (2020) 20–26, http://dx.doi.org/10.1108/AEAT-11- 2018-0285, publisher: Emerald Publishing Limited. [50] M. Migliorini, P.K. Zachos, D.G. MacManus, Novel method for evaluating intake unsteady flow distortion, J. Propuls. Power 38 (1) (2021) 135–147, http://dx.doi.org/10.2514/1.B38127. [75] M. Raffel, C.E. Willert, F. Scarano, C.J. Kähler, S.T. Wereley, J. Kompenhans, Particle Image Velocimetry, Springer International Publishing, 2018, http://dx. doi.org/10.1007/978-3-319-68852-7. [51] J.P. Murphy, D.G. MacManus, Ground vortex aerodynamics under crosswind conditions, Exp. Fluids 50 (1) (2011) 109–124, http://dx.doi.org/10.1007/ s00348-010-0902-4. [76] K.M. Hoopes, A New Method for Generating Swirl Inlet Distortion for Jet Engine Research, (Master thesis), Virginia Tech, 2013, URL https://vtechworks.lib.vt. edu/handle/10919/49545. [52] Ç. Atalayer, J. Friedrichs, D. Wulff, Aerodynamic investigation of S-duct intake for high power turboprop installed on a channel wing, Aeronaut. J. 121 (1242) (2017) 1131–1146, http://dx.doi.org/10.1017/aer.2017.46. [77] T. Guimaraes Bucalo, K.T. Lowe, W.F. O’Brien, An overview of recent results using the StreamVane method for generating tailored swirl distortion in jet engine research, in: 54th AIAA Aerospace Sciences Meeting, in: AIAA SciTech Forum, American Institute of Aeronautics and Astronautics, San Diego, USA, 2016, http://dx.doi.org/10.2514/6.2016-0534. [53] B. Bouldin, Y. Sheoran, Inlet flow angularity descriptors proposed for use with gas turbine engines, in: World Aviation Congress & Exposition, SAE International, Phoenix, USA, 2002, http://dx.doi.org/10.4271/2002-01-2919. [54] F. Aulehla, Intake swirl - a major disturbance parameter in engine/intake compatibility, in: 13th Congress of ICAS/AIAA, MBB-FE124-S-PUB–74, Seattle, WA, US, 1982, URL http://hdl.handle.net/10068/164048. [78] M. Nelson, K.T. Lowe, W.F. O’Brien, K.M. Hoopes, Stereoscopic PIV mea- surements of swirl distortion on a full-scale turbofan engine inlet, in: 52nd Aerospace Sciences Meeting, in: AIAA SciTech Forum, American Institute of Aeronautics and Astronautics, National Harbor, USA, 2014, http://dx.doi.org/ 10.2514/6.2014-0533. [55] A. Hamed, K. Numbers, Inlet distortion considerations for high cycle fatigue in gas turbine engine, in: AIAA/ASME/SAE/ASEE 33rd Joint Propulsion Confer- ence and Exhibit, American Institute of Aeronautics and Astronautics, Seattle, WA, US, 1997, http://dx.doi.org/10.2514/6.1997-3364. [79] T. Guimarães, K.T. Lowe, W.F. O’Brien, Streamvane turbofan inlet swirl dis- tortion generator: Mean flow and turbulence structure, J. Propuls. Power 34 (2) (2018) 340–353, http://dx.doi.org/10.2514/1.B36422, Publisher: American Institute of Aeronautics and Astronautics. [56] M. Lecht, H. Weyer, On the unsteady aerodynamic rotor blade loading in a transonic axial flow compressor with unsteady-state inlet distortion, in: IUTAM Symposium on Aeroelasticity in Turbomachines, Paris, France, 1976, pp. 1–16. [80] T. Guimarães, K. Todd Lowe, W.F. O’Brien, Complex flow generation and development in a full-scale turbofan inlet, J. Eng. Gas Turbines Power 140 (8) (2018) http://dx.doi.org/10.1115/1.4039179. References Glob. Power Propuls. Soc. 4 (2020) 226–237, http://dx. doi.org/10.33737/jgpps/130478. [87] U. Dierksheide, P. Meyer, T. Hovestadt, W. Hentschel, Endoscopic 2D particle image velocimetry (PIV) flow field measurements in IC engines, Exp. Fluids 33 (6) (2002) 794–800, http://dx.doi.org/10.1007/s00348-002-0499-3. [65] D. Kidman, J. Brownlow, B. Binkley, C. Morris, Application of extreme value statistics to reduce test and computational requirements for quantifying engine inlet total pressure distortion, in: Presentation for OA Forum DoD, Academia and Industry Statistics Workshop, 2016. [88] M. Kegalj, H.-P. Schiffer, Endoscopic PIV measurements in a low pressure turbine rig, Exp. Fluids 47 (4) (2009) 689, http://dx.doi.org/10.1007/s00348- 009-0712-8. [89] D. Kim, L. Rao, H. Oh, S. Kook, Endoscopic high-speed particle image velocime- try (eHS-PIV) in a high tumble production engine, Exp. Fluids 61 (10) (2020) 219, http://dx.doi.org/10.1007/s00348-020-03055-w. [66] S-16 Turbine Engine Inlet Flow Distortion Committee, An Assessment of Planar Waves, AIR5866A, Tech. rep, SAE International, 2021, p. 64, http://dx.doi.org/ 10.4271/AIR5866A. [90] R. Schlüßler, M. Bermuske, J. Czarske, A. Fischer, Simultaneous three- component velocity measurements in a swirl-stabilized flame, Exp. Fluids 56 (10) (2015) 183, http://dx.doi.org/10.1007/s00348-015-2055-y. [67] Y. Sheoran, B. Bouldin, R. Hoover, M. Matwey, A centrifugal compressor operability correlation with combined total pressure and swirl distortion, in: Volume 1: Aircraft Engine; Fans and Blowers; Marine; Honors and Awards, American Society of Mechanical Engineers, Charlotte, North Carolina, USA, 2017, http://dx.doi.org/10.1115/GT2017-63721. [91] A.J. Saltzman, K.T. Lowe, W.F. Ng, 250 kHz Three-component Doppler ve- locimetry at 32 simultaneous points: a new capability for high speed flows, Meas. Sci. Technol. 31 (9) (2020) 095302, http://dx.doi.org/10.1088/1361- 6501/ab8ee9, Publisher: IOP Publishing. [68] W.M. Braithwaite, R.H. Soeder, Combined pressure and temperature distortion effects on internal flow of a turbofan engine, J. Aircraft 17 (7) (1980) 468–472, http://dx.doi.org/10.2514/3.57927. 6501/ab8ee9, Publisher: IOP Publishing. [92] A.J. Saltzman, K.T. Lowe, W.F. Ng, 50 kHz Doppler global velocimetry for the study of large-scale turbulence in supersonic flows, Exp. Fluids 62 (9) (2021) 192, http://dx.doi.org/10.1007/s00348-021-03286-5. p g [69] G. Tanguy, D.G. MacManus, E. Garnier, P.G. Martin, Characteristics of unsteady total pressure distortion for a complex aero-engine intake duct, Aerosp. Sci. Technol. 78 (2018) 297–311, http://dx.doi.org/10.1016/j.ast.2018.04.031. [93] S.J. Thorpe, N. Quinlan, R.W. Ainsworth, The characterisation and application of a pulsed neodymium YAG laser DGV system to a time-varying high-speed flow, Opt. Laser Technol. 32 (7) (2000) 543–555, http://dx.doi.org/10.1016/ S0030-3992(00)00096-7. [70] D. Gil-Prieto, D.G. MacManus, P.K. Zachos, A. Bautista, Assessment methods for unsteady flow distortion in aero-engine intakes, Aerosp. Sci. Technol. 72 (2018) 292–304, http://dx.doi.org/10.1016/j.ast.2017.10.029. References 21 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 [94] M.A.F. Kendall, N.J. Quinlan, S.J. Thorpe, R.W. Ainsworth, B.J. Bellhouse, Measurements of the gas and particle flow within a converging-diverging nozzle for high speed powdered vaccine and drug delivery, Exp. Fluids 37 (1) (2004) 128–136, http://dx.doi.org/10.1007/s00348-004-0792-4. [116] D.T. Reese, R.J. Thompson, R.A. Burns, P.M. Danehy, Application of femtosecond-laser tagging for unseeded velocimetry in a large-scale transonic cryogenic wind tunnel, Exp. Fluids 62 (5) (2021) 99, http://dx.doi.org/10. 1007/s00348-021-03191-x. [95] R.L. McKenzie, M.S. Reinath, Three dimensional planar Doppler velocity mea- surements in a full-scale rotor wake, AIAA J. 43 (3) (2005) 489–499, http: //dx.doi.org/10.2514/1.12205. [117] J. Michael, M. Edwards, A. Dogariu, R. Miles, Velocimetry by femtosecond laser electronic excitation tagging (FLEET) of air and nitrogen, in: 50th AIAA Aerospace Sciences Meeting Including the New Horizons Forum and Aerospace Exposition, American Institute of Aeronautics and Astronautics, Nashville, USA, 2012, http://dx.doi.org/10.2514/6.2012-1053. [96] J.N. Forkey, Development and demonstration of filtered Rayleigh scattering: a laser based flow diagnostic for planar measurement of velocity, temperature, and pressure, (Ph.D. thesis), Princeton University, 1996. [118] M.R. Edwards, A. Dogariu, R.B. Miles, Simultaneous temperature and velocity measurements in air with femtosecond laser tagging, AIAA J. 53 (8) (2015) 2280–2288, http://dx.doi.org/10.2514/1.J053685. [97] J.N. Forkey, W.R. Lempert, R.B. Miles, Observation of a 100-MHz frequency variation across the output of a frequency-doubled injection-seeded unstable- resonator Q-switched nd:YAG laser, Opt. Lett. 22 (4) (1997) 230–232, http: //dx.doi.org/10.1364/OL.22.000230. [119] P.M. Danehy, B.F. Bathel, N.D. Calvert, A. Dogariu, R.B. Miles, Three com- ponent velocity and acceleration measurement using FLEET, in: 30th AIAA Aerodynamic Measurement Technology and Ground Testing Conference, in: AIAA AVIATION Forum, American Institute of Aeronautics and Astronautics, Atlanta, USA, 2014, http://dx.doi.org/10.2514/6.2014-2228. [98] C. Tropea, A.L. Yarin, J.F. Foss, Springer Handbook of Experimental Fluid Mechanics, in: Springer handbooks, Springer, Berlin, 2007, http://dx.doi.org/ 10.1007/978-3-540-30299-5. [99] R.K. Hanson, R.M. Spearrin, C.S. Goldenstein, Spectroscopy and Optical Diag- nostics for Gases, Springer International Publishing, Cham, 2016, http://dx.doi. org/10.1007/978-3-319-23252-2. [120] N.J. DeLuca, R.B. Miles, W.D. Kulatilaka, N. Jiang, J.R. Gord, Femtosecond laser electronic excitation tagging (FLEET) fundamental pulse energy and spectral response, in: 30th AIAA Aerodynamic Measurement Technology and Ground Testing Conference, American Institute of Aeronautics and Astronautics, Atlanta, USA, 2014, http://dx.doi.org/10.2514/6.2014-2227. [100] G. Massey, C. Lemon, Feasibility of measuring temperature and density fluctuations in air using laser-induced O2 fluorescence, event: IEEE J. Quan- tum Electron. 20 (5) (1984) 454–457, http://dx.doi.org/10.1109/JQE.1984. 1072427. [121] N. Calvert, Y. References Zhang, R.B. Miles, Characterizing FLEET for aerodynamic measurements in various gas mixtures and non-air environments, in: 32nd AIAA Aerodynamic Measurement Technology and Ground Testing Conference, American Institute of Aeronautics and Astronautics, Washington, D.C., USA, 2016, http://dx.doi.org/10.2514/6.2016-3206. [101] P. Andresen, A. Bath, W. Gröger, H.W. Lülf, G. Meijer, J.J. ter Meulen, Laser- induced fluorescence with tunable excimer lasers as a possible method for instantaneous temperature field measurements at high pressures: checks with an atmospheric flame, Appl. Opt. 27 (2) (1988) 365–378, http://dx.doi.org/10. 1364/AO.27.000365. [122] Y. Zhang, R.B. Miles, Femtosecond laser tagging for velocimetry in argon and nitrogen gas mixtures, Opt. Lett. 43 (3) (2018) 551–554, http://dx.doi.org/10. 1364/OL.43.000551, publisher: Optical Society of America. [102] R.B. Miles and, W.R. Lempert, Quantitative flow visualization in unseeded flows, Annu. Rev. Fluid Mech. 29 (1) (1997) 285–326, http://dx.doi.org/10.1146/ annurev.fluid.29.1.285. [123] R.A. Burns, C.J. Peters, P.M. Danehy, Unseeded velocimetry in nitrogen for high-pressure, cryogenic wind tunnels: part I. Femtosecond-laser tagging, Meas. Sci. Technol. 29 (11) (2018) 115302, http://dx.doi.org/10.1088/1361-6501/ aade1b, publisher: IOP Publishing. [103] R.B. Miles, J.J. Connors, P.J. Howard, E.C. Markovitz, G.J. Roth, Proposed single-pulse two-dimensional temperature and density measurements of oxygen and air, Opt. Lett. 13 (3) (1988) 195–197, http://dx.doi.org/10.1364/OL.13. 000195, publisher: Optical Society of America. [124] C.J. Peters, R.A. Burns, R.B. Miles, P.M. Danehy, Effect of low temperatures and pressures on signal, lifetime, accuracy and precision of femtosecond laser tagging velocimetry, Meas. Sci. Technol. (2020) http://dx.doi.org/10.1088/ 1361-6501/abc577. 000195, publisher: Optical Society of America. [104] G. Laufer, R.L. McKenzie, D.G. Fletcher, Method for measuring temperatures and densities in hypersonic wind tunnel air flows using laser-induced O2 fluorescence, Appl. Opt. 29 (33) (1990) 4873–4883, http://dx.doi.org/10.1364/ AO.29.004873, publisher: Optical Society of America. [125] N. Jiang, J.G. Mance, M.N. Slipchenko, J.J. Felver, H.U. Stauffer, T. Yi, P.M. Danehy, S. Roy, Seedless velocimetry at 100 khz with picosecond-laser electronic-excitation tagging, Opt. Lett. 42 (2) (2017) 239–242, http://dx.doi. org/10.1364/OL.42.000239, publisher: Optical Society of America. [105] M. Smith, W. Williams, L. Price, Applicability of dual-line LIF-O2 method for measurements in engine test facilities, in: 4th AlAA International Aerospace Planes Conference, American Institute of Aeronautics and Astronautics, Orlando, USA, 1992, http://dx.doi.org/10.2514/6.1992-5089. [126] R.A. Burns, P.M. Danehy, N. Jiang, M.N. Slipchenko, J. Felver, S. Roy, Unseeded velocimetry in nitrogen for high-pressure cryogenic wind tunnels: part II. Picosecond-laser tagging, Meas. Sci. Technol. 29 (11) (2018) 115203, http: //dx.doi.org/10.1088/1361-6501/aade15, publisher: IOP Publishing. [106] J. Kiefer, B. Zhou, J. Zetterberg, Z. Li, M. References Alden, Laser-induced fluorescence detection of hot molecular oxygen in flames using an alexandrite laser, Appl. Spectrosc. (2014) http://dx.doi.org/10.1366/14-07512, publisher: SAGE PublicationsSage UK: London, England. [127] Z. Zhang, N. Jiang, M.N. Slipchenko, J.G. Mance, S. Roy, Picosecond laser electronic excitation tagging velocimetry using a picosecond burst-mode laser, Appl. Opt. AO60 (15) (2021) C60–C67, http://dx.doi.org/10.1364/AO.419491. [107] M. Schultze, J. Mantzaras, F. Grygier, R. Bombach, Hetero-/homogeneous combustion of syngas mixtures over platinum at fuel-rich stoichiometries and pressures up to 14bar, Proc. Combust. Inst. 35 (2) (2015) 2223–2231, http: //dx.doi.org/10.1016/j.proci.2014.05.018. [128] J.M. Fisher, M.E. Smyser, M.N. Slipchenko, S. Roy, T.R. Meyer, Burst-mode femtosecond laser electronic excitation tagging for khz–MHz seedless velocime- try, Opt. Lett. 45 (2) (2020) 335–338, http://dx.doi.org/10.1364/OL.380109, publisher: Optical Society of America. [108] B.J. Kirby, R.K. Hanson, Linear excitation schemes for IR planar-induced fluorescence imaging of CO and CO2, Appl. Opt. 41 (6) (2002) 1190–1201, http: //dx.doi.org/10.1364/AO.41.001190, publisher: Optical Society of America. [129] P.M. Danehy, R.A. Burns, D.T. Reese, J.E. Retter, S.P. Kearney, FLEET ve- locimetry for aerodynamics, Annu. Rev. Fluid Mech. 54 (1) (2022) 525–553, http://dx.doi.org/10.1146/annurev-fluid-032321-025544. g p p y [109] B.J. Kirby, R.K. Hanson, CO2 imaging with saturated planar laser-induced vibrational fluorescence, Appl. Opt. 40 (33) (2001) 6136–6144, http://dx.doi. org/10.1364/AO.40.006136, publisher: Optical Society of America. [130] A. Ehn, J. Zhu, X. Li, J. Kiefer, Advanced laser-based techniques for gas-phase diagnostics in combustion and aerospace engineering, Appl. Spectrosc. 71 (3) (2017) 341–366, http://dx.doi.org/10.1177/0003702817690161. [110] D.A. Rothamer, R.K. Hanson, Temperature and pressure imaging using infrared planar laser-induced fluorescence, Appl. Opt. 49 (33) (2010) 6436–6447, http: //dx.doi.org/10.1364/AO.49.006436, publisher: Optical Society of America. [131] U. Doll, G. Stockhausen, C. Willert, Pressure, temperature, and three-component velocity fields by filtered Rayleigh scattering velocimetry, Opt. Lett. 42 (19) (2017) 3773–3776, http://dx.doi.org/10.1364/OL.42.003773. [111] C.S. Goldenstein, V.A. Miller, R.K. Hanson, Infrared planar laser-induced fluo- rescence with a CW quantum-cascade laser for spatially resolved CO2 and gas properties, Appl. Phys. B 120 (2) (2015) 185–199, http://dx.doi.org/10.1007/ s00340-015-6167-0. [132] X. Pan, M.N. Shneider, R.B. Miles, Coherent Rayleigh-Brillouin scattering, Phys. Rev. Lett. 89 (18) (2002) 183001, http://dx.doi.org/10.1103/PhysRevLett.89. 183001. [112] P.M. Danehy, B.F. Bathel, C.T. Johansen, M. Winter, S. O’Byrne, A.D. Cutler, Molecular-Based Optical Diagnostics for Hypersonic Nonequilibrium Flows, American Institute of Aeronautics and Astronautics, Reston, VA, 2015, http: //dx.doi.org/10.2514/4.103292. [133] X. Pan, P.F. Barker, A. Meschanov, J.H. Grinstead, M.N. Shneider, R.B. Miles, Temperature measurements by coherent Rayleigh scattering, Opt. Lett. 27 (3) (2002) 161, http://dx.doi.org/10.1364/OL.27.000161. [113] D.G. Bohl, M.M. Koochesfahani, B.J. References Olson, Development of stereoscopic molec- ular tagging velocimetry, Exp. Fluids 30 (3) (2001) 302–308, http://dx.doi.org/ 10.1007/s003480000178. [134] A. Gerakis, M.N. Shneider, Towards single shot gas flow velocity and tem- perature measurements with coherent Rayleigh-Brillouin scattering, in: AIAA Scitech 2019 Forum, American Institute of Aeronautics and Astronautics, San Diego, USA, 2019, http://dx.doi.org/10.2514/6.2019-1326. [114] R.B. Miles, J.J. Connors, E.C. Markovitz, P.J. Howard, G.J. Roth, Instanta- neous profiles and turbulence statistics of supersonic free shear layers by Raman excitation plus laser-induced electronic fluorescence (relief) velocity tagging of oxygen, Exp. Fluids 8 (1) (1989) 17–24, http://dx.doi.org/10.1007/ BF00203060. [135] R.G. Seasholtz, A.E. Buggele, M.F. Reeder, Flow measurements based on Rayleigh scattering and Fabry-Perot interferometer, Opt. Laser Eng. 27 (6) (1997) 543–570, http://dx.doi.org/10.1016/S0143-8166(96)00063-2. [115] J.B. Michael, M.R. Edwards, A. Dogariu, R.B. Miles, Femtosecond laser elec- tronic excitation tagging for quantitative velocity imaging in air, Appl. Opt. 50 (26) (2011) 5158–5162, http://dx.doi.org/10.1364/AO.50.005158, publisher: Optical Society of America. [136] A.F. Mielke, R.G. Seasholtz, K.A. Elam, J. Panda, Time-average measurement of velocity, density, temperature, and turbulence velocity fluctuations using Rayleigh and Mie scattering, Exp. Fluids 39 (2) (2005) 441–454, http://dx. doi.org/10.1007/s00348-005-0990-8. 22 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 [137] J. Panda, Spectrally-resolved Rayleigh scattering to measure velocity, tempera- ture, density, and density fluctuations in high-speed flows, Exp. Fluids 61 (3) (2020) 74, http://dx.doi.org/10.1007/s00348-020-2903-2. [158] R.G. Seasholtz, A.E. Buggele, Improvement in suppression of pulsed nd:YAG laser light with iodine absorption cells for filtered Rayleigh scattering mea- surements, in: Optical Technology in Fluid, Thermal, and Combustion Flow III, Vol.3172, International Society for Optics and Photonics, 1997, pp. 625–635, http://dx.doi.org/10.1117/12.293414. [138] A.D. Cutler, K. Rein, S. Roy, P.M. Danehy, N. Jiang, 100-Khz interferometric Rayleigh scattering for multi-parameter flow measurements, Opt. Express 28 (3) (2020) 3025–3040, http://dx.doi.org/10.1364/OE.380934, publisher: Optical Society of America. [159] P.M. Allison, T.A. McManus, J.A. Sutton, Quantitative fuel vapor/air mixing imaging in droplet/gas regions of an evaporating spray flow using filtered Rayleigh scattering, Opt. Lett. 41 (6) (2016) 1074, http://dx.doi.org/10.1364/ OL.41.001074. [139] H. Shimizu, S.A. Lee, C. She, High spectral resolution lidar system with atomic blocking filters for measuring atmospheric parameters, Appl. Opt. 22 (9) (1983) 1373–1381, http://dx.doi.org/10.1364/AO.22.001373. [160] C. Ground, L. Maddalena, Visualization of supersonic turbulent vortical flows with filtered Rayleigh scattering, J. Vis.-Jap. 20 (1) (2017) 75–86, http://dx. doi.org/10.1007/s12650-016-0382-6. [140] R. Miles, W. Lempert, Two-dimensional measurement of density, velocity, and temperature in turbulent high-speed air flows by UV Rayleigh scattering, Appl. Phys. References B: Photophys. Laser Chem. 51 (1) (1990) 1–7, http://dx.doi.org/10.1007/ BF00332317. [161] Y. Krishna, A.M. Elbaz, Y. Yue, G. Magnotti, Mole fraction measurement through a transparent quarl burner using filtered Rayleigh scattering, Appl. Opt. 58 (20) (2019) 5575, http://dx.doi.org/10.1364/AO.58.005575. [141] R. Miles, W. Lempert, J. Forkey, Instantaneous velocity fields and background suppression by filtered Rayleigh scattering, in: 29th Aerospace Sciences Meet- ing, American Institute of Aeronautics and Astronautics, Reno, USA, 1991, http://dx.doi.org/10.2514/6.1991-357. [162] R.A. Patton, J.A. Sutton, Seed laser power effects on the spectral purity of Q-switched nd:YAG lasers and the implications for filtered Rayleigh scattering measurements, Appl. Phys. B 111 (3) (2013) 457–468, http://dx.doi.org/10. 1007/s00340-013-5358-9. [142] R. Miles, J. Forkey, W. Lempert, Filtered Rayleigh scattering measurements in supersonic/hypersonic facilities, in: 28th Joint Propulsion Conference and Exhibit, American Institute of Aeronautics and Astronautics, Nashville, USA, 1992, http://dx.doi.org/10.2514/6.1992-3894. [163] J.A. Sutton, R.A. Patton, Improvements in filtered Rayleigh scattering mea- surements using Fabry–Perot etalons for spectral filtering of pulsed, 532-nm nd:YAG output, Appl. Phys. B 116 (3) (2014) 681–698, http://dx.doi.org/10. 1007/s00340-013-5752-3. [143] J. Forkey, S. Cogne, A. Smits, S. Bogdonoff, W. Lempert, R. Miles, Time- sequenced and spectrally filtered Rayleigh imaging of shock wave and boundary layer structure for inlet characterization, in: 29th Joint Propulsion Conference and Exhibit, American Institute of Aeronautics and Astronautics, Monterey, USA, 1993, http://dx.doi.org/10.2514/6.1993-2300. [164] U. Doll, E. Burow, G. Stockhausen, C. Willert, Methods to improve pressure, temperature and velocity accuracies of filtered Rayleigh scattering measure- ments in gaseous flows, Meas. Sci. Technol. 27 (12) (2016) 125204, http: //dx.doi.org/10.1088/0957-0233/27/12/125204. [165] U. Doll, G. Stockhausen, C. Willert, Endoscopic filtered Rayleigh scattering for the analysis of ducted gas flows, Exp. Fluids 55 (3) (2014) http://dx.doi.org/ 10.1007/s00348-014-1690-z. [144] R. Miles, W. Lempert, J. Forkey, N. Finkelstein, P. Erbland, Quantifying high speed air flows by light scattering from air molecules, in: Fluid Dynamics Conference, American Institute of Aeronautics and Astronautics, Colorado Springs, USA, 1994, http://dx.doi.org/10.2514/6.1994-2230. [166] D.S. Nobes, H.D. Ford, R.P. Tatam, Instantaneous, three-component planar Doppler velocimetry using imaging fibre bundles, Exp. Fluids 36 (1) (2004) 3–10, http://dx.doi.org/10.1007/s00348-003-0586-0. [145] J. Forkey, W. Lempert, S. Bogdonoff, R. Miles, G. Russell, Volumetric imaging of supersonic boundary layers using filtered Rayleigh scattering background suppression, in: 32nd Aerospace Sciences Meeting and Exhibit, American Institute of Aeronautics and Astronautics, Reno, USA, 1994, http://dx.doi.org/ 10.2514/6.1994-491. [167] M. Boyda, G. Byun, K.T. Lowe, Investigation of velocity and temperature mea- surement sensitivities in cross-correlation filtered Rayleigh scattering (CCFRS), Meas. Sci. Technol. References 30 (4) (2019) 044004, http://dx.doi.org/10.1088/1361- 6501/ab0350. [146] G.S. Elliott, N. Glumac, C.D. Carter, A.S. Nejad, Two-dimensional temper- ature field measurements using a molecular filter based technique, Com- bust. Sci. Technol. 125 (1–6) (1997) 351–369, http://dx.doi.org/10.1080/ 00102209708935663. [168] U. Doll, M. Dues, T. Bacci, A. Picchi, G. Stockhausen, C. Willert, Aero-thermal flow characterization downstream of an NGV cascade by five-hole probe and filtered Rayleigh scattering measurements, Exp. Fluids 59 (10) (2018) 150, http://dx.doi.org/10.1007/s00348-018-2607-z. [147] D. Hoffman, K.-U. Münch, A. Leipertz, Two-dimensional temperature determi- nation in sooting flames by filtered Rayleigh scattering, Opt. Lett. 21 (7) (1996) 525, http://dx.doi.org/10.1364/OL.21.000525. [169] U. Doll, E. Burow, M. Beversdorff, G. Stockhausen, C. Willert, C. Morsbach, D. Schlüß, M. Franke, The flow field inside a Ranque-Hilsch vortex tube part I: Experimental analysis using planar filtered Rayleigh scattering, in: 9th International Symposium on Turbulence and Shear Flow Phenomena, TSFP 2015, Vol.2, Melbourne, Australia, 2015. [148] D. Most, A. Leipertz, Simultaneous two-dimensional flow velocity and gas temperature measurements by use of a combined particle image velocimetry and filtered Rayleigh scattering technique, Appl. Opt. 40 (30) (2001) 5379, http://dx.doi.org/10.1364/AO.40.005379. [170] M. Schroll, U. Doll, G. Stockhausen, U. Meier, C. Willert, C. Hassa, I. Bagchi, Flow field characterization at the outlet of a lean burn single-sector combustor by laser-optical methods, J. Eng. Gas Turbines Power 139 (1) (2017) http: //dx.doi.org/10.1115/1.4034040. [149] S.P. Kearney, R.W. Schefer, S.J. Beresh, T.W. Grasser, Temperature imaging in nonpremixed flames by joint filtered Rayleigh and Raman scattering, Appl. Opt. 44 (9) (2005) 1548, http://dx.doi.org/10.1364/AO.44.001548. [171] U. Doll, G. Stockhausen, J. Heinze, U. Meier, C. Hassa, I. Bagchi, Temperature measurements at the outlet of a lean burn single-sector combustor by laser optical methods, J. Eng. Gas Turbines Power 139 (2) (2017) http://dx.doi.org/ 10.1115/1.4034355. [150] J. Zetterberg, Z. Li, M. Afzelius, M. Aldén, Two-dimensional temperature measurements in flames using filtered Rayleigh scattering at 254 nm, Appl. Spectrosc. 62 (7) (2008) 778–783, publisher: Society for Applied Spectroscopy. [151] D. Müller, R. Pagel, A. Burkert, V. Wagner, W. Paa, Two-dimensional temper- ature measurements in particle loaded technical flames by filtered Rayleigh scattering, Appl. Opt. 53 (9) (2014) 1750, http://dx.doi.org/10.1364/AO.53. 001750. [172] J. Heinze, U. Meier, T. Behrendt, C. Willert, K.-P. Geigle, O. Lammel, R. Lückerath, PLIF thermometry based on measurements of absolute concentrations of the OH radical, Z. Phys. Chem 225 (11–12) (2011) 1315–1341, http://dx. doi.org/10.1524/zpch.2011.0168. [152] T.A. McManus, J.A. References Sutton, Quantitative planar temperature imaging in turbu- lent non-premixed flames using filtered Rayleigh scattering, Appl. Opt. 58 (11) (2019) 2936, http://dx.doi.org/10.1364/AO.58.002936. [173] W. Koechner, Solid-State Laser Engineering, Vol. 1, 6, Springer, 2006, http: //dx.doi.org/10.1007/0-387-29338-8. [153] D. Most, F. Dinkelacker, A. Leipertz, Direct determination of the turbulent flux by simultaneous application of filtered Rayleigh scattering thermometry and particle image velocimetry, Proc. Combust. Inst. 29 (2) (2002) 2669–2677, http://dx.doi.org/10.1016/S1540-7489(02)80325-X. [174] M.G. Boguszko, Measurements In Fluid Flows Using Filtered Rayleigh Scattering, (Ph.D. thesis), Rutgers, The State University of New Jersey, 2003. [175] T.P. Jenkins, J. George, D. Feng, R.B. Miles, Filtered Rayleigh scattering for instantaneous measurements of pressure and temperature in gaseous flows, in: AIAA Scitech 2019 Forum, American Institute of Aeronautics and Astronautics, San Diego, USA, 2019, http://dx.doi.org/10.2514/6.2019-1324. [154] T.A. McManus, J.A. Sutton, Simultaneous 2D filtered Rayleigh scattering thermometry and stereoscopic particle image velocimetry measurements in turbulent non-premixed flames, Exp. Fluids 61 (6) (2020) 134, http://dx.doi. org/10.1007/s00348-020-02973-z. [176] Final Report Summary - AIM (Advanced in-flight measurement Techniques) \| Report Summary \| FP6, CORDIS \| European Commission, URL https://cordis. europa.eu/project/id/30827/reporting. [155] J.N. Forkey, N.D. Finkelstein, W.R. Lempert, R.B. Miles, Demonstration and characterization of filtered Rayleigh scattering for planar velocity mea- surements, AIAA J. 34 (3) (1996) 442–448, http://dx.doi.org/10.2514/3. 13087. [177] Final Report Summary - AIM2 (Advanced In-flight Measurement Techniques 2) \| Report Summary \| FP7, CORDIS \| European Commission, URL https: //cordis.europa.eu/project/id/266107/reporting. [156] J.N. Forkey, W.R. Lempert, R.B. Miles, Accuracy limits for planar measure- ments of flow field velocity, temperature and pressure using filtered Rayleigh scattering, Exp. Fluids 24 (2) (1998) 151–162, http://dx.doi.org/10.1007/ s003480050162. [178] F. Boden, N. Lawson, H.W. Jentink, J. Kompenhans (Eds.), Advanced in- flight measurement techniques, in: Research Topics in Aerospace, Springer Berlin Heidelberg, Berlin, Heidelberg, 2013, http://dx.doi.org/10.1007/978-3- 642-34738-2. [179] M. Beversdorff, W. Förster, R. Schodl, H.W. Jentink, In-flight laser anemometry for aerodynamic investigations on an aircraft, Opt. Laser Eng. 27 (6) (1997) 571–586, http://dx.doi.org/10.1016/S0143-8166(96)00064-4. [157] M. Boguszko, G.S. Elliott, On the use of filtered Rayleigh scattering for measurements in compressible flows and thermal fields, Exp. Fluids 38 (1) (2005) 33–49, http://dx.doi.org/10.1007/s00348-004-0881-4. 23 U. Doll et al. Progress in Aerospace Sciences 130 (2022) 100810 [180] R.K. Bogue, H.W. Jentink, Optical Air Flow Measurements in Flight, Tech. rep, National Aeronautics and Space Administration (NASA), 2004, p. 101. [183] C. Dunker, An in-flight investigation of a turbulent boundary layer at Reynolds numbers up to Re𝛷=49,400, Exp. Fluids 62 (1) (2021) 4, http://dx.doi.org/10. [182] C. Dunker, R. Geisler, Full-scale in-flight flow investigation of a high-lift vortex system by means of particle image velocimetry, in: New Results in Numerical and Experimental Fluid Mechanics XI, in: posts on Numerical Fluid Mechanics and Multidisciplinary Design, Springer International Publishing, Cham, Germany, 2018, pp. 523–532, http://dx.doi.org/10.1007/978-3-319- 64519-3_47. References 1007/s00348-020-03072-9. [181] H.W. Jentink, R.K. Bogue, Optical air flow measurements for flight tests and flight testing optical air flow meters, Tech. rep, National Aerospace Laboratory (NLR), 2005. [184] Bauhaus Luftfahrt https://www.bauhaus-luftfahrt.net/de/topthema/propulsive- fuselage/ (Accessed 16 April 2021). [182] C. Dunker, R. Geisler, Full-scale in-flight flow investigation of a high-lift vortex system by means of particle image velocimetry, in: New Results in Numerical and Experimental Fluid Mechanics XI, in: posts on Numerical Fluid Mechanics and Multidisciplinary Design, Springer International Publishing, Cham, Germany, 2018, pp. 523–532, http://dx.doi.org/10.1007/978-3-319- 64519-3_47. [185] D.P. Hand, J.D. Entwistle, R.R.J. Maier, A. Kuhn, C.A. Greated, J.D.C. Jones, Fibre optic beam delivery system for high peak power laser PIV illumination, Meas. Sci. Technol. 10 (3) (1999) 239–245, http://dx.doi.org/10.1088/0957- 0233/10/3/021. [186] J.E. Giuliani, J.-P. Chen, Fan response to boundary-layer ingesting inlet distortions, AIAA J. 54 (10) (2016) 3232–3243, http://dx.doi.org/10.2514/1. J054762. 24 24 Cranfield University CERES Research Repository https://dspace.lib.cranfield.ac.uk/ School of Aerospace, Transport and Manufacturing (SATM) Staff publications (SATM) School of Aerospace, Transport and Manufacturing (SATM) Staff publications (SATM) Doll U, Migliorini M, Baikie J, et al., (2022) Non-intrusive flow diagnostics for unsteady inlet flow distortion measurements in novel aircraft architectures, Progress in Aerospace Sciences, Volume 130, April 2022, Article number 100810 https://doi.org/10.1016/j.paerosci.2022.100810 Downloaded from CERES Research Repository, Cranfield University Non-intrusive flow diagnostics for unsteady inlet flow distortion measurements in novel aircraft architectures Doll, Ulrich 2022-03-09 Attribution 4.0 International
https://openalex.org/W4367593883	http://ejurnal.iaipd-nganjuk.ac.id/index.php/j-kis/article/download/553/340	Indonesian	null	The Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan	Jurnal Komunikasi Islam	2,023	cc-by	5,101	Analisis Program Siara Analisis Program Siar edan Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan 1Tifani Liusnimun, 2Nurul Hidayat, 3Zulfahmi Ariadi Nasution, 4Fahrur Rozi, 5Iqbal Saputra, 6Fadlan Sani Nasution 123456UIN Sumatera Utara 1tifaniliusnimun22@gmail.com, 2hdytnrl040@gmail.com, 3nasutionfahmi5@gmail.com, 4tornyrozy@gmail.com, 5saputraiqbal311@gmail..com, 5fadlansani175@gmail.com 1Tifani Liusnimun, 2Nurul Hidayat, 3Zulfahmi Ariadi Nasution, 4Fahrur Rozi, 5Iqbal Saputra, 6Fadlan Sani Nasution 123456UIN Sumatera Utara 1tifaniliusnimun22@gmail.com, 2hdytnrl040@gmail.com, 3nasutionfahmi5@gmail.com, 4tornyrozy@gmail.com, 5saputraiqbal311@gmail..com, 5fadlansani175@gmail.com INFO ARTIKEL Submit Revisi Diterima Publish : 12/11/2022 : 29/11/2022 : 25/12/2022 : 29/12/2022 INFO ARTIKEL Submit Revisi Diterima Publish : 12/11/2022 : 29/11/2022 : 25/12/2022 : 29/12/2022 Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 How to Cite: Hidayat, N. 2023. The Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan. J-KIs: Jurnal Komunikasi Islam. 3, 2 (Jan. 2023), 43-56. DOI:https://doi.org/10.53429/j- kis.v3i2.553. Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 ABSTRAK Radio Prambors sudah mengudara di delapan kota yang berada di Indonesia, salah satunya adalah Kota Medan dengan frekuensi 97.5 FM. Sebuah radio yang penyajiannya sangat memanjakan telinga dan mata dengan memberikan pengetahuan, kebutuhan untuk anak muda sekarang ini, sehingga anak muda mudah terpikat dan menyukai Radio Prambors. Banyaknya program siaran yang ada di Radio Prambors 97.5 FM Medan, Kumpul Kamu menjadi program yang sangat disukai pendengar, meskipun semua programnya sangat disukai namun Kumpul Kamu memiliki daya tarik yang kuat. Penelitian ini menggunakan pendekatan kualitatif dengan metode deskriptif. Teknik pengumpulan data pada penelitian ini melalui wawancara dan melengkapi dengan berbagai sumber melalui internet, buku dan jurnal yang membahas tentang Prambors. Teknik analisis data dalam penelitian ini ialah teknik analisis data kualitatif dari hasil wawancara dengan pihak radio Prambors 97.5 FM. Hasil penelitian menunjukkan bahwa sebelum program Kumpul Kamu disiarkan tentu melewati tiga tahap yaitu, pra produksi (Perencanaan program Kumpul Kamu sampai pelaksanaan kegiatan), produksi (Semua kegiatan yang dari programnya disiarkan) dan pasca produksi (Penyuntingan, evaluasi dll). Tujuan dari penelitian ini adalah untuk mengetahui bagaimana proses berlangsungnya program siaran Kumpul Kamu Radio Prambors 97.5 FM yang dimulai dari pra produksi, produksi dan pasca produksi. KATA KUNCI Radio Prambors 97.5 FM, Kumpul Kamu, Pra Produksi, Produksi, Pasca Produksi 121 Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 How to Cite: Hidayat, N. 2023. The Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan. J-KIs: Jurnal Komunikasi Islam. 3, 2 (Jan. 2023), 43-56. DOI:https://doi.org/10.53429/j- kis.v3i2.553. 122 Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 122 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 PENDAHULUAN Pada penelitian ini penulis meneliti tentang radio, karena pada saat ini radio sudah mulai dikesampingkan dengan berkembangnya teknologi komunikasi khususnya internet dan TV. Sejalan dengan perkembangan teknologi komunikasi, media komunikasi massa pun semakin canggih dan kompleks, serta memiliki kekuatan yang lebih dari masa-masa sebelumnya. Begitupun dengan media elektronik yang semakin berkembang dengan berbagai informasi yang disajikan dari berbagai sudut penjuru dunia. Berbicara mengenai media elektronik sebagai alat komunikasi massa pada hakikatnya merupakan suatu proses, sudah pasti memerlukan berbagai sistem atau komponen. Everett M. Rogers mengatakan bahwa dalam kegiatan komunikasi ada empat elemen yang harus diperhatikan, yaitu source, message, channel dan receiver. Komponen- komponen tersebut merupakan suatu syarat yang harus ada dalam suatu proses komunikasi, baik pada komunikasi interpersona, komunikasi kelompok maupun komunikasi massa. Kecanggihan teknologi komunikasi radio, juga turut serta mempengaruhi seluruh aspek kehidupan manusia, termasuk didalamnya kegiatan sosial. Dengan mengetahui kelebihan radio, maka alat tersebut dapat digunakan sebagai media bersosialisasi. Begitupun radio Prambors yang berdiri sejak tahun 1971, hadir dengan berbagai macam program siaran yang menyenangkan bagi kawula muda yang kreatif di Indonesia. Melakukan proses sistem sosial dari komunikator dalam hal ini penyiar dan tim yang berada di dalam radio Prambors sampai kepada pendengar (kawula muda). Seperti hal nya program yang disiarkan untuk kaum muda yang dimana program tersebut memilki nilai-nilai sosial yang terkandung di dalamnya, program tersebut diberi nama Kumpul Kamu. karena di dalamnya terdapat kegiatan yang mengumpulkan anak-anak muda dan melakukan interaksi bersama. Pada awal berdirinya, Radio Prambors menjadi radio kegemaran kawula muda di Indonesia, sesuai dengan misinya radio Prambors, yakni Radio Prambors hadir menjadi bagian dari kawula muda Indonesia, untuk menjadi bagian dari aktifitas kreatif remaja global. Sesuai dengan visi dan Misi prambors sendiri yaitu Menciptakan dan menjaga identitas sebagai Stasiun Radio untuk Generasi muda, Meningkatkan nilai-nilai dari penyiaran radio di Indonesia yang dimulai dari Jakarta. Menjadi Stasiun Radio nomor 1 dikalangan anak muda dan di tujuh kota lainnya, Menjadi panutan untuk anak muda di Jakarta dan di tujuh kota lainnya, Menjadi gaya tren di kalangan anak muda. Untuk tetap menjaga kelestarian identitas itu yakni sebagai radio untuk generasi muda, maka terciptalah program yang sangat identik dengan anak muda dan pastinya juga sangat diminati. PENDAHULUAN Program tersebut bernama Kumpul Kamu, merupakan program siaran yang bentuk dari kegiatannya adalah bertujuan untuk Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 123 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan mengumpulkan anak-anak muda yang berjiwa kreatif dari berbagai komunitas. Kumpul Kamu dengan tidak hanya sekedar mengumpulkan anak-anak yang kreatif saja namun juga menampilkan skill dari anak-anak kreatif yang hadir. Dan biasanya di acara tersebut di isi dengan sharing atau ngobrol bareng komunitas yang diundang, makan bareng, games bareng dan juga kegiatan seru lainnya, yang pastinya ngebuat anak muda itu senang karena kegiatan yang ada di dalamnya sangat menghibur dan juga bermanfaat untuk mereka yang di undang. Untuk itu pada penelitian ini penulis ingin lebih banyak mengetahui tentang program yang sangat identik dengan Prambors ini yaitu Program Kumpul Kamu, maka penulis akan menganalisis program tersebut dengan cara mendeskripiskan dan menggambarkannya tujuannya untuk mengetahui bagaimana kegiatan dari program siaran Kumpul Kamu pada radio Prambors Medan, mulai dari proses praproduksi, produksi sampai pasca produksi. METODE PENELITIAN Penelitian dilakukan untuk menganalisis artikel dan editorial tentang olahraga yang sesuai dengan kaidah kepenulisanya. Penelitian ini menggunakan pendekatan kualitatif dengan metode deskriptif, yang mana pendekatan kualitatif merupakan penelitian yang bersifat alamiah, dalam arti peneliti tidak berusaha memanipulasi setting penelitian, melainkan melakukan studi terhadap suatu fenomena. Data yang muncul dalam penelitian kualitatif ini berbentuk ungkapan kata (informasi) yang disampaikan narasumber dan responden kepada peneliti dan akan diakomodasi untuk mendapatkan hasil yang sesuai. Dalam penelitian ini, peneliti juga menggunakan beberapa metode selain menggunakan metode pendekatan kualitatif, yaitu: 1. Teknik Pengumpulan Data 1. Teknik Pengumpulan Data Adapun teknik pengumpulan data dalam penelitian ini adalah sebagai berikut: Adapun teknik pengumpulan data dalam penelitian ini adalah sebagai berikut: a. Metode Wawancara Narasumber pada penelitian ini diantaranya yaitu: a. Metode Wawancara Narasumber pada penelitian ini diantaranya yaitu: 1. Ihwan selaku Operasional Manager Radio Prambors 97.5 FM 2. Wina selaku Asisten Manager Radio Prambors 97.5 FM 3. Laskar selaku Koordinator Produksi Radio Prambors 97.5 FM 2. Teknik Analisis Data Teknik analisis data dalam penelitian ini ialah teknik analisis data kualitatif dari hasil wawancara dengan pihak radio Prambors 97.5 FM yang kemudian 2. Teknik Analisis Data Teknik analisis data dalam penelitian ini ialah teknik analisis data kualitatif dari hasil wawancara dengan pihak radio Prambors 97.5 FM yang kemudian 124 Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan dianalisis setiap jawaban yang diberikan kemudian diidentifikasi sesuai dengan maksud dan tujuan penelitian. 3. Populasi dan Sampel Subjek dari penelitian ini ialah program siaran di Radio Prambors 97.5 FM Medan. Adapun objek dari penelitian ini adalah Program siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan. Sedangkan, sampel pada penelitian ini ialah karyawan radio Prambors 97.5 FM. karyawan radio Prambors 97.5 FM. 1Laurencia dan Sugeng Wahjudi, Analisis Isi Pada Program Desta And Gina In The Morning Pada Radio Prambors Jakarta, Jurnal Semiotika, Vol. 13, No.1, (2019), hal. 44. HASIL DAN PEMBAHASAN Radio Prambors adalah stasiun penyiaran radio komersial yang mulai mendaftarkan badan siarannya kepada hukum di tahun 1970 dengan nama PT Radio Prambors Broadcasting Service.1 Seiring bergulirnya waktu, kini Radio Prambors sudah mengudara di beberapa daerah ibu kota salah satunya adalah Medan. Banyak program produksi Prambors yang masuk dalam siaran Radio Prambors 97.5 FM Medan. Padahal, semua program radio yang diluncurkan oleh Radio Prambors 97.5 FM Medan merupakan program radio spin-off dari Radio Prambors pusat 102.2 FM Jakarta. Selain memberikan hiburan dalam kehidupan sehari-hari, acara ini juga dapat mengisi waktu luang dengan mengisinya dengan hal-hal yang bermanfaat. Radio Prambors bukan hanya stasiun radio anak muda modern yang berfokus pada program-program seperti musik, tetapi juga menyiarkan program-program yang mendidik, menambah pengetahuan dan pergaulan, serta membangun hubungan sosial yang baik. Semuanya mengudara, menemani anak muda dalam kesehariannya. Program Kumpul Kamu di Radio Prambors pertama kali muncul pada tahun 2010 dengan nama Kumpul Kamu. Pertunjukan ini bisa dibilang satu-satunya yang sangat familiar di kalangan anak muda karena dipersembahkan untuk komunitas anak muda di kota Medan dan sekitarnya. Program Kumpul Kamu disiarkan dua bulan sekali dalam setahun. Ini karena lebih banyak persiapan dan dana yang dibutuhkan untuk menayangkan program tersebut, dan program Kumpul Kamu disiarkan secara off-air bertemu langsung dengan kawula muda. Kumpul Kamu merupakan program siaran yang sering terdengar ditelinga para pecinta radio Prambors yang berfrekuensi 97.5 FM. Program ini merupakan program mengumpulkan anak-anak yang kreatif, itulah sebabnya program ini disebut sebagai program yang identik dengan Prambors. Hal ini sesuai dengan logo Prambors yaitu berupa perempuan berambut keriting, rambut keriting ini dimaknai sebagai pemikiran yang bercabang-cabang atau pemikiran yang kreatif karena Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 125 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan banyak sekali cabang-cabangnya. Logo berambut keriting ini sendiri merupakan logo yang dibuat dari radio pusat yaitu Jakarta. Kumpul Kamu adalah program yang dibuat sendiri oleh Prambors, Pramborslah permulaan adanya Kumpul Kamu dengan tidak hanya sekedar mengumpulkan anak-anak yang kreatif saja namun juga menampilkan skill dari anak-anak kreatif yang hadir. Contoh penampilan mereka seperti band, menyanyi, kaligrafi, yang lagi havening yang ditampilkan pada saat itu. Selama pandemi Covid-19 dari tahun 2019 hingga 2022, program Kumpul Kamu belum dilanjutkan, karena pemerintah masih memberlakukan pembatasan kegiatan tertentu penduduk dalam suatu wilayah yang diduga masih terinfeksi Covid-19. HASIL DAN PEMBAHASAN Pada saat yang sama, banyak sponsor yang ingin menyelenggarakan program Kumpul Kamu di restoran atau kafe tempat mereka mendukung program tersebut. Dan Radio Prambors 97.5 FM berencana meluncurkan Kumpul Kamu pada akhir 2022. Untuk memahami program siaran Kumpul Kamu dibagi menjadi beberapa tahapan mulai dari pra produksi, produksi hingga pasca produksi. Dengan rincian sebagai berikut: 2 Intan Leliana, Analisis Program Siaran Balada Cerita Ramadhan di Radio Prambors 102.2 FM Jakarta, Jurnal Humaniora, Vol. 16, No. 1, (2016), hal. 11. Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 1. Pembuatan Proposal Langkah-langkah praproduksi untuk program Kumpul Kamu Radio Prambors 97.5 FM meliputi: 126 Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Membuat proposal merupakan tahap paling awal program Kumpul Kamu. Proposal ini bertujuan untuk mendapatkan sponsor. Program Kumpul Kamu selanjutnya dilakukan oleh sponsor ini. Biasanya yang menjadi sponsor acara Kumpul Kamu adalah restoran atau kafe yang ada di kota Medan. Para sponsor ini kerap meminta Radio Prambors untuk menggelar acara Kumpul Kamu di restoran atau kafe mereka. Sponsor-sponsor tersebut selanjutnya akan diberi kompensasi berupa logo sponsor di Radio Prambors dan kemudian ditampilkan di media sosial Radio Prambors Medan. Bahkan radio Parmbors akan memberikan ruang bagi mereka untuk memperkenalkan produknya. Berdasarkan hasil wawancara dengan pihak Radio Prambors 97.5 FM bahwa tidak terdapat ketetapan siapa yang membuat proposal disetiap hendak menjalan programnya. Sebab program Kumpul Kamu akan dijalankan yang bertugas membuat proposal bergantung dari atasan siapa yang dipilihnya untuk mengemban amanahnya tersebut. Umumnya buat melaksanakan langkah berikutnya untuk menunggu dari sponsor yang hendak menunjang. Serta persiapan buat proposal umumnya hanya membutuhkan waktu 2- 3 pekan, sehabis terdapat sponsor yang menunjang hingga langsung dijalankan langkah berikutnya. Namun persiapan ini dapat memakan waktu sepanjang satu bulan apabila tidak mendapatkan sponsor. Pra Produksi Program Kumpul Kamu Tahap praproduksi atau perencanaan suatu program adalah pengembangan dari rancangan program hingga rancangan produksi, atau dapat dikatakan semua kegiatan mulai dari pembahasan gagasan atau gagasan awal hingga penyiaran dan pelaksanaan penyiaran.2 Dalam pra prosuksi ditetapkan kebijakan menyeluruh tentang bagaimana pengaturan waktu, tempat dan distribusi materi siaran dalam jangka waktu hari, minggu, bulan atau tahun. Penyiaran dapat terjadi karena alat penyiaran. Sementara itu, tujuan penyiaran dalam arti luas adalah menyiarkan, dan akhirnya mencapai tujuan akhir penyiaran, yaitu hiburan, pendidikan, penyebarluasan, dan pengetahuan. Dalam perencanaan ini terjadi proses interaksi antara kreativitas manusia dengan dukungan dan peralatan yang tersedia. Kualitas proses penyiaran sangat ditentukan oleh perencanaan. Langkah-langkah praproduksi untuk program Kumpul Kamu Radio Prambors 97.5 FM meliputi: 1. Pembuatan Proposal Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 126 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan 3. Penentuan Tema serta Topik Setelah mendapatkan sponsor dan menentukan vanue untuk dilaksanakan program Kumpul Kamu, proses selanjutnya adalah menentukan tema dan topik yang akan diselenggarakan. Dan untuk menentukan tema dan topik ini juga bergantung pada sponsor yang menunjang. Misalnya, yang mensposori program siarannya merupakan Telkomsel, tentu topik yang hendak dibahas merupakan tentang telekomunikasi. Kumpul kalian bukan cuma hanya program yang mengumpulkan anak-anak kreatif maupun komunitas-komunitas anak muda yang terletak dikota Medan bukan untuk sekedar kumpul-kumpul, happy-happy serta yang lain. Namun, program kumpul kamu pula membagikan wawasan. Pada penentuan tema serta topik ini, para sponsor boleh tergabung kedalam penentuannya. Tetapi, tidak secara totalitas sebab senantiasa harus briefing dengan radio Parmbors 97.5 FM, perihal ini dilakukan sebab informasi akan disampaikan kepada kawula muda ialah generasi-generasi milenials dan generasi z. Tidak hanya lewat dari para sponsor, terkadang topik ditetapkan bersumber pada kawula muda yang diinvite. Misalnya merupakan komunitas anak motor hingga topiknya mangulas seputaran otomotif. 4. Materi Pada program Kumpul Kamu, materinya berisi tentang apa yang lagi digandrungi ditengah-tengah kawula muda seputar pengetahuan, pembelajaran, skill, berolahraga, otomotif, serta lain-lain bergantung dari sponsor serta kawula muda yang disinvite. Misalnya, yang mensponsori merupakan Telkomsel tentu materi yang dibahas komunikasi. Materi yang akan di informasikan dikemas dengan seapik bisa jadi serta di informasikan dengan bahasa yang awam ditelinga kawula muda sehingga merekan gampang bosan mencermati modul yang di informasikan. 5. Narasumber Narasumber yang didatangkan oleh radio Prambors 97.5 FM merupakan narasumber yang professional cocok dengan topik yang hendak dibahas. Saat sebelum Covid-19 radio Prambors 97.5 FM sempat memperkenalkan Musa Rajekshah ataupun yang biasa diketahui dengan Ijeck seseorang pembalap yang telah populer di kota Medan. Topik yang dibahas pada dikala itu merupakan otomotif dengan menggandeng komunitas motor antik, komunitas motor gede serta komunitas motor modif. 2. Penentuan Vanue Sehabis memperoleh sebagian sponsor yang menunjang, hingga langkah berikutnya merupakan memastikan vanue yang hendak digunakan buat melakukan program Kumpul Kalian secara off air. Umumnya vanue ini pula ditetapkan bersumber dari para sponsor, misalnya yang mensponsori merupakan Restoran Kembang, maka akan dilaksanakan di Restoran Kembang tergantung tema dan pendengar yang disinvite. Akan tetapi, jika vanue berdasarkan yang memberi sponsor, tentu pihak radio Prambors harus memberikan kompensasi kepada pihak yang memberikan sponsor. Seperti logo-logo dari Restoran Kembang akan ada di media sosial dan disiarkan oleh Prambors mengenai yang memberikan sponsor untuk program Kumpul Kamu. Selain logo, ada juga kompensasi yang lain yaitu dari segi makanan. Misalnya Restoran Kembang, kompensasinya merupakan mereka leluasa untuk mengcover santapan yang akan disajikan untuk pendengar, tetapi senantiasa dengan menu utama yang dikoordinasikan dengan radio Prambors 97. 5 FM. Tetapi, penentuan vanue ini tidak senantiasa tergantung dari para sponsor yang menunjang. Senantiasa radio Prambors 97. 5 FM mempunyai syarat serta kriteria vanuenya, sebab yang diinvite merupakan kawula muda yang banyak menggemari tempat- tempat yang aesthetic, aman serta bagus. Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 127 6. Menghadirkan Pendengar Pendengar yang diinvite dalam program siaran Kumpul Kamu di radio Prambors 97.5 FM merupakan kawula muda ialah anak-anak muda yang kreatif Pendengar yang diinvite dalam program siaran Kumpul Kamu di radio Prambors 97.5 FM merupakan kawula muda ialah anak-anak muda yang kreatif 128 Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan maupun komunitas-komunitas anak muda yang terdapat di kota Medan. Hal ini disebabkan sasaran dari radio Prambors 97.5 FM merupakan anak-anak muda supaya mereka menggemari radio. Disisi lain program Kumpul Kamu memanglah diperuntukkan kalangan pemuda yang sangat menyukai radio. Metode radio Prambors 97.5 FM dalam menginvite mereka merupakan dengan promo lewat on air tiap satu jam sekali. Sebab ini merupakan program radio Prambors 97.5 FM sendiri, hingga pasti pemberitahuannya tiap satu jam sekali sebab tidak membutuhkan bayaran untuk mengiklankannya. Umumnya penyiar hendak mengatakan komunitas anak muda yang hendak mereka undang, contohnya semacam komunitas motor, Blogger, Sport serta lain- lain. Disamping itu pula parallel dimedia sosial instagram. Menginvite pendengar diawali dari H-10 sampai H-2 program siaran Kumpul Kamu hendak dilaksanakan. Dalam perihal ini radio Prambors mempunyai target pendengarnya. Target pendengar program Kumpul Kamu 97.5 FM merupakan sekitar 250 orang, tetapi yang diiinvite oleh radio Prambors merupakan sekita 200 orang. Bersumber pada wawancara dengan Ihwan sebagai Operasional Manager, beliau melaporkan kalau dalam sesuatu event umumnya partisipan yang daftar dan hadir berbeda jumlahnya kala dihari siaran programnya dilaksanakan, perubahan ini bukan terus menjadi meningkat tetapi menurun apalagi tidak tidak sering dari pendengar yang tidak mendatangi event tersebut. 7. Biaya Produksi Perencana budget atau biaya produksi didasarkan sepenuhnya pada sponsor yang mendukung setiap program Kumpul Kamu dijalankan. Namun, radio Prambors 97.5 FM juga memiliki biaya sendiri yaitu untuk biaya promosi. Biaya produksi ini bisa berbentuk case dan berbentuk space. Biaya berbentuk case ini biasa dari pihak Prambors karena mempromosikan program Kumpul Kamu, namun biaya space ini berasal dari para sponsor yang ingin produk mereka di promosikan oleh pihak Prambors. Biasanya space ini Prambos hanya memberikan 20% untuk para sponsor mempromosikan produknya melalui Prambors 97.5 FM. Rata-rata nominal angka biaya produksi program Kumpul Kamu ini dimulia dari H-10 promosi, dalam sehari sekitar Rp.600.000. Akan tetapi, apabila dari internal dihitung sekitar Rp. 200.000, lalu dikalikan sekitar Rp. 2 juta dan dikali selama 10 hari. Maka, jika dijumlahkan nominalnya sekitar Rp. 4.000.000.000.000 3 Z. Arifin, Pembahasan, https://repository.dinamika.ac.id/id/eprint/2426/6/BAB_IV.pdf, diakses pada 23 Desember 2022. 4 Rahmawati Cindar Bumi, Skripsi: Analisis Produksi Program Radio Komunitas Pada Program Musik Studi Deskriptif Program Musik di Radio Love Jogja FM dan Radio Wijaya FM, (Yogyakarta: UII, 2017), hal. 27. Produksi Program Kumpul Kamu Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 129 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan 5 Intan Leliana, Analisis Program Siaran Balada Cerita Ramadhan di Radio Prambors 102.2 FM Jakarta, Jurnal Humaniora, Vol. 16, No. 1, (2016), hal. 11. Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Setelah perencanaan dan persiapan selesai, maka pelaksanaan produksi dimulai. Produksi merupakan kunci dalam aktivitas di radio siaran. Tahap produksi merupakan tahap untuk merealisasikan semua langkah yang ada pada tahap pra produksi.3 Suatu produksi siaran radio adalah hasil kerja sama antara penyiar dan operator, dan kerja sama ini menentukan baik tidaknya suatu produksi siaran. Suatu produksi siaran radio adalah hasil kerja sama antara penyiar dan operator, dan kerja sama ini menentukan baik tidaknya suatu produksi siaran.4 Tahap pelaksaan produksi dilakukan berdasarkan semua kegiatan materi siaran yang telah disiapkan, pelaksanaannya dari awal siaran hingga akhir siaran. Dalam proses produksi ini dapat dilihat bahwa pra produksi memang benar- benar dijalankan sesuai rencana atau tidak. Seperti halnya pihak Radio Prambors 97.5 FM yang menyatakan bahwa “Proses produksi itu kita lihat mulai dari start waktunya. Start program siaran Kumpul Kalian 97.5 FM umumnya diwaktu sore hari pada jam 18.00 Wib. Lama waktu program berjalan bergantung dari pihak radio Prambors 97.5 FM mau mengambil waktu berapa lama. Sebab start programnya mendekati waktu maghrib, buat eventnya diawali sehabis sholat maghrib berjamaah dengan pendengar yang muncul. Tetapi, buat pendaftaran senantiasa pada jam 18.00 Wib.”(Ihwan, wawancara, 30 November 2022). Pernyataan diatas dapat disimpulkan bahwa pada proses produksi yang paling utamanya adalah start waktu dimulai siaran programnya. Karena waktu menentukan suatu program siaran yang akan berjalan. “Pada sesi produksi program Kumpul Kamu, para kru pelaksana briefing terlebih dulu. Saat sebelum progam kegiatan diawali, kru telah mempersiapkan Rundown serta Cue card buat mengalurkan acaranya. Rundown digunakan buat memandang struktur pelaksana event nya. Tetapi cuma secara cover saja semacam start jam mulai sampai berakhir. Tetapi cue card dipaparkan secara terperinci, mulai dari seluruh pengisi dicantumkan didalamnya. Master of ceremoninya siapa, hadiah nya disiapkan, narasumber disiapkan. Apalagi PO nya memberitahukan kepada MC menimpa apa- apa yang hendak di informasikan tentang programnya serta gamesnya pada dikala opening, cue card menarangkan serinci itu. Sehabis seluruh persiapan telah matang, hingga kegiatan dilaksanakan cocok yang tertera di rundown serta cue card.”(Ihwan, wawancara, 30 November 2022). Dikala siaran Kumpul Kamu sedang berlangsung, terdapat segmen kuis serta game yang diberikan. Segmen kuis serta permainan dalam siaran Kumpul Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 130 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Kamu membuat kawula muda pendengar Prambors 97.5 FM lebih tertarik buat terus mencermati Kumpul Kamu tiap dua bulan sekali. Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Suatu prosuksi pelaksanaannya sesuai dengan koordinasi yang telah direncanakan oleh para kru serta pengisi siarannya dari radio Prambors. Tetapi bukan berarti perihal itu berjalan mudah sesuai dengan yang direncanakan, sebab terdapat hal- hal yang menghambat produksi siarannya. . Kamu membuat kawula muda pendengar Prambors 97.5 FM lebih tertarik buat terus mencermati Kumpul Kamu tiap dua bulan sekali. Suatu prosuksi pelaksanaannya sesuai dengan koordinasi yang telah direncanakan oleh para kru serta pengisi siarannya dari radio Prambors. Tetapi bukan berarti perihal itu berjalan mudah sesuai dengan yang direncanakan, sebab terdapat hal- hal yang menghambat produksi siarannya. . Dalam produksinya, aspek pendukung yang membuat program ini bisa terlaksana, yang awal merupakan terdapatnya sponsor yang sudah menunjang seluruhnya dengan terdapatnya program Kumpul Kamu ini, sponsor tersebut bermacam-macam serta berbeda tiap kali program Kumpul Kamu hendak dilaksanakan, tetapi terdapat sebagian yang kerap jadi sponsor dalam program Kumpul Kamu antara lain ialah Restoran Kembang, Restoran Sukardi, Budaya Resto, Lembur Kuning, Cinderalass, Neko-Neko serta Iga-Iga. kedua, para pengisi kegiatan serta pula para kru yang bekerja dengan optimal. Disamping materi yang di informasikan langsung oleh professional cocok dengan bidangnya membagikan kesan tertentu dihati pendengar, serta pesan yang ingin di informasikan oleh pihak radio Prambors tersampaikan dengan baik kepada kawula muda. Sehingga membuat kawula muda senantiasa mau lagi- lagi mencermati program siaran kumpul kalian sekalipun wajib menunggu dua bulan lamanya. Sebaliknya hambatan yang dialami tidak sangat banyak sebab tahun-tahun lebih dahulu penciptaan program hiburan serta bimbingan di Radio Prambors ini sudah dibuat serta sudah dipersiapkan sebaik bisa jadi. Hambatan yang dialami, terletak pada waktu untuk mendapatkan sponsor serta pekerjaannya para wadyabala ataupun kru dari produksi program Kumpul Kamu ini. Pada sponsor ini, progam akan cepat diproduksi apabila telah memperoleh sponsor sekitar dua hingga tiga pekan. Namun, apabila lebih dari itu belum memperoleh hingga wajib menunggu sepanjang satu bulan. Hingga akhirnya waktu terbuang habis hanya untuk menunggu mendapatkan sponsor. Kemudian pada bagian kru wadyabala ini merupakan minimnya sumber energi manusianya sehingga senantiasa meminta orang lain yang memanglah telah berpengalaman serta diketahui pihak prambors untuk jadi kru. Pasca Produksi Program Kumpul Kamu Pasca Prpduksi merupakan seluruh aktivitas sehabis penerapan program siaran hingga modul itu dinyatakan berakhir serta siap ditayangkan ataupun diputar kembali.5 Yang tercantum dalam pasca produksi siaran antara lain editing Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 131 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan (penyutingan). Partisipan yang mendaftar pasti terdapat databasenya, sehabis program berakhir dilaksanakan hingga radio Prambors 97.5 FM akan mengantarkan blass terima kasih kepada pendengar yang muncul di program kumpul kalian. Dan membuat testimoni kemudian ditayangkan melalaui on air serta pula di upload di sosial media instagram radio Prambors 97.5 FM. Hal ini dilakukan agar pendengar yang mendengar siarannya bahagia serta mau menjajaki program Kumpul Kamu kembali serta menginfluence kepada kawula muda yang lain buat menjajaki program kegiatan kumpul kalian yang terdapat di radio Prambors 97.5 FM. Tidak hanya itu dokumentasi dari kegiatan kumpul kalian di sediakan seapik bisa jadi dalam wujud video dokumenter kemudian dikirim ke prambors pusat yang terdapat di Jakarta untuk dinilai serta hendak disiarkan di radio Prambors Jakarta. Pada proses evaluasi produksi siaran Kumpul Kamu dilaksanakan oleh para wadyabala yang telah hadir serta pula dalam pra produlsi ataupun produksinya Kumpul Kamu. Penilaian dalam program Kumpul Kamu saat sebelum serta sehabis program ditayangkan oleh para kru. Ada pula metode mengevaluasi program tersebut merupakan: a. Source ataupun komunikator, dalam perihal ini pihak penciptaan dari Program Kumpul Kamu 97.5 FM. Pihak Prambors melaksanakan penilaian setiap tim dalam produksinya. Ialah dengan kurangi kesalahan-kesalahan dan terus memperbaiki untuk lebih baik lagi dalam menjalankan program siarannya. b. Message. Pada pengevaluasian dalam pesan yang terdapat di program Kumpul Kamu para regu penciptaan melaksanakan revisi buat kegiatan selanjutnya. c. Ikatan Wadyabala dengan kawula muda pendengar, receiver dengan metode memandang feedback dari kawula muda ataupun share dari segi tema ataupun dari segi liputan-liputannya dengan membuka peluang untuk memperhitungkan langsung lewat email serta dm (direct message) ke radio prambors. Dengan melihat komentar dan pendapat dari pendengar tersebut, tim produksi Kumpul Kamu bisa mengetahui segmen apa yang sangat diminati oleh pendengar dan memudahkan regu tim produksi dalam tingkatkan siaran-siarannya yang lebih menarik buat kedepannya. d. Dalam pasca produksi Kumpul Kamu ini pula, banyak yang untuk lebih mengenalkan kepada pendengar. Ada pula yang yang dari pihak promo production Kumpul Kamu, ialah pemutaran spot-spot iklan Kumpul Kalian disetiap program siaran yang lain, iklan dalam web diradio Prambors ialah www.pramborsfm.com serta iklan dalam media sosial lewat instagram @wadyabalamedan. d. Pasca Produksi Program Kumpul Kamu Dalam pasca produksi Kumpul Kamu ini pula, banyak yang untuk lebih mengenalkan kepada pendengar. Ada pula yang yang dari pihak promo production Kumpul Kamu, ialah pemutaran spot-spot iklan Kumpul Kalian disetiap program siaran yang lain, iklan dalam web diradio Prambors ialah www.pramborsfm.com serta iklan dalam media sosial lewat instagram @wadyabalamedan. Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 132 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 Kesimpulan Kumpul Kamu merupakan program siaran yang sering terdengar ditelinga para pecinta radio Prambors yang berfrekuensi 97.5 FM. Program ini merupakan program mengumpulkan anak-anak muda yang kreatif, itulah sebabnya program ini disebut sebagai program yang identik dengan Prambors. Kumpul Kamu adalah program yang dibuat sendiri oleh Prambors, Pramborslah permulaan adanya Kumpul Kamu dengan tidak hanya sekedar mengumpulkan anak-anak yang kreatif saja namun juga menampilkan skill dari anak-anak kreatif yang hadir. Contoh penampilan mereka seperti band, menyanyi, kaligrafi, yang lagi havening yang ditampilkan pada saat itu. Setelah melakukan penelitian dan menganalisis program siaran kumpul kamu mulai dari proses Pra Produksi, lalu proses Produksi sampai kepada proses Pasca Produksi. Maka dapat disimpulkan, sebagai berikut: a. Proses Pra Produksi, pada proses ini radio prambors yang memproduksi program siaran Kumpul Kamu mempersiapkan program dengan perencanaan yang sangat matang, hal-hal yang dilakukan seperti mempersiapkan proposal terlebih dahulu untuk mendapatkan sponsor, biasanya memerlukan 2-3 minggu waktu pengumpulannya. Setelah itu menentukan tempat (Vanue), dilanjutkan dengan menetukan tema dan topik yang akan dibahas, setelah itu mengudang peserta/pendengar yang akan mengikuti acara tersebut. b. Proses Produksi, pada tahap ini radio prambors dalam memproduksi program siaran Kumpul Kamu diawali dengan menentukan waktu siaran/acara dan akan dimulai pada waktu yang sudah ditentukan , setelah itu memepersiapkan materi dan juga narasumber, setelah itu masuk kepada tahap pelaksanaan produksi program Kumpul Kamu, para kru pelaksana briefing terlebih dahulu. Sebelum progam acara dimulai, kru sudah menyiapkan Rundown dan Cue card untuk mengalurkan acaranya. Rundown digunakan untuk melihat struktur pelaksana event nya. Namun hanya secara cover saja seperti start jam mulai hingga selesai. Namun cue card dijelaskan secara terperinci, mulai dari semua pengisi dicantumkan didalamnya. Master of ceremoninya siapa, hadiah nya disiapkan, narasumber disiapkan. c. Proses Pasca Produksi, pada proses pasca produksi program Kumpul kamu radio prambors melakukan beberapa hal seperti melakukan penyutingan/editing, setelah program selesai dilaksanakan maka radio Prambors 97.5 FM akan menyampaikan terima kasih kepada pendengar yang hadir di program kumpul kamu. Serta membuat testimoni lalu disiarkan melalaui on air dan juga di upload di sosial media instagram radio Prambors 97.5 FM. Hal ini dilakukan supaya c. Proses Pasca Produksi, pada proses pasca produksi program Kumpul kamu radio prambors melakukan beberapa hal seperti melakukan penyutingan/editing, setelah program selesai dilaksanakan maka radio Prambors 97.5 FM akan menyampaikan terima kasih kepada pendengar yang hadir di program kumpul kamu. Saran Dalam menyusun kajian penelitian ini, peneliti sadar bahwa masih terdapat banyak kekurangan dan kesalahan kata dalam penulisannya terlebih pada penyajian hasil penelitian dan pembahasannya. Maka dari itu diperlukannya masukan dari khalayak pembaca untuk memperbaiki penelitiannya, sehingga menghasilkan hasil yang baik dan berguna bagi bara pembaca mengetahui komunikasi internal yang terjadi didalam anggota organisasi melalui media. Kesimpulan Serta membuat testimoni lalu disiarkan melalaui on air dan juga di upload di sosial media instagram radio Prambors 97.5 FM. Hal ini dilakukan supaya c. Proses Pasca Produksi, pada proses pasca produksi program Kumpul kamu radio prambors melakukan beberapa hal seperti melakukan penyutingan/editing, setelah program selesai dilaksanakan maka radio Prambors 97.5 FM akan menyampaikan terima kasih kepada pendengar yang hadir di program kumpul kamu. Serta membuat testimoni lalu disiarkan melalaui on air dan juga di upload di sosial media instagram radio Prambors 97.5 FM. Hal ini dilakukan supaya Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 133 Analisis Program Siaran Kumpul Kamu di Radio Prambors 97.5 FM Medan pendengar yang hadir senang dan ingin mengikuti program kumpul kamu kembali dan menginfluence kepada kawula muda yang lain untuk mengikuti program acara kumpul kamu yang ada di radio Prambors 97.5 FM. Selain itu dokumentasi dari acara kumpul kamu di sajikan seapik mungkin dalam bentuk video dokumenter lalu dikirim ke prambors pusat yang ada di Jakarta untuk dinilai dan akan ditayangkan di radio Prambors Jakarta. Diakhir radio prambors melakukan proses evaluasi, yang dilakukan oleh beberapa dari wadyabala yang telah terlibat juga dalam pra produksi maupun produksinya program siaran Kumpul Kamu. Evaluasi dalam program Kumpul Kamu sebelum dan setelah program disiarkan oleh para kru. DAFTAR PUSTAKA Luaurencia, & Wahjudi, S. (2019). Analisis Isi Pada Program Desta and Gina In The Morning Pada Radio Prambors Jakarta. Jurnal Semiotika , 13(1); 43-67. Leliana, I. (2016). Analsis Program Siaran Balada Cerita Ramadhan di Radio Prambors 102.2 FM. Jurnal Humaniora, (16)1; 1-19. Arifin, Z. Pembahasan, https://repository.dinamika.ac.id/id/eprint/2426/6/BAB_IV.pdf, Diakses tanggal 23 Desember 2022. Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022 Z. Pembahasan, Diakses Diakses Bumi, R.C. (2017). Analisis Produksi Program Radio Komunitas Pada Program Musik Studi Deskriptif Program Musik di Radio Love Jogja FM dan Radio Wijaya FM. Skripsi. Program Studi Ilmu Komunikasi Universitas Islam Indonesia. 134 Jurnal Komunikasi Islam (J-KIs) I Vol.3, No.2, Desember 2022
https://openalex.org/W1876068125	https://europepmc.org/articles/pmc4549645?pdf=render	English	null	Overestimating the Effects of Healthy Aging	Frontiers in aging neuroscience	2,015	cc-by	2,217	Overestimating the effects of healthy aging University of the Basque Country (UPV/EHU), Vitoria & IKERBASQUE, Basque Foundation for Science, Bilbao, Spain Keywords: cognitive aging, Alzheimer’s disease, biomarkers, amyloidosis, neurodegeneration, neuroimaging Typical cognitive aging is often defined as aging free of dementia, and yet it does not seem to exclude dementia-related pathology. The most common type of dementia is Alzheimer’s disease (AD) – it is a progressive neurodegenerative disease that affects multiple cognitive domains, including memory, attention, reasoning, judgment, language, as well as behavior. AD accounts for 60–80% of dementia cases, currently affecting 35 million people worldwide and progressing toward an estimated 115 million by the year 2050 (Riedel, 2014). Since there is no cure for AD, the research focus has shifted from the symptomatic stage to the earlier stages of the disease, with the goal to develop treatments that would effectively delay the onset of symptoms and progression of this devastating disease (Selkoe, 2012; Bateman, 2015). The need to diagnose and begin treatment even before overt symptoms appear has generated in neurological researchers a strong interest in cognitively healthy people who are at risk for AD. Recent modifications of the research and diagnostic criteria for AD reflect this trend by including cognitively normal (CN) at-risk-for-AD persons as a preclinical stage in the AD continuum (Dubois et al., 2007, 2010; Sperling et al., 2011). ( p g ) Crucial in the modified criteria for AD is the concept of biomarkers of Alzheimer’s pathology, i.e., physiological and anatomical parameters that can be objectively measured to establish the presence of changes due to the disease (Jack and Holtzman, 2013). Since amyloid-β neuritic plaques and neurofibrillary tangles have been established as the pathological hallmarks of AD, biomarkers of amyloidosis and neurodegeneration are used as in vivo indicators of the presence of Alzheimer’s pathology (Dubois et al., 2007, 2010; Sperling et al., 2011; Jack and Holtzman, 2013). The most validated AD biomarkers are cerebrospinal fluid (CSF) amyloid-β1–42, total tau and phosphorylated tau as pathophysiological biomarkers of Alzheimer’s pathology, and brain regional structural and metabolic changes, as topographical biomarkers (Dubois et al., 2007; Jack and Holtzman, 2013). However, it has been recently recognized that the topographical markers lack pathological specificity necessary for diagnosis, and that they are more useful for measuring the disease progression (Dubois et al., 2014). Overestimating the effects of healthy aging According to this view, in vivo evidence of Alzheimer’s pathology, and thus preclinical AD stage, would constitute increased cerebral amyloid burden/decreased CSF amyloid level together with increased levels of total tau and phospho-tau. The general idea is that if a person with Alzheimer’s pathology lives long enough, they will eventually progress to AD dementia. Thus, biomarkers-based diagnosis provides an opportunity to introduce early treatments that will attempt to delay appearance of dementia symptoms and disease development. OPINION published: 26 August 2015 doi: 10.3389/fnagi.2015.00164 OPINION published: 26 August 2015 doi: 10.3389/fnagi.2015.00164 Edited by: Pranav J. Parikh, University of Houston, USA Reviewed by: Anusuyadevi M. Jayachandran, Bharathidasan University, India *Correspondence: Vanja Kljajevic vanja.kljajevic@gmail.com Received: 03 May 2015 Accepted: 10 August 2015 Published: 26 August 2015 Citation: Kljajevic V (2015) Overestimating the effects of healthy aging. Front. Aging Neurosci. 7:164. doi: 10.3389/fnagi.2015.00164 Edited by: Pranav J. Parikh, University of Houston, USA Reviewed by: Anusuyadevi M. Jayachandran, Bharathidasan University, India While the value of early diagnosis is beyond any dispute, the question of how to disentangle typical aging and preclinical AD remains open. This issue is further complicated by the heterogeneous nature of the preclinical AD. According to one model, preclinical AD comprises three stages: at stage 1, only brain amyloidosis is evident; at stage 2, both cerebral amyloidosis and neurodegeneration are present; and at stage 3, these features are further combined with subtle cognitive changes (Sperling et al., 2011). Two additional stages have been added to this model: stage 0, which represents cognitively intact people without brain amyloidosis, neurodegeneration, or subtle cognitive changes, and suspected non-amyloid pathology (SNAP), which is characterized by normal amyloid markers and abnormal neurodegeneration markers and which is not necessarily related to AD (Jack et al., 2012). While it is clear that preclinical AD is characterized by the absence of cognitive impairment Citation: Kljajevic V (2015) Overestimating the effects of healthy aging. Front. Aging Neurosci. 7:164. doi: 10.3389/fnagi.2015.00164 August 2015 \| Volume 7 \| Article 164 1 Frontiers in Aging Neuroscience \| www.frontiersin.org Overestimating the effects of healthy aging Kljajevic Kljajevic on objective measures of cognition, subtle cognitive changes that have been associated with this stage of disease remain poorly understood (Dubois et al., 2014). percentage of CN elderly subjects in such studies may represent preclinical AD. This problem has been occasionally recognized (Gold et al., 2013; Brier et al., 2014). It clearly requires a systematic change in approach, because subtle cognitive changes, reliance on cognitive strategies, and networks’ reorganization that one would interpret as the effects of healthy aging might actually reflect the disease progression. While the number of studies investigat- ing that the impact of atrophy, hypometabolism, white matter changes, and ApoE4 on cognitive processes across the AD stages is consistently growing, possible effects of β-amyloid, t-tau, and p-tau on cognitive processes in preclinical AD remain largely unexplored (Riedel, 2014). Another model of preclinical AD has recently been proposed in the context of a cross-sectional study that assessed a population- based sample of 985 CN people aged 50–89. The model combines only amyloidosis [positive (A+) or negative (A−)] and neurode- generation [positive (N+) or negative (N−)] status, leaving out subtle cognitive changes. The study revealed that the estimated population frequency of the A−N−sequence was 100% at age 50, but only 17% at age 89, with the A+N+ sequence reaching 42% by age of 89, whereas the frequency of the sequence A−N+ was 24% by that age (Jack et al., 2014). Examples of pathological sequences leading to AD dementia are as follows: A−N−to A+N−to A+N+, and A−N−to A−N+ to A+N+, whereas the two com- ponent sequence A−N−to A−N+ is linked to heterogeneous underlying pathology (Jack et al., 2014). However, the neurode- generative status in this study was determined based on topo- graphical biomarkers of AD, more specifically by an AD signature 18F-fluorodeoxyglucose (18F-FDG) PET and hippocampal volume on MRI. These topographical markers are not unique markers of Alzheimer’s pathology; for example, hippocampal volume is reduced in other conditions, such as frontotemporal demen- tia, hippocampal sclerosis, Lewy-related pathology, argyrophilic grain disease, diabetes, and bipolar disorder, among others (Jack et al., 2012; Dubois et al., 2014). The author gratefully acknowledges support from IKERBASQUE, Basque Foundation for Science (111407EMDD). The author gratefully acknowledges support from IKERBASQUE, Basque Foundation for Science (111407EMDD). The author gratefully acknowledges support from IKERBASQUE, Basque Foundation for Science (111407EMDD). Citation: Thus, although these findings still suggest that pathological aging is a predominant way of cog- nitive aging, they also indicate a need for consensus on which biomarkers are suggestive of Alzheimer’s pathology only and thus better suited for untangling preclinical AD and the healthy brain aging. Finally, in addition to apparently small percentage of CN per- sons who despite an advanced age resist Alzheimer’s pathology (Jack et al., 2012, 2014), there exist so-called SuperAgers. These are elderly people (80+) who appear to have healthy brains and well-preserved memory abilities. A recent study involving 12 SuperAgers found that their memory abilities were comparable to those of a group of healthy 50- and 65-year-old persons. When compared to a group of healthy age-matched peers on measures of cortical thickness, the SuperAgers had significantly thicker cerebral cortex. Furthermore, the left anterior cingulate was signif- icantly thicker in SuperAgers compared to both groups (Harrison et al., 2012). Thus, we find not only neuropathology but also healthy brains and preserved memory at well-advanced age. In conclusion, it is now possible to establish the pres- ence/absence of Alzheimer’s pathology in vivo by measuring parameters that indicate biological changes caused by AD, thereby determining if a person is at risk for developing AD. Incorporating such evidence into cognitive aging research allows a differenti- ation of possible influences of Alzheimer’s pathology from the effects of healthy aging on cognitive processes. Only by systemat- ically incorporating such evidence in research on cognitive aging, we will be able to make a progress in disentangling preclinical AD from healthy cognitive aging. As models of preclinical AD continue to develop, a challenge to the field is to reconcile the evidence of AD-related pathology found in a large number of CN elderly people (Jack et al., 2012, 2014) with the notion of “healthy” or “successful” aging (Rowe and Kahn, 1987). This evidence seems to question the research prac- tice of not considering possible presence of Alzheimer’s pathology in CN elderly participants when including healthy elderly per- sons in cognitive studies. However, without the actual evidence to exclude Alzheimer’s pathology, one can assume that some Riedel, W. J. (2014). Preventing cognitive decline in preclinical Alzheimer’s disease. Curr. Opin. Pharmacol. 14, 18–22. doi:10.1016/j.coph.2013.10.002 References Gold, B. T., Johnson, N. F., and Powell, D. K. (2013). Lifelong bilingualism contributes to cognitive reserve against white matter integrity declines in aging. Neuropsychologia 51, 2841–2846. doi:10.1016/j.neuropsychologia.2013. 09.037 Bateman, R. (2015). Alzheimer’s disease and other dementias: advances in 2014. Lancet Neurol. 14, 4–6. doi:10.1016/S1474-4422(14)70301-1 Harrison, T. M., Weintraub, S., Mesulam, M. M., and Rogalski, E. (2012). Supe- rior memory and higher cortical volumes in unusually successful cognitive aging. J. Int. Neuropsychol. Soc. 18, 1081–1085. doi:10.1016/j.neuropsychologia. 2013.09.037 Brier, M. R., Thomas, J. B., Snyder, A. Z., Wang, L., Fagan, A. M., Ben- zinger, T., et al. (2014). Unrecognized preclinical Alzheimer disease con- founds rs-fcMRI studies of normal aging. Neurology 83, 1–7. doi:10.1212/WNL. 0000000000000939 Jack, C. R. Jr, and Holtzman, D. M. (2013). Biomarker modeling of Alzheimer’s disease. Neuron 80, 1347–1358. doi:10.1016/j.neuron.2013.12.003 Dubois, B., Feldman, H. H., Jacova, C., Cummings, J. L., Dekosky, S. T., Barberger- Gateau, P., et al. (2010). Revising the definition of Alzheimer’s disease: a new lexicon. Lancet Neurol. 9, 1118–1127. doi:10.1016/S1474-4422(10)70223-4 Jack, C. R. Jr, Knopman, D. S., Weigand, S. D., Wiste, H. J., Vemuri, P., Lowe, V., et al. (2012). An operational approach to NIA-AA criteria for preclinical Alzheimer’s disease. Ann. Neurol. 71, 765–775. doi:10.1002/ana.22628 Dubois, B., Feldman, H. H., Jacova, C., Dekosky, S. T., Barberger-Gateau, P., Cummings, J., et al. (2007). Research criteria for the diagnosis of Alzheimer’s disease: revising the NINCDS-ADRDA criteria. Lancet Neurol. 6, 734–746. doi: 10.1016/S1474-4422(07)70178-3 Jack, C. R. Jr, Wiste, H. J., Weigand, S. D., Rocca, W. A., Knopman, D. S., Mielke, M. M., et al. (2014). Age-specific population frequencies of cerebral β-amyloidosis and neurodegeneration among people with normal cognitive function aged 50- 89 years: a cross-sectional study. Lancet Neurol. 13, 997–1005. doi:10.1016/ S1474-4422(14)70194-2 Dubois, B., Feldman, H. H., Jacova, C., Hampel, H., Molinuevo, J. L., Blennow, K., et al. (2014). Advancing research diagnostic criteria for Alzheimer’s disease: the IWG2 criteria. Lancet Neurol. 13, 614–629. doi:10.1016/S1474-4422(14)70090-0 August 2015 \| Volume 7 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 2 Overestimating the effects of healthy aging Kljajevic Kljajevic Riedel, W. J. (2014). Preventing cognitive decline in preclinical Alzheimer’s disease. Curr. Opin. Pharmacol. 14, 18–22. doi:10.1016/j.coph.2013.10.002 Conflict of Interest Statement: The author declares that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Rowe, J. H., and Kahn, R. L. (1987). Human aging: usual and successful. Rowe, J. H., and Kahn, R. L. (1987). Human aging: usual and successful. Science 237, 143–149. doi:10.1126/science.3299702 Sperling, R. A., Aisen, P. S., Beckett, L. A., Bennett, D. A., Craft, S., Fagan, A. M., et al. (2011). Toward defining the preclinical stages of Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s associa- tion workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement. 7, 280–292. doi:10.1016/j.jalz.2011.03.003 Selkoe, D. J. (2012). Preventing Alzheimer’s disease. Science 337, 1488–1492. doi:10. 1126/science.1228541 Riedel, W. J. (2014). Preventing cognitive decline in preclinical Alzheimer’s disease. Curr. Opin. Pharmacol. 14, 18–22. doi:10.1016/j.coph.2013.10.002 Rowe, J. H., and Kahn, R. L. (1987). Human aging: usual and successful. Science 237, 143–149. doi:10.1126/science.3299702 Selkoe, D. J. (2012). Preventing Alzheimer’s disease. Science 337, 1488–1492. doi:10. 1126/science.1228541 Sperling, R. A., Aisen, P. S., Beckett, L. A., Bennett, D. A., Craft, S., Fagan, A. M., et al. (2011). Toward defining the preclinical stages of Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s associa- tion workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement. 7, 280–292. doi:10.1016/j.jalz.2011.03.003 Frontiers in Aging Neuroscience \| www.frontiersin.org August 2015 \| Volume 7 \| Article 164 References Science 237, 143–149. doi:10.1126/science.3299702 Selkoe, D. J. (2012). Preventing Alzheimer’s disease. Science 337, 1488–1492. doi:10. 1126/science.1228541 Copyright © 2015 Kljajevic. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Sperling, R. A., Aisen, P. S., Beckett, L. A., Bennett, D. A., Craft, S., Fagan, A. M., et al. (2011). Toward defining the preclinical stages of Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s associa- tion workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement. 7, 280–292. doi:10.1016/j.jalz.2011.03.003 August 2015 \| Volume 7 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 3
https://openalex.org/W4210262932	https://journals.lub.lu.se/STK/article/download/23189/21055	Swedish	null	Ledare	Svensk teologisk kvartalskrift	2,021	cc-by	597	Svensk Teologisk Kvartalskrift 97 (2021), 103–104 p-ISSN 0039-6761 e-ISSN 2003-6248 DOI: https://doi.org/10.51619/stk.v97i2.23189 Svensk Teologisk Kvartalskrift 97 (2021), 103–104 p-ISSN 0039-6761 e-ISSN 2003-6248 DOI: https://doi.org/10.51619/stk.v97i2.23189 JAYNE SVENUNGSSON Vad händer när en stor tänkare tystnar? Jacques Derrida (1930–2004) ställde frågan i ett tal vid Emmanuel Levinas (1906–1995) begravning för många år sedan och svarade att det för hans egen del var en erfarenhet som skulle förbli lika outtömlig som det rika arv av tankar och texter som Levinas lämnade efter sig. Och nog är det så det förhåller sig med varje stor tänkare som går ur tiden. En tystnad och saknad, men också en tacksamhet över det intellektuella livsverk som består. Under detta års första månader har två betydande teologiska röster tystnat: James M. Gustafson (1925–2021) och Hans Küng (1928–2021). I och med detta nummer inleder vi en ny tradition av att mer regelbundet uppmärksamma när tongivande teologer och religionsvetare går ur tiden. Det är sålunda en glädje att få publicera Carl Reinhold Bråkenhielms och Mårten Björks fina minnesporträtt över Gustafson och Küng. g En glädje är det också att få kungöra resultatet av en annan ny tradition i tidskriftens regi. I fjol utlyste Svensk Teologisk Kvartalskrift en pristävling för bästa vetenskapliga artikel, riktad till nydisputerade forskare i teologi och humaniora. Bland de många välskrivna bidrag som inkom besluta- de redaktionen i mars att låta kyrkohistorikern Katarina Pålsson (Lunds universitet) och idéhistorikern Anton Jansson (Göteborgs universitet) dela på priset. Artiklarna kommer att publiceras i nästa nummer, men redan här vill vi rikta ett stort grattis till båda pristagarna! Att svensk teologi och religionsvetenskap i dag har många spännande yngre röster vittnar också flera av föreliggande nummers artiklar om. Johan Eddebos religionsfilosofiska reflektion över de sekulära eskatologiska före- ställningar som bar upp inflytelserika populärhistoriska verk i Sverige från 1970-tal till 1990-tal är här ett utmärkt exempel. Givande är det också när yngre forskare går i dialog med etablerade forskare, såsom i Samuel Åsbergs 103 stk ˙ 2 ˙ 2021 ledare ledare ansats att bryta upp några av låsningarna inom nutida kristen socialetik ge- nom en konstruktiv-kritisk läsning av Carl-Henric Grenholms trinitariskt grundade lutherska etik. g 2016 blev den amerikanska författarinnan Marilynne Robinson heders- doktor vid teologiska fakulteten i Lund och vi hade senare förmånen att publicera den tankeväckande föreläsning hon höll vid Centrum för teologi och religionsvetenskap under rubriken ”Theology for this Moment” (num- mer 3–4, 2016). Mot denna bakgrund är det roligt att konstatera att forsk- ningen kring Robinsons verk slagit rot i Norden. JAYNE SVENUNGSSON I sin artikel om dopets betydelse i Robinsons roman Lila visar Elina Takala på ett fascinerande sätt hur läsningen av verket kan fördjupas med ett teologiskt prisma. Den tema- tiska spridningen i detta nummer kröns till sist av den föreläsning David Davage höll hösten 2020 med anledning av att han blivit docent i Gamla testamentets exegetik vid teologiska fakulteten i Lund. Davage reflekterar här över det dilemma som ligger i att de flesta av Gamla testamentets texter är anonyma och diskuterar dilemmat i relation till olika författarförståelser. y I och med detta nummer av Svensk Teologisk Kvartalskrift sker ett par skif- ten i redaktionen. Sara Järlemyr, doktorand i Gamla testamentets exegetik vid Centrum för teologi och religionsvetenskap, blir ny recensionsredaktör efter Elisabet Nord, och undertecknad tar över redaktörskapet från Samuel Byrskog. Tillsammans med redaktionssekreterare Martin Nykvist och den övriga redaktionen ser vi fram emot att förvalta tidningens arv vidare in i 2020-talet. Vi vill också passa på att rikta vårt varma tack till Elisabet och Samuel för deras goda insatser under åren som gått. p 104 \| stk ˙ 2 ˙ 2021 ledare
https://openalex.org/W2608490192	https://digitalscholarship.unlv.edu/cgi/viewcontent.cgi?article=1165&context=edpsych_fac_articles	English	null	The effects of doodling on recall ability	Psychological thought	2,017	cc-by	6,702	Educational Psychology, Leadership, and Higher Education Faculty Publications Educational Psychology, Leadership, and Higher Education Educational Psychology, Leadership, and Higher Education Faculty Publications Educational Psychology, Leadership, and Higher Education Abstract Previous research has documented a positive effect of doodling on individuals’ ability to recall information. However, previous research is limited to structured doodling tasks, such as shading in basic shapes. The present study extends the extant research, and increases the external validity of the previous findings, by considering the effects of multiple forms of doodling on recall. In this experimental study, ninety- three undergraduate participants were randomly assigned to one of 4 conditions (control, structured doodling, unstructured doodling, or note-taking). Participants listened to a fictional dialogue between 2 friends discussing a recent earthquake and then completed a fill-in the blank quiz to test their recall of the conversational information. The results indicated that participants in the unstructured doodling condition performed significantly worse than those in the structured doodling and note-taking condition. Keywords: doodling, cognition, attention Psychological Thought, 2017, Vol. 10(1), 206–216, doi:10.5964/psyct.v10i1.217 Handling Editors: Marius Drugas, University of Oradea, Oradea, Romania; Stanislava Stoyanova, South-West University "Neo Bulgaria Handling Editors: Marius Drugas, University of Oradea, Oradea, Romania; Stanislava Stoyanova, South-West University "Neofit Rilski", Blagoevgrad, Bulgaria Corresponding author at: University of Nevada, Las Vegas, 4505 S Maryland Pkwy, Las Vegas, NV 89154, USA. E-mail: Jasonboggs@gmail.com This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Corresponding author at: University of Nevada, Las Vegas, 4505 S Maryland Pkwy, Las Vegas, NV 89154, USA. E-mail: Jas p g y g y y g gg @g This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. You are in class trying to listen to a classmate’s presentation but continually catch yourself daydreaming about what you will eat for lunch, why this class is required to graduate, why your friend has yet to return your text messages, etc. All the sudden the teacher asks the class a question about the presentation and you realize that not only were you not aware that the presentation had ended, but also that you cannot recall what the presentation was about in the first place. Jason Bruce Boggs* a, Jillian Lane Cohen a, Gwen C. Marchand a [a] Department of Educational Psychology and Higher Education, University of Nevada, Las Vegas, USA. Psychological Thought psyct.psychopen.eu \| 2193-7281 Psychological Thought psyct.psychopen.eu \| 2193-7281 Research Articles The Effects of Doodling on Recall Ability The Effects of Doodling on Recall Ability Jason Bruce Boggs University of Nevada, Las Vegas Jillian Lane Cohen University of Nevada, Las Vegas Follow this and additional works at: https://digitalscholarship.unlv.edu/edpsych_fac_articles Follow this and additional works at: https://digitalscholarship.unlv.edu/edpsych_fac_articles Part of the Educational Psychology Commons Part of the Educational Psychology Commons Part of the Educational Psychology Commons Repository Citation Repository Citation Boggs, J. B., Cohen, J. L., Marchand, G. C. (2017). The Effects of Doodling on Recall Ability. Psychological Thought, 10(1), 206-216. http://dx.doi.org/10.5964/psyct.v10i1.217 Repository Citation Repository Citation Boggs, J. B., Cohen, J. L., Marchand, G. C. (2017). The Effects of Doodling on Recall Ability. Psychological Thought, 10(1), 206-216. http://dx.doi.org/10.5964/psyct.v10i1.217 This Article is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Article in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Article has been accepted for inclusion in Educational Psychology, Leadership, and Higher Education Faculty Publications by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact digitalscholarship@unlv.edu. Abstract In her study, she showed that, compared to students who did not doodle, students who doodled while listening to a two- minute audio recording were able to recall significantly more information that they were told to attend to, as well as information that they were told they did not have to attend to. The researcher attributed this effect to a reduction in daydreaming that occurred in those who were doodling. This conclusion is supported by several studies showing that performance on information processing and/or recall tasks (e.g., memory tasks, listening to a lecture or video) can be increased when one engages in low attention-demanding tasks simultaneously, such as doodling, in situations where the learner experiences boredom (Roche et al., 2007; Smallwood, O’Connor, Sudbery, & Obonsawin, 2007). This occurs because doodling maintains arousal while consuming a limited amount of available cognitive resources compared to daydreaming, which consumes a high amount of cognitive resources (Teasdale, Proctor, Lloyd, & Baddeley, 1993). There were, as the researcher mentions in the study, a few limitations in her study, as well as opportunities to extend her findings, and this is the purpose of the current research study. The main limitation of the Andrade (2010) study was the way that participants doodled. Participants were given sheets of paper with objects to shade in (squares and circles) while they listened to the audio recording. Although this is certainly a form of doodling, this method certainly lacks ecological validity. It is reasonable to assume that those who doodle, whether students in class or workers in a business meeting, would be engaged in more of a freestyle form of doodling. It is also reasonable to assume that those who doodle would exert various amounts of their attention and other cognitive resources into their doodling activities. For example, a detailed drawing of another classmate or person would most likely require more cognitive resources than doodling random shapes or squiggly lines, and thus it is possible that the type of doodling may moderate the relationship between doodling and increased recall ability. An additional limitation of Andrade’s (2010) study, which the author mentioned is the article, is that there were no measures of daydreaming that could confirm the hypothesis that students who doodled engaged in daydreaming less than students who listened and took notes. The present study was designed to address some of the limitations of the existing work on doodling. Abstract First, we focused on whether different types of doodling would yield the same effect on recall ability. Our second question centered on a more practical issue: How does doodling compare to other proven tools such as note-taking? In Andrade’s study, participants in the “control” group took notes but only on part of the information on which they were to be assessed. Finally, we sought to compare different types of doodling to note-taking. Abstract This or a similar scenario has most likely happened to all of us at one point or another whether we were purposefully trying to daydream or whether we indeed were trying to attend to the lecture/presentation. Engaging in task unrelated thoughts (TUTs), such as daydreaming, commonly happens when we are bored and/or disengaged from the stimuli whether a business, academic, or social setting (Smallwood & Schooler, 2006). We do many things to combat boredom including playing with our phones, chatting with neighbors, doodling, or taking notes (Harris, 2000; London, Schubert, & Washburn, 1972). There is no shortage of literature showing the positive effects and correct way to take notes for acquiring and storing information (Di Vesta & Gray, 1972; Kiewra, 1985; Kobayashi, 2005), but there are times when we simply want to listen to the lecture or presentation without taking notes while still being able to tend to the information being delivered. Boggs, Cohen, & Marchand 207 Jackie Andrade published a study in 2010 that examined the effects of doodling on recall ability. In her study, she showed that, compared to students who did not doodle, students who doodled while listening to a two- minute audio recording were able to recall significantly more information that they were told to attend to, as well as information that they were told they did not have to attend to. The researcher attributed this effect to a reduction in daydreaming that occurred in those who were doodling. This conclusion is supported by several studies showing that performance on information processing and/or recall tasks (e.g., memory tasks, listening to a lecture or video) can be increased when one engages in low attention-demanding tasks simultaneously, such as doodling, in situations where the learner experiences boredom (Roche et al., 2007; Smallwood, O’Connor, Sudbery, & Obonsawin, 2007). This occurs because doodling maintains arousal while consuming a limited amount of available cognitive resources compared to daydreaming, which consumes a high amount of cognitive resources (Teasdale, Proctor, Lloyd, & Baddeley, 1993). There were, as the researcher mentions in the study, a few limitations in her study, as well as opportunities to extend her findings, and this is the purpose of the current research study. Jackie Andrade published a study in 2010 that examined the effects of doodling on recall ability. Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 The Current Study In the current study we sought to further assess the potential benefits of doodling on recall ability. We wanted to look specifically at whether allowing participants to doodle whatever they choose would yield a comparable effect to Andrade’s doodling condition (i.e., shading in shapes). This, as stated previously, was done to increase external validity since most people who doodle in the “real world” would be doing so in an unstructured manner. An additional difference between the current study and Andrade’s is that we added a note-taking condition whereas Andrade permitted both the control and doodling groups to take notes. Although taking notes and reviewing then going over them at a later point in time has been shown to be more beneficial than not doing so, the act of taking notes itself has also been shown to yield significant encoding effects compared to simply listening to or watching incoming stimuli (Di Vesta & Gray, 1972). We chose not to allow participants in the note- Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 The Effects of Doodling on Recall Ability 208 taking condition to use their notes for the recall task so that we could compare the process of note-taking to the process of doodling rather than the participants’ note-taking quality. taking condition to use their notes for the recall task so that we could compare the process of note-taking to the process of doodling rather than the participants’ note-taking quality. Our assessment was guided by three hypotheses. First, we hypothesized that both the structured and unstructured doodling groups as well as the note-taking group would perform significantly better on the recall test than the control group. Second, we hypothesized that the type of doodling would not have a significant effect so that those in the unstructured doodling condition would perform as well as those in the structured doodling condition on the recall test. Our third hypothesis was that those in the doodling conditions would perform as well as those in the note-taking condition on the recall task. These hypotheses are consistent with the previous literature and would serve as a replication and extension of Andrade’s study (2010). The Current Study Specifically our hypotheses were designed to replicate Andrade’s (2010) results and extend them by showing that: 1) doodling can be a beneficial tool for staying engaged during lectures and other similar settings, 2) the effects of doodling can be achieved with unstructured doodling in addition to the structured doodling that Andrade assessed, and 3) doodling can be as effective as the process of note-taking on recall ability. Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 Procedure Participants signed up through the university’s online research system under the department of education to fulfill research requirements for various courses in the education department. The study description informed participants that they would “listen to an audio recording while either doodling, taking notes, or just listening, and then be given a quiz assessing how much information you are able to recall. This study will take approximately 40 minutes of your time”. Participants were then randomly assigned to the control, structured doodle, unstructured doodle, or note-taking groups. After allowing participants time to complete a demographic questionnaire, participants were instructed as to what they would be doing during the audio recording depending on their group assignment (taking notes, doodling, or just listening) until the audio recording started. Participants were observed during the 5-minute audio recording to ensure that they were on task by a member of the research team. Following the audio recording the participants were told to turn over their notes or doodling sheets and all participants were given a copy of the quiz and told to try their best to answer all items from memory and that those who took notes were not to use them. Participants and Design There were 93 participants who signed up and completed the study. Of those, 20 were male and the ages ranged from 18-50 years with the mean age being 22. The vast majority of students who participated were studying elementary education and this was due to the requirement for participants to sign up for the study through the college of education online research participation system. As for ethnicity, the majority of participants were Caucasian (55%) and Latino (21%). Participants were randomly assigned to one of the four conditions (control, structured doodling, unstructured doodling, and note-taking) with each group having 23 people except for the note-taking group with 24. All participants monitored a conversation between two people and then attempted to recall information from the recording. A mock conversation between two friends was recorded at a relaxed conversational pace and played for participants at a comfortable volume (see Appendix for a copy of the conversation transcript). We chose to use an audio conversation rather than a lecture, song, or video-taped conversation to be consistent with Andrade’s (2010) design. In addition, we wanted to assess recall on fictitious information to control for previous knowledge that participants may have on specific subject areas. The fictional conversation was about a couples’ vacation to Hawaii and lasted approximately 5 minutes. Participants in the control condition just listened to the conversation. Those in the structured doodling condition used a pencil or pen to shade in 42 shapes (squares, circles, stars, crosses, and triangles) approximately 1 inch in diameter. Those in the unstructured doodling condition were provided with a white 8 by 11 inch sheet of blank paper and instructed to doodle anything they wanted, with the exception of inappropriate material or notes, during the audio recording. Those assigned to the note-taking condition were provided with a blank sheet of 8 by 11 inch white paper to take notes while listening to the recording (copies of all instruments are in the Appendix). All participants completed the recall quiz which consisted of 13 short-answer items (see the Appendix for the quiz). Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 Boggs, Cohen, & Marchand 209 Results We first verified that there were no significant differences according to age (F(8,69) = 1.51, p = .169) or gender (F(1,70) = 0.24, p = .626), and there were none. We then analyzed the data using a standard ANOVA and found significant group differences (F(3,65) = 6.21, p = .001, η2 = .22). Using Tukey HSD tests for post-hoc analyses, we found significant differences in test performance between those in the unstructured doodling group and those in the structured doodling and note-taking groups. Specifically, those in the unstructured doodling condition scored significantly lower on the recall quiz compared to those in the structured doodling (Mean diff. = 2.24, Std. error = 0.81, p = .04), and those in the note-taking condition (Mean diff. = 3.12, Std. error = 0.77, p = .001). We also found a significant difference between those in the note-taking group and those in the control group (Mean diff. = 1.99, Std. error = 0.72, p = .04) (see Table 1 below for all means and standard deviations). Table 1 Means and Standard Deviations for each condition on the 13 point recall quiz Group M SD Control 6.56 1.72 Structured Doodling 7.67 2.00 Unstructured Doodling 5.43 2.14 Note-taking 8.55 2.84 Discussion Means and Standard Deviations for each condition on the 13 point recall quiz Group M SD Control 6.56 1.72 Structured Doodling 7.67 2.00 Unstructured Doodling 5.43 2.14 Note-taking 8.55 2.84 Means and Standard Deviations for each condition on the 13 point recall quiz Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 Discussion As an extension of Andrade’s (2010) study, we expected to find that both the unstructured and structured forms of doodling would be beneficial to recall ability, but this was not the case in our study. We also predicted that the type of doodling, whether structured (as Andrade examined) or unstructured, would not make a significant difference on performance, but this was again shown to be an erroneous prediction. Lastly, we predicted that both types of doodling conditions would perform as well as those in the note-taking condition on the quiz, and Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 The Effects of Doodling on Recall Ability 210 this was partly confirmed. We found no support that doodling improved participants’ recall ability beyond just listening although we did find a significant effect for note-taking. this was partly confirmed. We found no support that doodling improved participants’ recall ability beyond just listening although we did find a significant effect for note-taking. The significant results we did find showed that there was a drop in recall performance for those who were in the unstructured doodling condition compared to those in the note-taking and structured doodling conditions. We believe that this may be due to participants putting more attention, thought, and/or effort into their doodles compared to those who were just shading in shapes (those in the structured doodling condition). This hypothesis is consistent with previous studies that have shown a “bottleneck” effect (de Jong, 1993; McCann & Johnston, 1992) where tasks requiring different competing cognitive resources (e.g., attention, short-term memory, decision making) can only be completed one at a time. For example, with the unstructured doodling task participants must actively decide what to doodle, reproduce a mental image on the paper, and attend to the audio recording. In contrast, participants in the structured doodling need only to attend to shading shapes and the audio recording. We conducted a post-hoc analysis of the participants’ unstructured doodles to see if those who put more effort into them did poorer on the recall exam. We separated the doodles in half and placed those which appeared to have more effort put into them in one group and the other half in another. We compared the groups and found that they were not significantly different, but with our limited sample of 8 participants in each group this was expected. Discussion Although non-significant, those who put less effort into their doodles had an average recall test score of 6.14 (SD = 1.95) compared to the recall test average of 4.71 (SD = 2.22) of those who put more effort into their doodles. It is significant that a structured doodling had a statistically similar effect on recall ability compared to a thoroughly documented encoding aid such as note-taking. We were not surprised that participants in the note- taking group performed the best on the recall quiz since the activity of taking notes would serve, just as doodling, as a form of arousal to prevent mind wandering. In addition, through dual-coding, the act of taking notes would help the encoding process. The authors have no support to report. The authors have no support to report. The authors have no support to report. Funding The authors have no funding to report. Competing Interests The authors have declared that no competing interests exist. Conclusion It is important to point out that this was not a direct replication of Andrade’s procedure. Participants in her study were told to attend to only part of the information that they were later asked to recall, and both the doodling and control participants were allowed to take notes on the information that they were expecting to be asked to recall. In our study, we only permitted participants in the note-taking condition to take notes, and we did not employ and form of deception about what participants would be asked to recall. These differences could possibly affect our inability to replicate Andrade’s findings, but mind wandering, as the mechanism postulated as disrupting attention, was indirectly assessed using the same design in both the current and Andrade’s (2010) study. This being said, our data suggest that there may not be an advantage for doodling as a tool for increasing recall performance even if daydreaming is avoided. Even if one avoids the attentional drain of daydreaming, the concurrent task needs to detract as little attention as possible to be beneficial. There were several limitations in our study. First, we did not measure participants’ doodling and/or drawing habits. It is possible that those who already doodle during classes or in similar environments would perform differently than those who do not. The second limitation applies to both Andrade’s (2010) and the current study; in neither study was there a direct measure of boredom and/or mind wandering. Andrade (2010) used an audio recording that lasted less than 3 minutes, and in the current study we utilized an audio recording that lasted approximately 5 minutes, but the question remains whether this is sufficient time for participants to cognitively Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 Boggs, Cohen, & Marchand 211 disengage from the task. The third limitation was sample size. There were between 20 and 25 participants in each of the 4 conditions, but to detect additional group differences it would have helped to increase the sample size. Future studies could provide further insight into the possible benefits of doodling by increasing the duration of the stimulus (e.g., recording or lecture) so that there would be a greater probability of mind wandering. Future studies could also further measure the effort placed into participants’ doodles to assess whether effort moderates the effects of unstructured doodling on preserving attention. The Effects of Doodling on Recall Ability 212 Smallwood, J., O’Connor, R. C., Sudbery, M. V., & Obonsawin, M. (2007). Mind-wandering and dysphoria. Cognition and Emotion, 21(4), 816-842. doi:10.1080/02699930600911531 Smallwood, J., O’Connor, R. C., Sudbery, M. V., & Obonsawin, M. (2007). Mind-wandering and dysphoria. Cognition and Emotion, 21(4), 816-842. doi:10.1080/02699930600911531 Smallwood, J., & Schooler, J. W. (2006). The restless mind. Psychological Bulletin, 132(6), 946-958. Teasdale, J. D., Proctor, L., Lloyd, C. A., & Baddeley, A. D. (1993). Working memory and stimulus-independent thought: Effects of memory load and presentation rate. The European Journal of Cognitive Psychology, 5(4), 417-433. doi:10.1080/09541449308520128 References Andrade, J. (2010). What does doodling do. Applied Cognitive Psychology, 24(1), 100-106. doi:10.1002/acp.1561 Andrade, J. (2010). What does doodling do. Applied Cognitive Psychology, 24(1), 100-106. doi:10.1002/acp.1561 de Jong, R. (1993). Multiple bottlenecks in overlapping task performance. Journal of Experimental Psychology: Human Perception and Performance, 19(5), 965-980. doi:10.1037/0096-1523.19.5.965 de Jong, R. (1993). Multiple bottlenecks in overlapping task performance. Journal of Experimental Psychology: Human Perception and Performance, 19(5), 965-980. doi:10.1037/0096-1523.19.5.965 Di Vesta, F. J., & Gray, G. S. (1972). Listening and note taking. Journal of Educational Psychology, 63(1), 8-14. doi:10.1037/h0032243 Harris, R. L. (2000). Batting 1,000: Questioning techniques in student-centered classrooms. The Clearing House: A Journal of Educational Strategies, Issues and Ideas, 74(1), 25-26. doi:10.1080/00098655.2000.11478634 Kiewra, K. A. (1985). Investigating notetaking and review: A depth of processing alternative. Educational Psychologist, 20(1), 23-32. doi:10.1207/s15326985ep2001_4 Kobayashi, K. (2005). What limits the encoding effect of note-taking? A meta-analytic examination. Contemporary Educational Psychology, 30(2), 242-262. doi:10.1016/j.cedpsych.2004.10.001 London, H., Schubert, D. S., & Washburn, D. (1972). Increase of autonomic arousal by boredom. Journal of Abnormal Psychology, 80(1), 29-36. doi:10.1037/h0033311 McCann, R. S., & Johnston, J. C. (1992). Locus of the single-channel bottleneck in dual-task interference. Journal of Experimental Psychology: Human Perception and Performance, 18(2), 471-484. doi:10.1037/0096-1523.18.2.471 Roche, R. A. P., Commins, S., Agnew, F., Cassidy, S., Corapi, K., Leibbrand, S., . . . O’Mara, S. M. (2007). Concurrent task performance enhances low-level visuomotor learning. Perception & Psychophysics, 69(4), 513-522. doi:10.3758/BF03193908 Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 The Effects of Doodling on Recall Ability Audio Script Let’s go over our itinerary before we go to sleep. Sound good? Jake: Ok. But we only have a few hours before our flight to Maui, so we can’t venture too far from the airport. So let’s see the harbor and then we can grab some lunch since I’m sure we will be starving. I guess we can head back to the airport after that. We can’t check in to our hotel until 4 so we may have some time to kill when we land. Jake: Ok. But we only have a few hours before our flight to Maui, so we can’t venture too far from the airport. So let’s see the harbor and then we can grab some lunch since I’m sure we will be starving. I guess we can head back to the airport after that. We can’t check in to our hotel until 4 so we may have some time to kill when we land. Kira: Sometimes they have the rooms ready early, so we may as well go to the hotel and see since we will have all of our luggage with us. I’m sure we will be tired anyway since we have to wake up so early. Kira: Sometimes they have the rooms ready early, so we may as well go to the hotel and see since we will have all of our luggage with us. I’m sure we will be tired anyway since we have to wake up so early. Jake: If you say so…I’m just excited to lay on the beach and do absolutely nothing…Working 10 hour days has taken its toll over the past few months. I don’t even want to think about work…when I get back I have that presentation to give to the board. You will probably have to drag me to the airport in two weeks. Jake: If you say so…I’m just excited to lay on the beach and do absolutely nothing…Working 10 hour days has taken its toll over the past few months. I don’t even want to think about work…when I get back I have that presentation to give to the board. You will probably have to drag me to the airport in two weeks. Kira: Well, remember you agreed to take scuba lessons with me, so you can sleep in every morning except for one day since the lessons start at 8am. Psychological Thought 2017, Vol. Audio Script Jake: Kira, good news…..I found out that we will earn double miles for this flight. Kira: Oh yeah? How many miles do we have so far? Don’t we need like 50,000 to earn a free flight? Jake: I think it’s only 20,000, but I will check when we get back. I think we have 15,000 already so maybe we will be able to go somewhere nice this Christmas instead of your parents. Kira: Don’t even start, it’s only July and your already planning a way to get out of spending the holidays with my family. Jake: I’m only joking. So what do you want to eat for dinner? Kira: Don’t even start, it’s only July and your already planning a way to get out of spending the holidays with my family. Jake: I’m only joking. So what do you want to eat for dinner? Kira: Well, we have leftovers from last night? Or we could have salads….afterall, we could both use a little bit of dieting since we’re going to be wearing our bathing suits everyday. Kira: I’m just about finished…just need to figure out what I want to wear if we go anywhere fancy, have you checked the weather report? Jake: It’s Hawaii….I’m sure it will be nice. Kira: Just check please, maybe it is supposed to rain or some freak cold front is coming. Jake: Ok, I will- relax. Kira: Did you borrow the suitcase from Jim? Jake: Yes, I took care of everything, don’t worry. Kira: Ok, remember to check in online before we leave in the morning. Jake: I still can’t believe that we got such a good deal on this trip, I didn’t think we would ever be able to afford to stay for two weeks. Jake: I still can’t believe that we got such a good deal on this trip, I didn’t think we would ever be able to afford to stay for two weeks. Jake: I still can’t believe that we got such a good deal on this trip, I didn’t think we would ever be able to afford to stay for two weeks. Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 213 Boggs, Cohen, & Marchand Kira: Yeah, but you know that we’re still on a budget, so don’t get any crazy ideas, haha. Kira: Yeah, but you know that we’re still on a budget, so don’t get any crazy ideas, haha. Audio Script We can’t check in to our hotel until 4 so we may have some time to kill when we land. Kira: Sometimes they have the rooms ready early, so we may as well go to the hotel and see since we will have all of our luggage with us. I’m sure we will be tired anyway since we have to wake up so early. Jake: Good point, no need to rush since we will have two weeks. So what are you most excited about doing while we’re there? Kira: Seriously? You’ve known me for 9 years and you have to ask? Jake: Haha, I know, I know…swimming with the turtles- I can’t believe they don’t scare you..they are as big as cars! Kira: Don’t be silly, they’re not that big and they don’t eat humans- they’re gentle. Jake: If you say so…I’m just excited to lay on the beach and do absolutely nothing…Working 10 hour days has taken its tol Kira: True, but that doesn’t make me less nervous right now. I do wish that we would’ve been able to leave back in January; the weather has been so nice here lately it doesn’t seem as special. Kira: True, but that doesn’t make me less nervous right now. I do wish that we would’ve been able to leave back in January; the weather has been so nice here lately it doesn’t seem as special. Jake: Maui is special no matter what time of year you go. Trust me, you’ve never been there before. You know that if I didn’t have my back surgery back In January I would have been miserable the entire time, so even though we had to wait it will be better for both of us since we will be able to do so much more. Jake: Maui is special no matter what time of year you go. Trust me, you’ve never been there before. You know that if I didn’t have my back surgery back In January I would have been miserable the entire time, so even though we had to wait it will be better for both of us since we will be able to do so much more. Jake: We have plenty of time to see Paris Kira, plus a four hour flight is probably a little more manageable for than a ten hour one. Jake: Ok, subject change. Audio Script Jake: Kira, you only live once. I didn’t save up for two years to penny-pinch. e you’re right, I just don’t want to get back and pass out when the credit card bill comes. Jake: I promise we won’t get too crazy, and the promotion I received at work does take a lot of pressure off. Are you still nervous about the flight tomorrow? Jake: I promise we won’t get too crazy, and the promotion I received at work does take a lot of pressure off. Are you still nervous about the flight tomorrow? Jake: I promise we won t get too crazy, and the promotion I received at work does take a lot of pressure off. Are you still nervous about the flight tomorrow? Kira: Ughh..don’t bring it up- you know I hate flying. Jake: At least you have those pills Dr. Snyder gave you this time. Kira: True, but that doesn’t make me less nervous right now. I do wish that we would’ve been able to leave back in January the weather has been so nice here lately it doesn’t seem as special. Jake: Maui is special no matter what time of year you go. Trust me, you’ve never been there before. You know that if I didn’ have my back surgery back In January I would have been miserable the entire time, so even though we had to wait it will be better for both of us since we will be able to do so much more. Kira: It better be. We did give up Paris Jake: We have plenty of time to see Paris Kira, plus a four hour flight is probably a little more manageable for than a ten hour one. Kira: Ok, ok…don’t’ remind me of flying anymore tonight. Jake: Ok, subject change. Let’s go over our itinerary before we go to sleep. Sound good? Kira: Alright. The first place I want to visit when we land in Honolulu is Pearl Harbor. Jake: Ok. But we only have a few hours before our flight to Maui, so we can’t venture too far from the airport. So let’s see the harbor and then we can grab some lunch since I’m sure we will be starving. I guess we can head back to the airport after that. Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 Audio Script 10(1), 206–216 doi:10.5964/psyct.v10i1.217 214 The Effects of Doodling on Recall Ability Jake: At least we were lucky enough to get a beach view in our hotel room- I’ve always said that I sleep best when I can hear the ocean at night. I am also really looking forward to eating fresh seafood…let’s eat a light lunch so we can have a big seafood dinner tomorrow night. Kira: I know, the hotel room seems like it will be perfect but I am still a little disappointed that they only had a queen size bed instead of a king. You know how I like to stretch out when I sleep- how can they not have a single room available with a king size bed? Can you ask them again when we get there if something has opened up? Please? For 200 dollars a night, we should at least get a king size bed. Jake: Ok, I can ask but I doubt they will. I hope you won’t complain the whole time if they don’t have anything but the queen. If that’s the only thing that we have to complain about the entire time we will be lucky. Jake: Ok, I can ask but I doubt they will. I hope you won’t complain the whole time if they don’t have anything but the queen. If that’s the only thing that we have to complain about the entire time we will be lucky. Kira: Ok, but you still better ask. Is your brother still taking us to the airport in the morning? Why don’t you call him and remind him he needs to be here by 6? You know how busy Phoenix can get and I really think we should get there at least 90 minutes before our flight. Kira: Ok, but you still better ask. Is your brother still taking us to the airport in the morning? Why don’t you call him and remind him he needs to be here by 6? You know how busy Phoenix can get and I really think we should get there at least 90 minutes before our flight. Jake: Don’t worry, Jim wakes up at 5 every morning, but I will call him anyway. And don’t complain about traffic since you’re the one who begged to move to Phoenix. Kira: Just stating the facts. Audio Script And you were the one who complained about the snow every year back in Vermont so don’t act like you weren’t content to move. By the way, did you give Jane the spare key so she can get in to feed Zack and Zoey? Jake: Darnit! I knew I forgot something! Do you think she would still be up? Kira: I knew you would forget. I don’t know…you better call her. Did you think those two dogs were going to feed them- selves? Jake: Take it easy, I’ve had a lot going on and I will take care of it…even if she is asleep I can put the spare key under the rock outside and call her tomorrow. The dogs won’t starve. Kira: Just one more thing to worry about…At least we packed yesterday and can get some sleep. Jake: No kidding, I just hope I’ll be able to fall sleep. Do you think I should take an ambient? Kira: No, I wouldn’t…you know how drowsy that will make you in the morning. Maybe if you take the dogs for a walk you’ll be tired by the time you get back? Kira: No, I wouldn’t…you know how drowsy that will make you in the morning. Maybe if you take the dogs for a walk you’ll be tired by the time you get back? Jake: Nice try, it’s your turn and you know it. Jake: Nice try, it’s your turn and you know it. Jake: I’m gonna jump in the shower while you’re gone. Kira: Ok, but before you do remember to call your brother and Jane. Kira: Ok, but before you do remember to call your brother and Jane. Boggs, Cohen, & Marchand 215 Recall Quiz Recall Quiz Please answer the following questions: 1. What is the first place Kira wants to visit on their trip to the Hawaiian islands? 2. On which of the Hawaiian islands will they be staying? 3. How long did it take Jake and Kira to save for their vacation? 4. What activity did Kira say she was most excited about doing? 5. What does Kira not want on her pizza? 6. What are they borrowing from Jake’s brother? 7. How long have Jake and Kira known each other? 8. Why is Jake happy about their room? 9. Why did Kira complain about the hotel room? 10. Where did Jake and Kira originally plan to go on vacation back in January? 11. Why did Jake need to wait until June to leave to go on vacation? 12. Name one of Kira and Jake’s dogs. 13. What do Kira and Jake want to wake up early for while on vacation? 13. What do Kira and Jake want to wake up early for while on vacation? y Structured Doodle Sheet Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 Structured Doodle Sheet The Effects of Doodling on Recall Ability 216 Psychological Thought 2017, Vol. 10(1), 206–216 doi:10.5964/psyct.v10i1.217 PsychOpen is a publishing service by Leibniz Institute for Psychology Information (ZPID), Trier, Germany. www.zpid.de/en About the Authors Jason Boggs is a doctoral student in educational psychology with interests in attention, perception, and criminal thinking assessment. He currently works as a corporate research manager, and is working on several projects that assess the effec- tiveness of various programs designed to reduce recidivism. Jillian Cohen received her PhD in Educational Psychology with a specialization in School Psychology from the University of Nevada, Las Vegas. She has national and state certification and is currently working as a school psychologist on the assessment team in Baltimore City Public Schools. She is interested in evidenced based interventions for children with ADHD and specific learning disabilities. Dr. Gwen Marchand is an Associate Professor in Educational Psychology at the University of Nevada, Las Vegas. Her re- search interests include topics such as motivation and engagement in formal educational systems; student mobility and school transitions from a complex systems perspective; evaluation of collaborative research and teaching networks.
https://openalex.org/W2616761626	https://escholarship.org/content/qt1dz7270w/qt1dz7270w.pdf?t=pztve0	English	null	Effect of Age on the Association of Vascular Access Type with Mortality in a Cohort of Incident End-Stage Renal Disease Patients	The Nephron journals/Nephron journals	2,017	cc-by	6,111	Permalink Permalink https://escholarship.org/uc/item/1dz7270w Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Effect of Age on the Association of Vascular Access Type with Mortality in a Cohort of Incident End-Stage Renal Disease Patients. Effect of Age on the Association of Vascular Access Type with Mortality in a Cohort of Incident End-Stage Renal Disease Patients. Effect of age on the association of vascular access type with mortality in a cohort of incident ESRD patients Tarek Saleh1, Keiichi Sumida1,2,3, Miklos Z Molnar1, Praveen K Potukuchi1, Fridtjof Thomas1, Jun Ling Lu1, Geeta Gyamlani4, Elani Streja5, Kamyar Kalantar-Zadeh5, and Csaba P Kovesdy1,4 1Division of Nephrology, University of Tennessee Health Science Center, Memphis, TN, United States Author Manuscript 2Nephrology Center, Toranomon Hospital Kajigaya, Kanagawa, Japan p gy , p j g y , g , p 3Department of Nephrology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan 4Nephrology Section, Memphis VA Medical Center, Memphis, TN, United States 5Harold Simmons Center for Chronic Disease Research and Epidemiology, Division of Nephrology and Hypertension, University of California-Irvine, Orange, CA, United States 3Department of Nephrology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan 4Nephrology Section, Memphis VA Medical Center, Memphis, TN, United States 5Harold Simmons Center for Chronic Disease Research and Epidemiology, Division of Nephrology and Hypertension, University of California-Irvine, Orange, CA, United States Address for correspondence: Csaba P. Kovesdy, MD, Nephrology Section, Memphis VA Medical Center, 1030 Jefferson Ave., Memphis, TN 38104, Phone: 901-523-8990, Fax: 901-577-7539, ckovesdy@uthsc.edu. DISCLOSURES HHS Public Access Author Manuscript Published in final edited form as: Nephron. 2017 ; 137(1): 57–63. doi:10.1159/000477271. Powered by the California Digital Library University of California eScholarship.org eScholarship.org Address for correspondence: Csaba P. Kovesdy, MD, Nephrology Section, Memphis VA Medical Center, 1030 Jefferson Ave., Memphis, TN 38104, Phone: 901-523-8990, Fax: 901-577-7539, ckovesdy@uthsc.edu. DISCLOSURES None. INTRODUCTION The number of elderly patients with end-stage renal disease (ESRD) in the United States has been increasing in recent decades.[1] The National Institute on Aging reports that the United States population age 65 and over is expected to double in size within the next 25 years, and by 2030 more than 70 million people will be 65 years or older and more than 2.24 million patients will have ESRD.[1] Each year, more elderly patients transition to ESRD in the United States, the majority of whom are treated with in-center hemodialysis and require a vascular access, such as an arteriovenous fistula (AVF) or graft (AVG), or a tunneled central venous catheter (TDC).[2] Author Manuscript The type of vascular access for hemodialysis is one of the major factors known to be associated with mortality in ESRD patients. Clinical guidelines, including the Fistula First Breakthrough Initiative, endorse the use of AVF over AVG and TDC because it is associated with better survival and lower complications rates.[3–5] Despite higher initial successful function rates compared with AVFs and lower infection rates compared with TDCs, AVGs are the least used form of vascular access at hemodialysis initiation because they tend to require more interventions and are more likely to fail compared with AVFs.[4,6] On the other hand, multiple studies have shown that older patients are at greatest risk of having AVF maturation failure, potentially leaving them dependent on TDCs for vascular access.[7–9] Author Manuscript With these conflicting results about the different types of vascular access in elderly hemodialysis patients, it is unclear if elderly hemodialysis patients derive a survival benefit from an AVF over an AVG. Furthermore, knowledge is scarce about the association of access type with outcomes in the oldest hemodialysis patients (e.g. >80 years old), in whom it is possible that even a TDC could be equivalent to an AVF or AVG. We hypothesized that patients with advanced age and may not experience a survival benefit from an AVF over an AVG or a TDC during the immediate post-transition period to hemodialysis. Given that placement and maintenance are arguably more invasive for fistulas and grafts they take a longer time to mature, older individuals may not garner the same benefit from the “fistula first” approach as younger patients who have lower comorbidity levels and longer survival. Abstract Background/Aims—Fistula first is the general recommendation for all hemodialysis (HD) patients, but creating a mature arteriovenous fistula (AVF) can be challenging in elderly individuals. It is unclear if elderly incident HD patients derive a survival benefit from an AVF over an arteriovenous graft (AVG) or a tunneled catheter (TDC). Author Manuscript Methods—We examined the association of vascular access type (AVF, AVG, and TDC with and without a maturing AVF/AVG at dialysis transition) at hemodialysis initiation with all-cause, cardiovascular and infection-related mortality in 46,786 US veterans, using Cox models with adjustment for confounders. Effect modification by age was examined by examining associations in pre-specified age subgroups (<60, 60–<70, 70–<80 and ≥80 years old), and by including interaction terms. Results—8,940 (19%) patients started HD with an AVF, 1,090 (3%) with an AVG, 8,262 (18%) with a TDC and a maturing AVF/AVG and 28,494 (61%) with a TDC without a maturing AVF/ AVG. A total of 13,303 all-cause, 4,392 cardiovascular, and 1,058 infection-related deaths were observed in the first year after hemodialysis transition. Compared to patients with AVF, those with AVG and TDC with and without maturing AVF/AVG had incrementally higher overall risk of all- cause mortality and cardiovascular mortality. Only TDC use was associated with higher infection- associated mortality. These associations were not modified by age. Author Manuscript Author Manuscript Page 2 Saleh et al. Conclusion—Although most of our patients consisted of male veterans and the results may not be generalized to the general population, the use of TDCs is associated with poor outcomes even in the most elderly incident hemodialysis patients. Author Manuscript Nephron. Author manuscript; available in PMC 2018 May 18. MATERIALS and METHODS Author Manuscript Cohort Definition We analyzed data from the Transition of Care in Chronic Kidney Disease (TC-CKD) study, a retrospective cohort study examining US veterans transitioning to renal replacement therapy from October 1, 2007 through September 30, 2011.[10] A total of 52,172 US veterans were identified from the US Renal Data System (USRDS)[1] as the initial cohort. We used the vascular access type listed on the USRDS Patient and Medical Evidence Form 2728 to identify patients who initiated hemodialysis using either an AVF, an AVG, or a TDC with and without a maturing AVF or AVG (treated as separate categories). Patients with missing or unknown information about hemodialysis vascular access (n=4,909), those with missing age or age <18 years old (n=7), and those with no follow-up information (n=470) were excluded, resulting in a final analytical sample of 46,786 patients. Patients were categorized by their age at hemodialysis start into prespecified groups of <60, 60–<70, 70– <80 and ≥80 years old. Author Manuscript INTRODUCTION Therefore, we investigated the association of vascular access type with all-cause and cause- specific patient mortality among four different age subgroups in a large national cohort of incident hemodialysis patients from the US Department of Veterans Administration (VA). Author Manuscript Page 3 Page 3 Saleh et al. Nephron. Author manuscript; available in PMC 2018 May 18. Data collection Data from the USRDS Patient and Medical Evidence Form 2728 were used to determine baseline demographic characteristics at the time of dialysis initiation, laboratory variables prior to hemodialysis (estimated GFR, albumin and hemoglobin), and presence or absence of Nephrology subspecialty care received prior to hemodialysis. Information about comorbidities was extracted from the VA Inpatient and Outpatient Medical SAS Datasets, [11] using ICD-9-CM diagnostic and procedure codes and CPT codes, as well as from CMS Data files, as previously described.[12] Cardiovascular disease was defined as the presence of diagnostic codes for coronary artery disease, angina, myocardial infarction, or cerebrovascular disease. We calculated the Charlson Comorbidity Index score using the Deyo modification for administrative data sets, without including kidney disease.[13] Author Manuscript Statistical analysis Data are presented as number (percent) for categorical variables and mean ± standard deviation or median (interquartile range [IQR]) as appropriate. Continuous variables were compared using ANOVA and categorical variables were compared with χ2 test. The association of various demographic and clinical characteristics with the type of vascular access was examined in multinomial logistic regression analyses. The co-primary outcomes were all-cause, cardiovascular, and infection-related mortality during the first year after dialysis initiation. Information about all-cause mortality was obtained from the VA Vital Status Files, [14] and causes of death were obtained from the USRDS. We examined the association of vascular access type with outcomes during the first year following hemodialysis initiation using the Kaplan-Meier method and the log-rank test. Hazard ratios of all-cause mortality were calculated in unadjusted and multivariable adjusted Cox models. All multivariable models were adjusted based on a priori considerations for age, gender, race, comorbid conditions (history of diabetes mellitus, myocardial infarction, congestive heart failure, peripheral vascular disease, cerebrovascular disease, dementia, chronic lung disease, liver disease and malignancies), and for the Charlson comorbidity Author Manuscript Saleh et al. Page 4 index as an omnibus measure of illness. The effect of age on the association of vascular access type with the various outcomes was additionally examined by performing analyses in subgroups of patients categorized by their baseline age, and by including multiplicative interaction terms. Author Manuscript Of the variables included in the main multivariable model, data points were missing for race (<0.1%) and for comorbid conditions (11%). 41,578 patients (89%) had complete data for multivariable analysis; due to the relatively low proportion of missingness, missing data was not imputed. In sensitivity analyses we examined the same associations over the first 6-month post- transition time period, and after additional adjustments for the last estimated glomerular filtration rate (eGFR), hemoglobin and serum albumin levels before transition to hemodialysis (obtained from USRDS Form 2728). Analyses were conducted using STATA MP Version 14 (STATA Corporation, College Station, TX). The study was approved by the Institutional Review Boards of the Memphis and Long Beach VA Medical Centers, with exemption from informed consent. Author Manuscript Nephron. Author manuscript; available in PMC 2018 May 18. RESULTS Overall, patients were 70±12 years old, 94% were male, 25% were African-American, and 58% had diabetes. 8,940 (19%) started hemodialysis with an AVF; 1,090 (2.3%) with an AVG; 8,262 (18%) with a TDC and a maturing AVF or AVG indicating prior attempts to create a permanent vascular access (of which 7,549 had a maturing AVF and 713 had a maturing AVG); and 28,494 (61%) with a TDC and no maturing AVF or AVG (indicating no attempts to create a permanent vascular access in the pre-dialysis time period). Patients’ baseline characteristics at the time of dialysis initiation by their initial vascular access type are presented in Table 1. Compared to patients with a mature AVF, patients with an AVG were more likely to be female and black, and to not have received Nephrology care during the pre-dialysis period. Compared to patients with a mature AVF, patients with a TDC and no maturing AVF/AVG had a higher prevalence of cardiovascular and chronic lung disease, and were substantially less likely to have received Nephrology care during prelude. Patients with a TDC who had a maturing AVF/AVG had similar or fewer comorbidities as those with a TDC and no maturing AVF/AVG, with the exception of diabetes mellitus, which was more common in the former group. Furthermore, the group with TDC and maturing AVF/AVG had a higher likelihood of receiving pre-dialysis Nephrology care compared to those with a TDC and no maturing AVF/AVG (although it was still lower compared to the group with a mature AVF). Author Manuscript Author Manuscript 13,303 patients died (mortality rate 343/1000 patient-years, 95%CI: 338–350) during the first year after transition to hemodialysis. Figure 1 shows cumulative unadjusted event curves for overall all-cause (Panel A), cardiovascular (Panel B) and infection-related (Panel C) mortality, in patients grouped by their initial vascular access type, indicating incrementally higher risk of all-cause and cardiovascular mortality in patients with an AVG, a TDC and maturing AVF/AVG and a TDC without maturing AVF/AVG, compared to patients with a mature AVF. Table 2 shows the crude and multivariable adjusted hazard Saleh et al. Page 5 ratios of all-cause, cardiovascular and infection-related mortality associated with AVG and TDC (compared to AVF) in patient subgroups of various ages. All-cause mortality trended higher in patients with an AVG vs. RESULTS AVF in all age groups; patients with a TDC and a maturing AVF/AVG experienced higher mortality than those with a mature AVF or AVG; and patients with a TDC and no maturing AVF/AVG experienced the worst mortality of all four groups in all age categories. The association of vascular access type with all-cause mortality was not significantly modified by age (p value for interaction=0.08). The associations of vascular access type with cardiovascular mortality were similar in nature and magnitude to those seen for all-cause mortality, with incremental risk associated with AVG, TDC with and without maturing AVF/AVG, and with no effect modification by age (p value for interaction=0.40) (Table 2). Compared to patients with AVF, the risk of infection-related mortality was not significantly higher in patients with AVG, although the number of events precluded assessment in some age groups. The risk of infection-related mortality was highest in patients with TDC and no maturing AVF/AVG (multivariable adjusted hazard ratios ranging from 2.07 to 8.92 in various age subgroups), followed by those with TDC and maturing AVF/AVG (multivariable adjusted hazard ratios ranging from 1.09 to 4.28 in various age subgroups), without significant effect modification by age (p value for interaction=0.45) (Table 2). Author Manuscript Aut Author Manuscript Author Manuscript Results remained unchanged in sensitivity analyses examining outcomes occurring over the first 6 months following hemodialysis transition, and after additional adjustments for pre- transition eGFR, hemoglobin and serum albumin (data not shown). Nephron. Author manuscript; available in PMC 2018 May 18. DISCUSSION [5,8,17] Author Manuscript Autho Author Manuscript On the other hand, AVGs are the least used form of vascular access at hemodialysis initiation, presumably due to the lower long term patency rates and an increased association with morbidity and mortality.[4,6] The cumulative primary patency at 1 and 2 years was 81% and 65% in a study of 67 elderly patients over the age of 70 years, and the secondary patency was 65% and 58% at 1 and 2-years, respectively.[18] In another study the 1 and 2- year total patency rate among elderly patients with synthetic grafts was 44.2% and 38.6%.23 However, AVGs are considered viable options in patients with failed fistulas, exhausted, unsuitable, or damaged veins, or when there is late Nephrology referral and need for urgent cannulation with avoidance of central venous catheters.[19] Overall, AVF survival is no better than AVG when primary failures are included in access survival analyses.[19,20] In a study comparing access survival by access type among those >65 years old using data from the USRDS, [21] use of a AVF vs. AVG was not associated with increased patency among non-diabetic (OR 1.48, 95% CI 0.95–2.3) or diabetic elderly (OR 1.49, 95% CI 0.76–2.89). DISCUSSION In this large and nationally representative retrospective cohort study of incident hemodialysis patients, we compared the association of AVG and TDC (vs. AVF) with all- cause and cause-specific mortality in US veterans aged <60, 60–<70, 70–<80, and ≥80 years old. We found that using a TDC is associated with higher mortality in all age groups, the risk being highest for infection-related mortality. Patients with a TDC and no maturing AVF/AVG had higher mortality rates compared to patients with a TDC and a maturing AVF/ AVG, but the latter had worse outcomes compared to patients with a mature AVF or AVG. Even though the risk of various outcomes associated with AVG vs. AVF was not statistically significantly different in all age groups, use of an AVG was associated with an overall modestly increased risk of all-cause and cardiovascular mortality, with no effect modification by age upon statistical testing. Author Manuscript Older age is often considered a challenge to the creation and use of vascular access. Given the complicated process of establishing a successful vascular access, clinicians are faced with difficult decisions regarding the choice of an optimal access in elderly CKD patients transitioning to hemodialysis, who face a high chance of having an access created but not used (due to either succumbing to death prior to reaching end stage renal disease, or due to primary maturation failure).[15,16] Recent studies showed a high rate of primary maturation failure in native AVFs, [15,17] yet there are no standard patient eligibility criteria to guide AVF placement. Currently, the primary and secondary fistula patency rate in the elderly at 1 year have ranged from 43% to 74% and 56% to 82%, respectively.[15] Older age has been Author Manuscript Page 6 Saleh et al. associated with lower rates of fistula use which is attributable to a decrease in referral for AVFs as well as increased rates of failure to mature among those who do get a fistula. Nephron. Author manuscript; available in PMC 2018 May 18. DISCUSSION Placing an AVG first may in fact dramatically lengthen the proportion of the patient’s lifespan with freedom from catheter dependence and its potential complications.[19] Author Manuscript Similar to our study, in a previous observational study of 66,595 elderly (>67 years old) Medicare USRDS patients, the use of AVF as initial access for long-term dialysis therapy was associated with the best survival, followed by AVG and TDC use.[21] In another large analysis of patients >67 years old from USRDS, incident catheter use was associated with significantly higher long-term mortality compared to AVF use, but the association of AVG use with mortality was only significant in relatively younger patients (<80 years old), and was not present in patients ≥80 years old.[22] The seemingly contradictory results regarding the association of AVG with mortality in these studies may be caused by differences in sample size and analytical approaches, as the point estimates for the risk were fairly modest and similar in magnitude in these two studies.[4,22] Our study supports and extends these findings, by examining cause-specific mortality and emphasizing the major importance of infection-related deaths for TDC use, and by including a full spectrum of patient age and thus establishing that age is not a significant effect modifier of the associations between access type and mortality. In our study TDC use was associated with the worst outcomes even when accounting for pre-dialysis attempts to create a permanent vascular access (AVF or AVG), although patients with such attempts experienced better outcomes compared to patients with no prior attempts. A recent study examining 2,300 incident hemodialysis patients from five Canadian dialysis centers also found that patients who do not undergo attempts to place pre-dialysis AVF had worse outcomes compared to those who had such attempts, independent of age, and suggested that the mortality associated with TDC use may not be caused by catheters, but by the underlying comorbid conditions necessitating catheter placement [23]. Author Manuscript These results are difficult to compare to our findings, due to marked differences in sample size, in the nature of the two study populations (Canadian vs. US), and the different Saleh et al. Page 7 analytical approaches (we examined immediate post-dialysis mortality, as opposed to longer term mortality in the study by Quinn et al.). DISCUSSION However, the fact that in our study even patients with TDC who had attempts at creating an AVF or AVG experienced significantly worse outcomes compared to patients with AVF, and the fact that associations were particularly strong with infection-related mortality suggests that a direct effect of catheter use on adverse outcomes cannot be ruled out. Findings similar to our study were also reported recently by Brown et al., who showed that patients initiating hemodialysis with a TDC after failed AVF placement had significantly lower all-cause mortality rates than the TDC-only group, but higher mortality than patients initiating dialysis using an AVF.[24] Author Manuscript Au Author Manuscript Our study is notable for its large sample size of late-stage non-dialysis dependent CKD patients transitioning to dialysis, for representing the full age spectrum of such patients, and for being representative of veterans in the entire geographic United States. However, several limitations need to be acknowledged. This study was observational, and therefore susceptible to residual confounding and confounding by indication. Even though we adjusted for numerous available confounders, the possibility of residual confounding remains. This may be especially important in the case of patients who transitioned to dialysis using a TDC who have no maturing AVF/AVG, as catheters are the sole form of vascular access in patients who need emergent initiation of renal replacement therapy, a group that is known to experience worse outcomes.[25] Most of our patients consisted of male veterans; therefore, the results may not be generalized to other cohorts or the general population. We used administrative data for our study, which could be prone to a sampling bias and inaccurate measurement of the predictor variables. Author Manuscript In conclusion, an AVF at hemodialysis transition is associated with lower all-cause, cardiovascular and infection-related mortality across all age categories, including even the oldest incident hemodialysis patients. Using a TDC is associated with higher mortality in all age groups, even in patients with prior attempts to creating an AVF or an AVG. Our study thus suggests that a mature AVF at the time of hemodialysis transition is the preferable access even in all incident hemodialysis the most elderly patients. Further studies are needed to inform the field about the best strategies to increase AVF placement and maturation in those with advanced age. DISCUSSION AVGs may offer an acceptable and in some cases even desirable alternative to AVFs in elderly individuals, due to their more predictable and shorter maturation, thus minimizing the duration of TDC exposure and potentially decreasing the potential risks associated with the latter. Author Manuscript Acknowledgments American journal of kidney diseases : the official journal of the National Kidney Foundation. 2016; 67:Svii, S1–305. [PubMed: 26925525] 2. Vascular Access Work G. Clinical practice guidelines for vascular access. American journal of kidney diseases : the official journal of the National Kidney Foundation. 2006; 48(Suppl 1):S176– 247. [PubMed: 16813989] 3. Lacson E Jr, Wang W, Hakim RM, Teng M, Lazarus JM. Associates of mortality and hospitalization in hemodialysis: potentially actionable laboratory variables and vascular access. American journal of kidney diseases : the official journal of the National Kidney Foundation. 2009; 53:79–90. [PubMed: 18930570] Author Manuscript 4. Xue JL, Dahl D, Ebben JP, Collins AJ. The association of initial hemodialysis access type with mortality outcomes in elderly Medicare ESRD patients. American journal of kidney diseases : the official journal of the National Kidney Foundation. 2003; 42:1013–1019. [PubMed: 14582045] 5. Pisoni RL, Arrington CJ, Albert JM, Ethier J, Kimata N, Krishnan M, Rayner HC, Saito A, Sands JJ, Saran R, Gillespie B, Wolfe RA, Port FK. Facility hemodialysis vascular access use and mortality in countries participating in DOPPS: an instrumental variable analysis. American journal of kidney diseases : the official journal of the National Kidney Foundation. 2009; 53:475–491. [PubMed: 19150158] 6. Lok CE, Sontrop JM, Tomlinson G, Rajan D, Cattral M, Oreopoulos G, Harris J, Moist L. Cumulative patency of contemporary fistulas versus grafts (2000–2010). Clinical journal of the American Society of Nephrology : CJASN. 2013; 8:810–818. [PubMed: 23371955] 7. Monroy-Cuadros M, Yilmaz S, Salazar-Banuelos A, Doig C. Risk factors associated with patency loss of hemodialysis vascular access within 6 months. Clinical journal of the American Society of Nephrology : CJASN. 2010; 5:1787–1792. [PubMed: 20576823] Author Manuscript 8. Lok CE, Allon M, Moist L, Oliver MJ, Shah H, Zimmerman D. Risk equation determining unsuccessful cannulation events and failure to maturation in arteriovenous fistulas (REDUCE FTM I). Journal of the American Society of Nephrology : JASN. 2006; 17:3204–3212. [PubMed: 16988062] 9. Miller CD, Robbin ML, Allon M. Gender differences in outcomes of arteriovenous fistulas in hemodialysis patients. Kidney international. 2003; 63:346–352. [PubMed: 12472802] 10. Sumida K, Molnar MZ, Potukuchi PK, Thomas F, Lu JL, Jing J, Ravel VA, Soohoo M, Rhee CM, Streja E, Kalantar-Zadeh K, Kovesdy CP. Association of Slopes of Estimated Glomerular Filtration Rate With Post-End-Stage Renal Disease Mortality in Patients With Advanced Chronic Kidney Disease Transitioning to Dialysis. Mayo Clin Proc. 2016; 91:196–207. [PubMed: 26848002] 11. Acknowledgments Author Manuscript This study is supported by grant 5U01DK102163 from the National Institute of Health (NIH) to KKZ and CPK, and by resources from the US Department of Veterans Affairs. The data reported here have been supplied by the United States Renal Data System (USRDS). Support for VA/CMS data is provided by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Health Services Research and Development, VA Information Resource Center (Project Numbers SDR 02-237 and 98-004). CPK and KKZ are employees of the Department of Veterans affairs. The interpretation and reporting of these data are the responsibility of the authors and in no way should be seen as official policy or interpretation of the Department of Veterans Affairs or the US government. The results of this paper have not been published previously in whole or part. Nephron. Author manuscript; available in PMC 2018 May 18. Page 8 Page 8 Saleh et al. Author Manuscript 1. Saran R, Li Y, Robinson B, Abbott KC, Agodoa LY, Ayanian J, Bragg-Gresham J, Balkrishnan R, Chen JL, Cope E, Eggers PW, Gillen D, Gipson D, Hailpern SM, Hall YN, He K, Herman W, Heung M, Hirth RA, Hutton D, Jacobsen SJ, Kalantar-Zadeh K, Kovesdy CP, Lu Y, Molnar MZ, 1. Saran R, Li Y, Robinson B, Abbott KC, Agodoa LY, Ayanian J, Bragg-Gresham J, Balkrishnan R, Chen JL, Cope E, Eggers PW, Gillen D, Gipson D, Hailpern SM, Hall YN, He K, Herman W, Heung M, Hirth RA, Hutton D, Jacobsen SJ, Kalantar-Zadeh K, Kovesdy CP, Lu Y, Molnar MZ, Morgenstern H, Nallamothu B, Nguyen DV, O’Hare AM, Plattner B, Pisoni R, Port FK, Rao P, Rhee CM, Sakhuja A, Schaubel DE, Selewski DT, Shahinian V, Sim JJ, Song P, Streja E, Kurella Tamura M, Tentori F, White S, Woodside K, Hirth RA. US Renal Data System 2015 Annual Data Report: Epidemiology of Kidney Disease in the United States. American journal of kidney diseases : the official journal of the National Kidney Foundation. 2016; 67:Svii, S1–305. [PubMed: 26925525] Morgenstern H, Nallamothu B, Nguyen DV, O’Hare AM, Plattner B, Pisoni R, Port FK, Rao P, Rhee CM, Sakhuja A, Schaubel DE, Selewski DT, Shahinian V, Sim JJ, Song P, Streja E, Kurella Tamura M, Tentori F, White S, Woodside K, Hirth RA. US Renal Data System 2015 Annual Data Report: Epidemiology of Kidney Disease in the United States. Nephron. Author manuscript; available in PMC 2018 May 18. Acknowledgments VIReC Research User Guide. VHA Medical SAS Inpatient Datasets FY2006–2007. 2007. 12. Kovesdy CP, Norris KC, Boulware LE, Lu JL, Ma JZ, Streja E, Molnar MZ, Kalantar-Zadeh K. Association of Race With Mortality and Cardiovascular Events in a Large Cohort of US Veterans. Circulation. 2015; 132:1538–1548. [PubMed: 26384521] Author Manuscript 13. Deyo RA, Cherkin DC, Ciol MA. Adapting a clinical comorbidity index for use with ICD-9-CM administrative databases. J Clin Epidemiol. 1992; 45:613–619. [PubMed: 1607900] 14. Sohn MW, Arnold N, Maynard C, Hynes DM. Accuracy and completeness of mortality data in the Department of Veterans Affairs. Popul Health Metr. 2006; 4:2. [PubMed: 16606453] 15. Moist LM, Lok CE, Vachharajani TJ, Xi W, AlJaishi A, Polkinghorne KR, Vazquez M, Lee TC. Optimal hemodialysis vascular access in the elderly patient. Seminars in dialysis. 2012; 25:640– 648. [PubMed: 23173892] Saleh et al. Page 9 Page 9 16. O’Hare AM. Vascular access for hemodialysis in older adults: a “patient first” approach. Journal of the American Society of Nephrology : JASN. 2013; 24:1187–1190. [PubMed: 23813217] Author Manuscript 17. Ethier J, Mendelssohn DC, Elder SJ, Hasegawa T, Akizawa T, Akiba T, Canaud BJ, Pisoni RL. Vascular access use and outcomes: an international perspective from the Dialysis Outcomes and Practice Patterns Study. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 2008; 23:3219–3226. 18. Staramos DN, Lazarides MK, Tzilalis VD, Ekonomou CS, Simopoulos CE, Dayantas JN. Patency of autologous and prosthetic arteriovenous fistulas in elderly patients. Eur J Surg. 2000; 166:777– 781. [PubMed: 11071164] 19. Allon MLC. Dialysis fistula or graft: the role for randomized clinical trials. Clinical journal of the American Society of Nephrology : CJASN. 2010; 5:2348–2354. [PubMed: 21030576] 20. Schild AF, Perez E, Gillaspie E, Seaver C, Livingstone J, Thibonnier A. Arteriovenous fistulae vs. arteriovenous grafts: a retrospective review of 1,700 consecutive vascular access cases. The journal of vascular access. 2008; 9:231–235. [PubMed: 19085891] 21. Chan MR, Sanchez RJ, Young HN, Yevzlin AS. Vascular access outcomes in the elderly hemodialysis population: A USRDS study. Seminars in dialysis. 2007; 20:606–610. [PubMed: 17991212] Author Manuscript 22. DeSilva RN, Patibandla BK, Vin Y, Narra A, Chawla V, Brown RS, Goldfarb-Rumyantzev AS. Fistula first is not always the best strategy for the elderly. Journal of the American Society of Nephrology : JASN. 2013; 24:1297–1304. [PubMed: 23813216] 23. Quinn RR. Nephron. Author manuscript; available in PMC 2018 May 18. Nephron. Author manuscript; available in PMC 2018 May 18. Acknowledgments The Effect of Predialysis Fistula Attempt on Risk of All-Cause and Access-Related Death. JASN. 2016 24. Brown RS, Patibandla BK, Goldfarb-Rumyantzev AS. The Survival Benefit of “Fistula First, Catheter Last” in Hemodialysis Is Primarily Due to Patient Factors. Journal of the American Society of Nephrology : JASN. 2017; 28:645–652. [PubMed: 27605542] 25. Lorenzo V, Martn M, Rufino M, Hernandez D, Torres A, Ayus JC. Predialysis nephrologic care and a functioning arteriovenous fistula at entry are associated with better survival in incident hemodialysis patients: an observational cohort study. American journal of kidney diseases : the official journal of the National Kidney Foundation. 2004; 43:999–1007. [PubMed: 15168379] Author Manuscript Author Manuscript Page 10 Saleh et al. Figure 1. Cumulative event curves for overall all-cause (Panel A), cardiovascular (Panel B) and infection-related (Panel C) mortality, in patients divided by their initial vascular access type. Author Manuscript Figure 1. Figure 1. Figure 1. Cumulative event curves for overall all-cause (Panel A), cardiovascular (Panel B) and infection-related (Panel C) mortality, in patients divided by their initial vascular access type. Author Manuscript Author Manuscript Author Manuscript Saleh et al. Acknowledgments AVF) P value All-cause 1.22 (0.74–2.00) 0.4 1.77 (1.40–2.23) <0.001 2.93 (2.41–3.55) <0.001 1.33 (0.79–2.22) 0.3 1.80 (1.40–2.33) <0.001 2.76 (2.24–3.40) <0.001 CV 1.42 (0.64–3.20) 0.4 1.75 (1.17–2.62) 0.006 2.36 (1.69–3.30) <0.001 1.53 (0.64–3.68) 0.3 1.92 (1.23–3.99) 0.004 2.50 (1.72–3.65) <0.001 Infection-related - - 4.69 (1.56–13.94) 0.005 7.98 (2.93–21.71) <0.001 - - 4.28 (1.19–15.36) 0.026 8.92 (2.81–28.33) <0.001 70 All-cause 1.68 (1.19–2.36) 0.003 1.82 (1.54–2.15) <0.001 3.19 (2.78–3.67) <0.001 1.55 (1.08–2.21) 0.016 1.80 (1.50–2-13) <0.001 2.93 (2.53–3.39) <0.001 CV 1.15 (0.56–2.22) 0.7 1.83 (1.39–2.42) <0.001 2.70 (2.13–3.40) <0.001 1.10 (0.55–2.20) 0.8 1.75 (1.31–2.32) <0.001 2.37 (1.87–3.023) <0.001 Infection-related 1.51 (0.53–4.35) 0.4 1.15 (0.66–2.00) 0.6 2.23 (1.46–3.41) <0.001 1.52 (0.52–4.42) 0.4 1.09 (0.61–1.94) 0.8 2.07 (1.33–3.22) 0.001 80 All-cause 1.52 (1.20–1.94) 0.001 1.64 (1.45–1.85) <0.001 2.94 (2.61–3.41) <0.001 1.54 (1.20–1.97) 0.001 1.52 (1.34–1.73) <0.001 2.70 (2.43–3.00) <0.001 CV 1.92 (1.28–2.90) 0.002 2.01 (1.61–2.50) <0.001 3.30 (2.74–3.96) <0.001 1.96 (1.29–2.97) 0.002 1.80 (1.44–2.25) <0.001 2.94 (2.44–3.55) <0.001 Infection-related 0.96 (0.22–4.15) 0.9 3.55 (2.09–6.03) <0.01 5.78 (3.60–9.31) <0.001 1.06 (0.24–4.63) 0.9 3.56 (2.03–6.20) <0.001 5.73 (3.46–9.50) <0.001 All-cause 1.34 (1.06–1.69) 0.012 1.57 (1.39–1.76) <0.001 2.74 (2.49–3.01) <0.001 1.33 (1.05–1.68) 0.018 1.51 (1.34–1.70) <0.001 2.56 (2.32–2.82) <0.001 CV 1.13 (0.74–1.74) 0.567 1.62 (1.32–1.99) <0.001 2.77 (2.35–3.27) <0.001 1.16 (0.75–1.79) 0.5 1.51 (1.23–1.86) <0.001 2.48 (2.10–2.94) <0.001 Infection-related 1.38 (0.58–3.31) 0.5 1.98 (1.28–3.06) 0.002 3.05 (2.12–4.40) <0.001 1.27 (0.53–3.05) 0.6 1.65 (1.06–2.56) 0.027 2.58 (1.79–3.73) <0.001 are from unadjusted and multivariable adjusted Cox models (for all-cause mortality) and competing risk regression models (for cause-specific mortality). The number of events for infection-related deaths was too low in the <60 year old age group for reliable estimation b-hazard ratios. diovascular; HR, hazard ratio; CI, confidence interval; AVG, arteriovenous graft; AVF, arteriovenous fistula; TDC, tunneled dialysis catheter Author Manuscript Author Manuscript Author Manuscript Author Manuscript Table 2 Crude and multivariable adjusted hazard/subhazard ratios of all-cause, cardiovascular and infection-related mortality associated with AVG and TDC with and without matu in patient subgroups of various ages Age Group Mortality Unadjusted HR/SHR (95% CI) Adjusted HR/SHR (95% CI) AVG (vs. AVF) P value TDC, maturing AVF/AVG (vs. AVF) P value TDC, no maturing AVF/AVG (vs. AVF) P value AVG (vs. AVF) P value TDC, maturing AVF/AVG (vs. AVF) P value TDC, no maturing AVF/AVG (vs. Acknowledgments Page 11 Author Manuscript or Manuscript Author Manuscript Author Manuscript Author Manuscript Table 1 Baseline characteristics of patients categorized according to the type of vascular access used to initiate hemodialysis AVF N=8,940) AVG N=1,090 TDC, maturing AVF/AVG N=8,262 TDC, no maturing AVF/AVG N=28,494 P value Age (years) 70.2±11.5 70.7±11.9 70.3±11.7 71.0±12.0 <0.001 Sex (male) 8,582 (96) 1,121 (91) 7,797 (94) 26,903 (94) <0.001 Race (black) 2,148 (24) 462 (37) 2,073 (25) 6,814 (24) <0.001 Ethnicity (Non-Hispanic) 8,348 (93) 1,168 (95) 7,746 (94) 28,861 (94) 0.009 Pre-dialysis Nephrology care 8,187 (96) 1,020 (87) 6,101 (81) 13,596 (56) <0.001 Diabetes mellitus 5,277 (66) 743 (67) 5,138 (71) 16,308 (64) <0.001 Myocardial infarction 1,937 (24) 284 (26) 2,150 (30) 7,964 (31) <0.001 Congestive heart failure 3,984 (50) 577 (52) 4,406 (61) 15,596 (61) <0.001 Peripheral vascular disease 3,102 (39) 441 (40) 3,079 (42) 10,327 (41) 0.004 Cerebrovascular disease 2,360 (30) 378 (34) 2,348 (32) 8,219 (32) <0.001 Dementia 160 (2) 60 (5) 197 (3) 850 (3) <0.001 Chronic lung disease 3,079 (39) 470 (43) 3,282 (45) 12,137 (48) <0.001 Liver disease 819 (10) 138 (13) 819 (11) 3,199 (13) <0.001 Malignancy 1,965 (25) 291 (26) 1,749 (24) 6,803 (27) <0.001 Last estimated GFR prior to dialysis (ml/minute/1.73 m2) 11.5±4.14 12.0 ±4.4 11.7±4.7 12.4 ±5.4 <0.001 Charlson comorbidity index 4.4±2.8 4.9±3.1 4.9±2.8 4.9±3.0 <0.001 Last serum albumin prior to dialysis (g/dl) 3.4±0.60 3.3±0.63 3.2±0.63 3.0±0.67 <0.001 Last hemoglobin prior to dialysis (g/dl) 10.3±1.5 10.1±1.5 9.9±1.5 9.9±1.6 <0.001 Results presented as means ± standard deviations and number (percent). AVF, arteriovenous fistula; AVG, arteriovenous graft; TDC, tunneled dialysis catheter Nephron. Author manuscript; available in PMC 2018 May 18. Author Manuscript Nephron. Author manuscript; available in PMC 2018 May 18. Page 12 Page 12 Saleh et al. A th M i t A th M i t A th M i t A th M i t Saleh et al. Table 2 e and multivariable adjusted hazard/subhazard ratios of all-cause, cardiovascular and infection-related mortality associated with AVG and TDC with and without maturing AVF/AVG (compared to AVF) ient subgroups of various ages p Mortality Unadjusted HR/SHR (95% CI) Adjusted HR/SHR (95% CI) AVG (vs. AVF) P value TDC, maturing AVF/AVG (vs. AVF) P value TDC, no maturing AVF/AVG (vs. AVF) P value AVG (vs. AVF) P value TDC, maturing AVF/AVG (vs. AVF) P value TDC, no maturing AVF/AVG (vs. Nephron. Author manuscript; available in PMC 2018 May 18. Acknowledgments AVF) P value <60 All-cause 1.22 (0.74–2.00) 0.4 1.77 (1.40–2.23) <0.001 2.93 (2.41–3.55) <0.001 1.33 (0.79–2.22) 0.3 1.80 (1.40–2.33) <0.001 2.76 (2.24–3.40) <0.001 CV 1.42 (0.64–3.20) 0.4 1.75 (1.17–2.62) 0.006 2.36 (1.69–3.30) <0.001 1.53 (0.64–3.68) 0.3 1.92 (1.23–3.99) 0.004 2.50 (1.72–3.65) <0.001 Infection-related - - 4.69 (1.56–13.94) 0.005 7.98 (2.93–21.71) <0.001 - - 4.28 (1.19–15.36) 0.026 8.92 (2.81–28.33) <0.001 60–<70 All-cause 1.68 (1.19–2.36) 0.003 1.82 (1.54–2.15) <0.001 3.19 (2.78–3.67) <0.001 1.55 (1.08–2.21) 0.016 1.80 (1.50–2-13) <0.001 2.93 (2.53–3.39) <0.001 CV 1.15 (0.56–2.22) 0.7 1.83 (1.39–2.42) <0.001 2.70 (2.13–3.40) <0.001 1.10 (0.55–2.20) 0.8 1.75 (1.31–2.32) <0.001 2.37 (1.87–3.023) <0.001 Infection-related 1.51 (0.53–4.35) 0.4 1.15 (0.66–2.00) 0.6 2.23 (1.46–3.41) <0.001 1.52 (0.52–4.42) 0.4 1.09 (0.61–1.94) 0.8 2.07 (1.33–3.22) 0.001 70–<80 All-cause 1.52 (1.20–1.94) 0.001 1.64 (1.45–1.85) <0.001 2.94 (2.61–3.41) <0.001 1.54 (1.20–1.97) 0.001 1.52 (1.34–1.73) <0.001 2.70 (2.43–3.00) <0.001 CV 1.92 (1.28–2.90) 0.002 2.01 (1.61–2.50) <0.001 3.30 (2.74–3.96) <0.001 1.96 (1.29–2.97) 0.002 1.80 (1.44–2.25) <0.001 2.94 (2.44–3.55) <0.001 Infection-related 0.96 (0.22–4.15) 0.9 3.55 (2.09–6.03) <0.01 5.78 (3.60–9.31) <0.001 1.06 (0.24–4.63) 0.9 3.56 (2.03–6.20) <0.001 5.73 (3.46–9.50) <0.001 ≥80 All-cause 1.34 (1.06–1.69) 0.012 1.57 (1.39–1.76) <0.001 2.74 (2.49–3.01) <0.001 1.33 (1.05–1.68) 0.018 1.51 (1.34–1.70) <0.001 2.56 (2.32–2.82) <0.001 CV 1.13 (0.74–1.74) 0.567 1.62 (1.32–1.99) <0.001 2.77 (2.35–3.27) <0.001 1.16 (0.75–1.79) 0.5 1.51 (1.23–1.86) <0.001 2.48 (2.10–2.94) <0.001 Infection-related 1.38 (0.58–3.31) 0.5 1.98 (1.28–3.06) 0.002 3.05 (2.12–4.40) <0.001 1.27 (0.53–3.05) 0.6 1.65 (1.06–2.56) 0.027 2.58 (1.79–3.73) <0.001 Results are from unadjusted and multivariable adjusted Cox models (for all-cause mortality) and competing risk regression models (for cause-specific mortality). The number of events for infection-related deaths was too low in th of all sub-hazard ratios. CV, cardiovascular; HR, hazard ratio; CI, confidence interval; AVG, arteriovenous graft; AVF, arteriovenous fistula; TDC, tunneled dialysis catheter Nephron. Author manuscript; available in PMC 2018 May 18. Nephron. Author manuscript; available in PMC 2018 May 18.
https://openalex.org/W4313364515	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0279421&type=printable	English	null	Green credit and market expansion strategy of high pollution enterprises—Evidence from China	PloS one	2,022	cc-by	13,178	RESEARCH ARTICLE Green credit and market expansion strategy of high pollution enterprises—Evidence from China RESEARCH ARTICLE Qian ZhongID1, Xuemeng Ding2, Xiaoke SunID3, Hairui Zhao4 1 Department of Financial Engineering, School of Finance, Guangdong University of Foreign Studies, Guangzhou, Guangdong, China, 2 Department of Public Administration, School of Humanities and Laws, Hebei University of Technology, Tianjin, Hebei, China, 3 Department of Insurance, School of Finance, Guangdong University of Foreign Studies, Guangzhou, Guangdong, China, 4 Department of Finance, School of Economics and Law, University of Science and Technology Liaoning, Anshan, Liaoning, China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Ning Du, DePaul University, UNITED STATES Editor: Ning Du, DePaul University, UNITED STATES Received: November 4, 2021 Accepted: December 6, 2022 Published: December 30, 2022 Copyright: © 2022 Zhong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: November 4, 2021 Accepted: December 6, 2022 Published: December 30, 2022 Copyright: © 2022 Zhong 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 datas are available from the CSMAR database and we have uploaded our study’s minimal underlying data set as Supporting information files. People who need data can use Stata to open the data in the zip. Abstract This paper uses the Difference-in-Differences method to test the impact of the promulgation of Green Credit Guidelines, a market-oriented environmental regulation, on the enterprise market expansion strategy, based on the panel data of Chinese A-share listed companies from 2008 to 2015. We find that the promulgation of Green Credit Guidelines significantly inhibited the market expansion strategy of high pollution enterprises. Two channels through which the Green Credit Guidelines affect the market expansion strategies of high polluters are increasing the cost of financing and promoting green R&D. Heterogeneity analysis finds that the impact of Green Credit Guidelines on the market expansion of highly polluting enter- prises is more significant in non-state-owned enterprises and enterprises without equity incentive. Further analysis shows that the promulgation of Green Credit Guidelines dam- ages the corporate image and profitability of high polluting enterprises, but it doesn’t increase the risk of high polluting enterprises. The results of this research could help rele- vant government departments to formulate practical environmental regulations and promote sustainable economic development. OPEN ACCESS Citation: Zhong Q, Ding X, Sun X, Zhao H (2022) Green credit and market expansion strategy of high pollution enterprises—Evidence from China. PLoS ONE 17(12): e0279421. https://doi.org/10.1371/ journal.pone.0279421 Citation: Zhong Q, Ding X, Sun X, Zhao H (2022) Green credit and market expansion strategy of high pollution enterprises—Evidence from China. PLoS ONE 17(12): e0279421. https://doi.org/10.1371/ journal.pone.0279421 Editor: Ning Du, DePaul University, UNITED STATES Received: November 4, 2021 Accepted: December 6, 2022 Published: December 30, 2022 Copyright: © 2022 Zhong 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. sunxiaoke@gdufs.edu.cn PLOS ONE PLOS ONE 1. Introduction Over the past decade, China’s contribution to world economic growth has exceeded 30%, ranking first in the world. However, the long-term industrial development model of high investment, high consumption and high pollution has led to a series of problems such as exces- sive consumption of resources and environmental degradation, which has seriously affected the sustainable and healthy development of economy and social harmony [1]. As the largest carbon emitter, China’s electricity consumption is still dominated by traditional energy gener- ation, with thermal power generation contributing more than 70%. Chinese government has actively explored an effective model for green economic development but has not fundamen- tally reversed China’s development model of high energy consumption and high emission [2]. Funding: Qian Zhong would like to acknowledge the support of the Guangdong Society Project Planning (Project code: GD22YYJ02) and the Scientific Research Project of Guangdong University of Foreign Studies (Project code: 21QN22). Xiaoke Sun would like to acknowledge the support of the Ministry of Education Scientific PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 1 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises (Project code: 22YJC840025) and the Guangdong Basic and Applied Basic Research Foundation (Project code: 2022A1515111007). The key question is whether China can develop appropriate environmental regulations to align firms’ profit maximization strategy choices with the country’s environmental protection strategy choices. In other words, whether there is an appropriate environmental regulation that makes Porter’s hypothesis valid. Competing interests: The authors have declared that no competing interests exist. In essence, green finance is a way to guide funds from the pollution field to the green field with the help of the effective allocation of financial resources, so as to realize the sustainable development of ecological environment and economy. Green credit is the main form of green finance in China, which is a bank dominated economy. Since 2007, the Chinese government has been building a green credit policy system and started small-scale pilot projects. In 2012, the China Banking Regulatory Commission issued Green Credit Guidelines, marking the full implementation of green credit policies in China. In recent years, China’s major banking insti- tutions have gradually intensified their implementation of green credit. By the end of the first quarter of 2021, the green balance of 21 major Chinese banks reached 12.5 trillion yuan, rank- ing first in the world. 1. Introduction The non-performing loan ratio of green credit remaining below 0.7% in the past five years, much lower than the overall non-performing level of various loans in the same period. China’s green credit policies are already bearing fruit, and commercial banks are supporting the upgrading of industrial structure and optimizing the allocation of resources through funds. The green credit policies strongly promote the transformation and develop- ment of China’s economy. The enterprises, as for-profit organizations, aim to maximize profits. Green credit, a mar- ket-based environmental regulation, can only guide high-polluting enterprises to make a green transition by adjusting the amount of funds allocated among different enterprises [3]. How- ever, Chinese high polluters are accustomed to relying on rent-seeking to obtain monopolistic positions in their industries [4, 5]. The differential allocation policy of green credit may inten- sify the rent-seeking behavior of high-polluting enterprises and make the market strategy choice of high-polluting enterprises contradict with the government’s sustainable development strategy choice. That such a scenario is logically possible is by now beyond doubt. That such a mechanism is of quantitative importance, however, remains an empirical question. Unfortunately, the ‘micro’ observation that green credit reduces bank lending to high-polluting companies [3] and promotes innovation in high-polluting companies [5] do not constitute sufficient evidence of this mechanism. Instead, we examine this mechanism at a relatively macro level by examining the behavior of enterprise market expansion strategy selection. Specifically, this paper takes the promulga- tion of Green Credit Guidelines in 2012 as a quasi-natural experiment, we assemble a firm- level dataset including 7272 samples from 2008 to 2015 and employ the difference-in-differ- ences (DID) approach to identify causality. We find that high polluting firms reduce the rate of market expansion after green credit guidelines are issued. This implies that even if the government adopts non-coercive environ- mental regulations, high polluters still adjust their corporate strategies to match the national strategic needs. This paper also provides empirical evidence from China for the validity of Por- ter’s hypothesis. We are interested in the channels through which green credit guidelines influ- ence the strategic choices of high-polluting firms. We find that the Green Credit Guidelines increase the financing constraints and promote green R&D of high-polluting firms, all of which may inhibit high-polluting firms from adopting market-expanding strategies. We find that the Guidelines would be more influential if the firm is non-SOE or does not have equity incentives. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 (Project code: 22YJC840025) and the Guangdong Basic and Applied Basic Research Foundation (Project code: 2022A1515111007). Competing interests: The authors have declared that no competing interests exist. 1. Introduction At the same time, we also found that although the green credit guidelines reduced the prof- itability of highly polluting enterprises, they did not increase the risk of highly polluting 2 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Green credit and market expansion strategy of high pollution enterprises enterprises. This shows that a well-designed policy, such as the Green Credit Guidelines, can realize the green transformation of the economy on the premise of stabilizing market risk. This paper contributes to our understanding of the role of the Green Credit Guidelines. We study the impact of green credit policy on the company from the perspective of enterprise stra- tegic deployment. The existing literature mainly investigates the impact of green credit policy on corporate behavior at specific levels [6, 7]. This paper focuses on the choice of corporate strategy in the strategic management theory. We investigate the impact of market-oriented environmental regulation on the company’s strategic choice, reveal the internal relationship between environmental protection policy and company behavior from a deeper level, and enrich the research in the field of economic consequences of environmental protection policy. At the same time, this paper is also a beneficial attempt to the interdisciplinary research of finance, management and environmental protection. This paper links the research on the influencing factors of the company’s strategic choice with the national strategy of environmental protection. The external environment and the company’s strategic choice have been discussed for a long time. Existing literature has focused on the impact of political, economic, institutional and other macro-environments as well as the industrial environment on the company’s strategic choices. This article takes the promul- gation of China’s Green Credit Guidelines as the starting point, and extends the research on the factors affecting the company’s strategic choices to the fields of environmental protection and world sustainable development. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 2. Institutional background As early as the beginning of reform and opening up, the Chinese government has paid atten- tion to environmental protection issues. In 1995, the People’s Bank of China issued the "Notice on Issues Related to Implementing Credit Policies and Strengthening Environmental Protec- tion Work". The Notice requires financial institutions to include the environmental friendli- ness of enterprises in credit review conditions. This is China’s first document on green credit policies. However, the primary development goal at that time was economic growth, the con- tradiction between economic growth and environmental protection was not yet prominent. The green credit was not given enough attention. In 2007, the People’s Bank of China and the environmental protection department jointly issued the "Opinions on Implementing Environ- mental Protection Policies and Regulations to Prevent Credit Risks". The Opinions clearly stated that commercial banks should support environmental protection and control credit to polluting enterprises as an important part of fulfilling their social responsibilities. However, the green credit policy has not been effectively implemented, due to the lack of corresponding supporting measures and supervision mechanisms. On February 24, 2012, the China Banking Regulatory Commission issued the "Green Credit Guidelines". The Guidelines make specific arrangements for the green credit business of bank- ing financial institutions and the supervision of regulators from six aspects: organization man- agement, policy and system construction, process management, internal control management and information disclosure, supervision and inspection. The Green Credit Guidelines are applicable to policy banks, commercial banks, rural cooperative banks, and rural credit coop- eratives legally established in China. The Guidelines require banking financial institutions to formulate their own green credit policies in order to strengthen the management of enter- prises’ environmental and social responsibility. The Guidelines also ask banking financial institutions to invest more in key areas such as green economy, low-carbon economy and cir- cular economy. The Guidelines clearly states that the regulatory authorities must comprehen- sively evaluate the green credit performance of banking financial institutions, and take the PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 3 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises evaluation results as an important basis for the regulatory rating, institutional access, business access and senior executives’ performance evaluation of banking financial institutions in accordance with relevant laws and regulations. The "Green Credit Guidelines" is China’s first normative document specifically aimed at green credit policies. 3.1 Literature review More and more studies have found that environmental pollution will affect climate change and human health [8–11], so countries have promulgated policies to promote sustainable development. The promulgation of the Green Credit Guidelines in 2012 provided practical guidance for Chinese financial institutions on how to effectively carry out green credit and support the establishment of a low-carbon circular development industrial system. So far, a large number of literatures have carried out research on the effect of green credit. The existing literature mainly focuses on the following two aspects: One is to analyze the risks and uncer- tainties of green credit from the macro level. For example, Shen and Ma [12] believe that the promotion of green credit must solve the contradiction between environmental protection and the maximization of GDP performance. Zhao et al. [13] find that the relationship between environmental regulation and green total factor productivity is complex, which is reflected in the differences and nonlinearity between cities with different monitoring levels and different economic development levels. Liu et al. [14] calculate the total factor productivity of the green economy and find that green finance promotes high-quality economic development through technological innovation and industrial structure upgrading. The other is to analyze the effect of green credit policy implementation from a micro level. For example, Liu et al. [15] finds that green credit policy can help reduce the output of papermaking and chemical enterprises in the short to medium term by using CGE model. Wang et al. [7] use the DID model to find that green credit policies inhibit the financing of high-polluting companies. Much of the exist- ing literature focuses on the impact of Green Credit Guidelines on corporate financing behav- ior, investment behavior, and R&D behavior at the micro level. But all these behaviors are only a slice of the corporate strategy. The existing literature does not sufficiently explain what impact the Guidelines will have on the strategic choices of high polluting firms. In management, there are many ways to divide corporate strategy. Among them, the classi- fication method of Miles and Snow [16] can cover other mainstream strategic classifications and is easy to measure. The classification method of Miles and Snow [16] is widely accepted by scholars as a result. According to the definition of Miles and Snow [16], corporate strategy is divided into offensive, analytical and defensive. 2. Institutional background It defines the framework of China’s green credit policy system and is of great significance to the development of green credit and green finance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 3.1 Literature review Companies implementing different strategies have significant differences in business models and organizational structures. Offensive com- panies are keen on product creation, technology research, and market development. The offensive company’s management team changes frequently and the employee turnover rate is relatively high. Unlike offensive companies, defensive companies usually focus on several existing products or services, and pay more attention to how to reduce product costs and improve production efficiency, lack of enthusiasm for new business or new market expansion. The management team in defensive companies is more stable, and the average tenure of employees is relatively long. The characteristics of analytical companies are somewhere in between. In fact, the company’s strategy is neither good nor bad, only suitable or not [16]. If the com- pany wants to achieve sustainable development, it needs to make its own strategic choices. The 4 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Green credit and market expansion strategy of high pollution enterprises external environment is an important factor affecting the company’s strategic choice. Chandler [17] first proposes that the company cannot change the external environment, and its strategy must adapt to the current situation and change trend of the external environment. Learned et al. [18] further points out on the basis of Chandler [16] that the process of strategic selection is essentially a process of matching the company’s internal characteristics with the external environment, and proposes the SWOT model. Porter [19] put forward the theory of competi- tive strategy, holding that the industrial structure is the key factor determining the company’s profitability. Institutional theory (Meyer and Rowan [20]) also believes that organizational behavior actively adapts to the external institutional environment, so the company’s strategic choice will also be affected by the institutional environment. China’s 14th Five-Year Plan puts forward the long-term goal of basically realizing socialist modernization by 2035: widely forming a green production and lifestyle, steadily decreasing carbon emissions after reaching the peak, fundamentally improving the ecological environ- ment, and basically realizing the goal of building a beautiful China. It shows that environmen- tal protection and sustainable development have been highly valued by the Chinese government. Therefore, environmental protection is also an external environmental factor that cannot be ignored when the management chooses the company’s strategy in recent years. 3.2 Theoretical analysis Rational enterprises will make discretionary choices according to their own conditions, facing the constraints brought by market-oriented environmental regulation. The self-selection behavior of enterprises is heterogeneous: reduce production scale or even withdraw from the market, upgrade products to reduce pollution or transform [21]. Due to financial and technical constraints, small enterprises cannot internalize the costs caused by environmental regulation. Shutdown has become a common means to deal with environmental regulation for small enterprises. The enterprises, with a certain capital and technological foundation, may adjust their production lines and actively innovate technology to achieve energy conservation, emis- sion reduction and industrial upgrading. The promulgation of green credit guidelines shows the Chinese government’s determination to develop green finance and govern the environ- ment. The Guidelines strengthens the bank’s supervision of highly polluting enterprises, increases the probability of negative information from high-polluting companies being exposed, and restrains the occurrence of opportunism [22]. All in all, the promulgation of the Green Credit Guidelines, as a market-oriented environmental regulation, will change the sur- vival mode of high polluting enterprises—making profits by polluting the environment. At this time, the company’s management needs to review the situation and make adjustments to its strategic choices to fit the current China’s national strategy of achieving a beautiful China. Regardless of whether companies choose to shut down high-polluting industries or carry out green upgrades to the industrial chain, they will not adopt a radical market expansion strategy in the short term. Therefore, we propose the following hypothesis: Hypothesis 1 (H1): The promulgation of the Green Credit Guidelines inhibits the market expan- sion strategy of high-polluting companies. Hypothesis 1 (H1): The promulgation of the Green Credit Guidelines inhibits the market expan- sion strategy of high-polluting companies. According to the green credit guidelines, commercial banks strictly control the credit threshold and take the environmental compliance of enterprises as an important condition for granting loans. The supply of credit to highly polluting enterprises decreased. The financing cost of highly polluting enterprises increased, and bank loans, especially long-term loans, decreased significantly [23, 24]. Modigliani Miller theory [25, 26] points out that the company value depends on the company’s investment decision, and has nothing to do with the financing 5 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Green credit and market expansion strategy of high pollution enterprises decision and capital structure. However, in actual business activities, enterprises often have over investment and under investment [27]. The existing literature find that enterprises can supplement free cash flow through debt financing [28], overinvest with free cash flow [29, 30]. Environmental protection regulation reduces the over investment of high pollution enterprises by limiting the financing capacity [31]. In other words, after the promulgation of the green credit guidelines, the financing costs of high polluting enterprises have increased, over invest- ment has decreased, and the market expansion strategy has become defensive. Therefore, we propose the following hypothesis: Hypothesis 2 (H2): The promulgation of Green Credit Guidelines inhibits the market expansion of high polluting enterprises by increasing the financing cost. As a market-based environmental regulation, the Green Credit Guidelines can provide more flexible and effective innovation incentives [32, 33]. The Green Credit Guidelines require banks to regularly conduct internal control inspections and evaluations of green concepts such as environmental protection, conservation, and sustainable development on loans. The Guide- lines also require banks to provide preferential lending policies to companies that meet the conditions for environmental protection and sustainable development. In order to obtain the economic benefits of preferential loans, companies seeking to maximize profits will increase the R & D of green innovative technologies. Green innovation can bring new competitive advantages to enterprises, because green environmental protection and sustainable develop- ment are advocated all over the world [34, 35]. For companies with strong innovation capabili- ties, their innovation activities often have the characteristics of sustainability. The innovation activities of enterprises with strong innovation ability are often sustainable. Hypothesis 1 (H1): The promulgation of the Green Credit Guidelines inhibits the market expan- sion strategy of high-polluting companies. Enterprises with relatively weak innovation ability are more vulnerable to external factors such as subsidies and rewards due to limited relevant innovation resources [36]. All in all, the promulgation of the Green Credit Guidelines will guide companies to increase green innovation R & D. Technol- ogy research and development business enhance the company’s growth, but also increase the company’s performance risk [37, 38]. To reduce the uncertainty of the company in the future, companies that increase R & D may reduce the scale of market expansion in the short term, that is, the R & D tendency will crowd out the company’s market expansion tendency. Based on this, we propose following hypothesis: Hypothesis 3 (H3): The promulgation of Green Credit Guidelines inhibits enterprise market expansion by encouraging green R & D of highly polluting enterprises. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 4.2. Model setting and definition of variables We use a Difference-in-Differences model to test the influence of Green Credit Guidelines on enterprises’ market expansion strategy, following Liu et al. (2019). A standard Difference-in- Differences (DID) model is as follows: This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. ð1Þ yi;t ¼ a0 þ a1du þ a2dt þ a3ðdu dtÞ þ a4control þ εi;t ð1Þ du is a dummy variable to measure whether enterprises are affected by policies. If firm i is affected by the policy du = 1, otherwise du = 0. dt is a dummy variable representing whether the policy is implemented. dt is 0 before policy is implemented and 1 after policy is imple- mented. εi,t is the error term, which is assumed to be normally distributed with zero mean value and constant variance according to the existing literature [39–41]. When we use the bidirectional fixed effects DID model, du will be absorbed by individual fixed effect, dt will be absorbed by the time fixed effect. Therefore, the DID model is set as fol- lows: ð2Þ Strategyi;t ¼ b0 þ b1ðPollutei PolicytÞ þ b2control þ li þ ut þ εi;t ð2Þ Strategy is the variable we use to measure the company’s market expansion strategy. Refer- ring to the existing literature [42, 43], we use this method to measure the company’s market expansion strategy. In the first step, we calculate the ratio of the sum of the company’s sales expenses and management expenses to the operating revenue as the proxy index of the com- pany’s market expansion tendency. In the second step, we calculate the average value of the company’s market expansion tendency in the past five years and sort it into six groups from small to large by industry-year. Assign 1 to the minimum value group, 2 to the secondary small value group. . .. . . and 6 to the maximum value group. The higher the score, the more offensive the company’s expansion strategy. Compared with the method of directly using financial indicators, this method has the fol- lowing advantages: 1. This indicator covers the company’s resource allocation and operating results in the past five years, and reflects the company’s long-term action plan and imple- mented strategy. 2. 4.1. Data source This paper takes Chinese A-share listed companies from 2008 to 2015 as research samples, and the data required are from the CSMAR database. In this paper, the samples are processed as follows: 1. Eliminate the sample of financial listed companies; 2. Eliminate the sample of com- panies that have been listed for less than five years, because we need five years of financial data to calculate the company’s market expansion tendency. 3. Eliminate the samples with missing data in variables. 4. Eliminate companies with a leverage ratio of more than 1. 5. Winsorize the continuous variables at the 1% level. The promulgation of the Green Credit Guidelines is a completely exogenous event for enterprises. According to the specific content of the Green Credit Guidelines, the Guidelines only affect the credit policies and corporate social responsibilities of high-polluting companies. Therefore, we take high-polluting companies as the treated group and other companies as the PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 6 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises control group, use the DID (Difference-in-Difference) model to analyze the impact of green credit policies on corporate expansion strategies. We consider these companies as high-pollut- ing companies: companies in the 14 high-polluting industries disclosed in the "List of Listed Companies Environmental Inspection Industry Classification Management Directory" issued by the Ministry of Environmental Protection in June 2008. The 14 heavily polluting industries are: thermal power, steel, cement, electrolytic aluminum, coal, metallurgy, building materials, mining, chemicals, petrochemicals, pharmaceuticals, light industry, textiles and tanning. We use the "List of Listed Companies Environmental Inspection Industry Classification Manage- ment Directory" promulgated in June 2008 for the following reasons: First, this is the classifica- tion issued by the Ministry of Environmental Protection of China, which is authoritative. Second, this is a classification issued before the Green Credit Guidelines and before the sample research time of this article. Using this classification can effectively solve the problem of sam- ple selection. We finally identified 7272 enterprise-annual samples, including 1032 high-polluting enter- prise samples and 6,240 control group samples. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 4.2. Model setting and definition of variables Using this indicator can avoid the possible endogenous problems caused by the direct use of financial indicators. 3. We can calculate the corporate strategy of each PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 7 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises Table 1. Descriptions of variables. Variable Description Pollute 1 for highly polluting enterprises, 0 for non-highly polluting enterprises Policy 0 for policies issued during 2008–2011, 1 for policies issued during 2012–2015 Strategy Score on the company’s market expansion tendency. Calculate the ratio of the sum of the company’s sales expenses and administrative expenses to the company’s operating revenue as the company’s market expansion tendency. Calculate the average value of the company’s market expansion tendency in the past five years, sort the values from small to large and divide them into 6 groups by industry-year. Assign 1 to the minimum value group, 2 to the secondary small value group, and so on, and 6 to the maximum group. size The natural logarithm of the enterprise’s assets at the end of the year age The year of the current year minus the year of listing. leverage Total liabilities/total assets. ROE Net income/equity. SOE 1 for a state-owned enterprise, 0 for others. Top5Hold Shareholding ratio of the top five shareholders. board Number of directors Indep Number of independent directors / number of board members MgAge Average age of non-independent directors and executives Indpos Operating income of the company / operating income of the whole industry https://doi.org/10.1371/journal.pone.0279421.t001 https://doi.org/10.1371/journal.pone.0279421.t001 company, which is conducive to large sample empirical research. In the robustness test part, we also use the market position in the next period and the growth rate of sales scale in the next period to measure the enterprise’s market expansion strategy. Pollute × Policy is the main explanatory variable in the Difference-in-Difference model. Pol- lute is a virtual variable that indicates whether the company is high-polluting. Pollute = 1 when a company is high-polluting, otherwise Pollute = 0. Policy is a virtual variable that measures the promulgation of Green Credit Guidelines. Policy = 0 if samples are in 2008 to 2011, Policy = 1 if samples are in 2012 to 2015. The coefficient of Pollute × Policy measures the net impact of Green Credit Guidelines on the market expansion strategy choice of high-pollution enterprises. control is the control variable. 4.2. Model setting and definition of variables We control the following main characteristics that may affect corporate strategy in Formula 2: company size (size), leverage (leverage), return on equity (ROE), listing years (age), state owned enterprise (SOE), ownership concentration (Top5Hold) and industry position (Indpos). In addition, corporate strategic decisions are mainly decided by the board of directors and the management. Therefore, we control the variables related to the characteristics of the board of directors and management: board size (Board), proportion of independent directors (Indep) and mean age of the management (MgAge). See Table 1 for a description of the variables. PLOS ONE Green credit and market expansion strategy of high pollution enterprises PLOS ONE Table 2. Descriptive statistics. variable N mean p50 sd min max Strategy 7272 3.043 3.000 1.344 1.000 6.000 size 7272 22.288 22.150 1.226 18.831 25.827 age 7272 12.171 12.000 4.594 5.000 23.000 leverage 7272 0.504 0.512 0.195 0.073 0.993 ROE 7272 0.077 0.076 0.138 -1.033 0.570 SOE 7272 0.595 1.000 0.491 0.000 1.000 Top5Hold 7272 49.832 49.790 15.271 16.920 86.320 board 7272 9.068 9.000 1.815 5.000 15.000 Indep 7272 0.368 0.333 0.053 0.250 0.571 MgAge 7272 47.848 47.944 3.075 39.273 55.133 Indpos 7272 0.079 0.013 0.221 0.000 1.529 Note: All monetary terms are denominated in Chinese Yuan (CNY) p50 refers to the 50% quantile sd represents the standard deviation max and min represent the Note: All monetary terms are denominated in Chinese Yuan (CNY). p50 refers to the 50% quantile, sd represents the standard deviation, max and min represent the maximum and minimum values respectively. value is 0.595, indicating that China’s state-owned enterprises are slightly more than non- state-owned enterprises. The median of Top5hold is 49.79, indicating that nearly half of the companies’ top five shareholders hold more than 50% of the shares. The mean of board is 9.068, indicating that the average number of shareholders of listed companies is 9. The mean of MgAge is 47.8, indicating that most of the directors and executives of listed companies are middle-aged. The median of Indpos is 0.013 and the average is 0.079, indicating that most industries in China are close to a perfectly competitive market. p y p Table 3 shows the differences between the treated group and the control group before and after the policy is promulgated. We can see from the table that the mean value of Strategy in high polluting enterprises after the promulgation is 2.769, which is significantly lower than 3.126, the mean value before the promulgation. This shows that high polluting enterprises have significantly reduced the market expansion tendency after the promulgation of Green Credit Guidelines. However, there is no significant difference in the Strategy of non-highly Table 3. Two-group testing of the difference of the means. , and represent significance levels of 1%, 5% and 10%, respectively. MeanDiff represents the difference of means. https://doi.org/10.1371/journal.pone.0279421.t002 Note: , and represent significance levels of 1%, 5% and 10%, respectively. MeanDiff represents the difference of means. https://doi.org/10.1371/journal.pone.0279421.t003 https://doi.org/10.1371/journal.pone.0279421.t003 Note: Note: , and represent significance levels of 1%, 5% and 10%, respectively. MeanDiff represents the difference of means. 5.1 Descriptive statistics Table 2 reports the descriptive statistical results of the main variables. The average value of strategy is slightly larger than the median, indicating that there are certain companies with a strong tendency to expand. The standard deviation of strategy is 1.347, indicating that different companies have different market expansion propensities. The standard deviation of size is 1.226, indicating that the size of the assets of each enterprise is quite different. The mean of age is 12.17, indicating that most listed companies have been established for more than 10 years. The mean of lev is 0.504, indicating that most companies have low debt ratios. The average soe 8 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 Note: All monetary terms are denominated in Chinese Yuan (CNY). p50 refers to the 50% quantile, sd represents the standard deviation, max and min represent the maximum and minimum values respectively. nt significance levels of 1%, 5% and 10%, respectively. MeanDiff represents the difference of means. %, 5% and 10%, respectively. MeanDiff represents the difference of means. PLOS ONE Treated Control Before After MeanDiff Before After MeanDiff Strategy 3.126 2.769 0.357 3.037 3.085 -0.048 size 22.290 22.675 -0.385 22.166 22.429 -0.263 age 11.311 13.055 -1.744 12.147 12.645 -0.498 leverage 0.519 0.478 0.041 0.519 0.467 0.052 ROE 0.075 0.028 0.047 0.088 0.071 0.016 SOE 0.680 0.588 0.092 0.631 0.444 0.187 Top5Hold 49.761 52.346 -2.585 49.409 49.974 -0.565 board 9.324 8.968 0.356 9.134 8.737 0.397 Indep 0.363 0.372 -0.010 0.367 0.372 -0.005 MgAge 47.731 49.060 -1.329 47.557 48.188 -0.632 Indpos 0.038 0.054 -0.016 0.083 0.116 -0.033 Table 3. Two-group testing of the difference of the means. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 9 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises polluting companies before and after the promulgation of the policy, only from 3.037 to 3.085. So non-highly polluting enterprises have a slight tendency of market expansion, but the change is not significant. The above information shows that the promulgation of the Green Credit Pol- icy does have a negative impact on the market expansion strategy of the experimental group companies. This impact needs to be further studied using the DID model. , and represent significance levels of 1%, 5% and 10%, respectively. nd represent significance levels of 1%, 5% and 10%, respectively. , and represent significance levels of 1%, 5% and 10%, respectively. https://doi.org/10.1371/journal.pone.0279421.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 https://doi.org/10.1371/journal.pone.0279421.t004 5.2. Analysis of regression results To verify Hypothesis 1: Green Credit Guidelines will inhibit the market expansion strategy of high-polluting companies, we conduct an empirical study according to Formula 2. The regres- sion results are shown in Table 4. The first column is the two-way fixed effect model with only Pollute × Policy. The coefficient of Pollute × Policy is -0.2453, statistically significant at the 1% level. This shows that the promulgation of the Green Credit Guidelines has significantly inhib- ited the market expansion strategy of high-polluting companies. Hypothesis 1 holds. The green credit policy is an effective environmental protection policy. The second to fourth col- umns are the regression results after adding other control variables, the coefficient of Pollute × Policy is still significantly negative. Table 4. Green Credit Guide and enterprise market expansion strategy. (1) (2) (3) (4) Pollute×Policy -0.2453 -0.1229 -0.1630 -0.1638 (0.064) (0.062) (0.062) (0.062) size 0.5176 0.4470 0.4509 (0.042) (0.045) (0.045) age -1.1952 -1.0016 -0.9577 (0.267) (0.268) (0.268) leverage 0.8297 0.9685 0.9530 (0.157) (0.157) (0.157) ROE 1.4472 1.3990 1.3905 (0.117) (0.117) (0.117) SOE -0.0767 -0.0600 (0.117) (0.117) Top5Hold 0.0162 0.0159 (0.002) (0.002) board 0.0090 0.0079 (0.015) (0.016) Indpos -0.5110 -0.4912 (0.206) (0.206) Indep -0.2077 (0.400) Mgage -0.0360 (0.011) Constant 3.3308 1.4134 0.4498 1.7656 (0.042) (2.379) (2.383) (2.417) Observations 7,272 7,272 7,272 7,272 R-squared 0.037 0.108 0.117 0.119 Number of stkcd 1,727 1,727 1,727 1,727 Firm&Year YES YES YES YES Table 4. Green Credit Guide and enterprise market expansion strategy. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 10 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises We can also find from the regression results that the coefficient of size is significantly positive, indicating that the larger the scale of the company, the more motivated it is to adopt a radical market expansion strategy. The coefficient of age is significantly negative, which means that the younger the company is, the more likely it is to adopt aggressive expansion strategies. There is a significant positive correlation between leverage and Strategy, which means that external financ- ing is an important source of funds for the market expansion of the Chinese enterprise. The more profitable companies are, the more motivated they are to adopt aggressive strategies, because ROE and Strategy are significantly positively correlated. 5.3 Parallel trend test and placebo test The DID method needs to satisfy the parallel trend assumption. That is, the trend of Strategy in the treated group is not statistically significant different from the trend in the control group, while there is a significant difference in the trends of the two after the policy. Refer to Moser and Voena [44], new time dummy variables are set respectively: from 2 years before the policy to 3 years after the intervention (the dummy variable for time in the first year before the policy, as the baseline variable, has been dropped), multiply the new time dummy variable by the dummy variable stand for the high-polluting companies, then it is incorporated into the DID regression model. The regression results are plotted in Fig 1. From Fig 1, we can see that before the Green Credit Guidelines are issued, there is no significant difference between the market expansion strategies of the control group and the treated group. However, the market expan- sion strategy tendency of the treated group is significantly lower than that of the control group from the year the Guidelines are issued. This shows that the DID model in this paper satisfies the assumption of parallel trend testing. The text continues here. p p g We also performed a placebo test to exclude other factors. We consider a possibility: the decline in the aggressiveness of enterprise market expansion strategy is caused by the decline of China’s economic growth, rather than the promulgation of Green Credit Guidelines. To eliminate this potential possibility, we constructed a virtual policy occurrence time before the actual policy occurrence (before 2012). Columns 1 and 2 of Table 5 are the regression results using did two-way fixed effect when the virtual policy time is 2010 and 2011 respectively. We can see that when the construction time of the virtual policy is 2010 and 2011, the coefficient of the interaction term is not significant. Further, we split the sample into 2008–2012 and 2013–2015, and assume that there are two virtual exogenous shocks in 2010 and 2014 respec- tively. We retest whether the market expansion strategy of high polluting enterprises has changed after the virtual exogenous impact. The results are listed in columns (3) and (4) of Table 5 respectively. The two exogenous virtual shocks still have no significant impact on the market expansion strategy of high pollution enterprises. 5.2. Analysis of regression results The Top5hold’s coefficient is sig- nificantly positive, that is, a company with a higher degree of equity concentration is more likely to aggressively expand the market. Interestingly, the negative coefficient of MgAge tells us that the older the managements, the more defensive a company’s business strategy, which may be related to the calm and moderate personality of the Chinese elders. Indpos and Strategy are sig- nificantly negatively correlated, indicating that companies with a higher market share tend to be more prudent in operation, while companies with a lower market share have a strong tendency to market expansion. The regression results in Table 4 are consistent with the actual situation. 5.3 Parallel trend test and placebo test The results in Table 5 show that there are no other time factors driving the benchmark results in this paper. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Table 5. Placebo test. (1) (2) (3) (4) policy = 2010 policy = 2011 2008–2012 2013–2015 Pollute×Policy -0.0105 -0.0263 0.0460 -0.2747 (0.047) (0.049) (0.050) (0.420) size 0.4579 0.4578 0.4981 0.2032 (0.045) (0.045) (0.067) (0.117) age -0.9854 -0.9834 -0.1639 0.0319 (0.268) (0.268) (0.017) (0.420) leverage 0.9617 0.9589 0.7290 0.8864 (0.158) (0.158) (0.224) (0.378) ROE 1.4054 1.4038 0.7586 -0.3866 (0.117) (0.117) (0.140) (0.288) SOE -0.0659 -0.0660 0.1594 -0.6674 (0.117) (0.117) (0.144) (0.471) Top5Hold 0.0154 0.0155 0.0077 0.0012 (0.002) (0.002) (0.004) (0.006) board 0.0089 0.0088 -0.0141 0.0283 (0.016) (0.016) (0.021) (0.043) Indpos -0.4619 -0.4658 -0.3931 -0.1101 (0.206) (0.206) (0.279) (0.834) Indep -0.2301 -0.2299 -0.2778 0.1639 (0.400) (0.400) (0.521) (0.977) Mgage -0.0358 -0.0358 -0.0144 0.0202 (0.011) (0.011) (0.015) (0.031) Constant 1.8525 1.8363 -6.0324 -3.1714 (2.418) (2.419) (1.431) (5.908) Observations 7,272 7,272 4,062 3210 R-squared 0.118 0.118 0.084 0.051 Number of stkcd 1,727 1,727 1,221 1,577 Firm&Year YES YES YES YES Table 5. Placebo test. , and represent significance levels of 1%, 5% and 10%, respectively. market expansion strategy is also significantly negative, indicating the looser the monetary policy, the more aggressive market expansion strategy is. market expansion strategy is also significantly negative, indicating the looser the monetary policy, the more aggressive market expansion strategy is. 3. We also try to eliminate the interference of other factors on causal identification in this paper. We consider such a situation: the market expansion strategy of high pollution enter- prises is different from that of non-high pollution enterprises. This difference may not be brought about by the green credit policy, but an inevitable choice in the process of enter- prise development. Since it is mainly up to the management to decide whether the company will carry out an aggressive market expansion strategy, we use the PSM-DID to try to match individuals similar to the management indicators of high polluting enterprises in non-high polluting enterprises as the control group, and then conduct did test. The regression results are listed in column (4) of Table 6. we use the propensity matching score method (PSM) to find the non-high pollution enterprises, which match the control group in non-high pollu- tion enterprises as much as possible, whose management indicators are similar to those of 3. We also try to eliminate the interference of other factors on causal identification in this paper. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 5.4 Robustness test To ensure the robustness and reliability of the research conclusions, we replace the proxy vari- ables, change the sample, and do PSM-DID (propensity score matching-DID). 11 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Green credit and market expansion strategy of high pollution enterprises Fig 1. Parallel trend test. https://doi.org/10.1371/journal.pone.0279421.g001 Fig 1. Parallel trend test. https://doi.org/10.1371/journal.pone.0279421.g001 1. Replace the variables that measure the market expansion strategy. In Table 4, we calculate the average value of the market expansion tendency of each company in the past five years, rank them from small to large by industry-year, and divide them into six groups to measure the market expansion strategy. Here, we measure the market expansion strategy according to the market share of enterprises in the future. That is, we use the market position (F. Indpos) and sales scale (F. ln(revenue)) of enterprises in the next period to measure the mar- ket expansion strategy of enterprises in the current period. The definition of market posi- tion (lndpos) is shown in Table 1. The sales scale of an enterprise is measured by the logarithm of its operating income. The results are listed in columns (1) and (2) of Table 6, respectively. The coefficient of Pollute × Policy is still significantly negative. 2. After the financial crisis in 2008, China launched a series of economic stimulus policies, such as the implementation of the "four trillion" fiscal stimulus policy, the reduction of loan interest rates for five times and deposit interest rates for four times. In order to eliminate the potential impact of macroeconomic factors on the enterprise’s market expansion strat- egy, we add the SHIBOR (Shanghai interbank offered rate) as the control variable and re estimated formula 2, shown in column (3) of Table 6. The coefficient of Pollute × Policy is significantly negative, hypothesis 1 holds. The impact of interest rate on the company’s PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 12 / 24 Green credit and market expansion strategy of high pollution enterprises PLOS ONE Table 6. Robustness test. (1) (2) (3) (4) F.IndPos F. ln(revenue) 2009–2015 PSM-DID Pollute×Policy -0.0185 -0.1332 -0.1638 -0.1682 (0.004) (0.029) (0.062) (0.062) size 0.0292 0.4370 0.4515 0.4515 (0.003) (0.021) (0.046) (0.046) age 0.0288 -0.3038 -0.9614 -0.9614 (0.033) (0.232) (0.268) (0.268) leverage 0.0025 0.5232 1.0415 1.0415 (0.010) (0.070) (0.162) (0.162) ROE 0.0401 0.3128 1.3651 1.3651 (0.008) (0.054) (0.124) (0.124) SOE 0.0137 0.0789 -0.0870 -0.0870 (0.007) (0.051) (0.122) (0.122) Top5Hold 0.0004 0.0006 0.0154 0.0154 (0.000) (0.001) (0.002) (0.002) board -0.0022 -0.0027 0.0067 0.0067 (0.001) (0.007) (0.017) (0.017) Indpos 0.4075 -0.4820 -0.4820 (0.105) (0.230) (0.230) Indep 0.0791 -0.3809 -0.1148 -0.1148 (0.023) (0.166) (0.417) (0.417) Mgage 0.0005 0.0042 -0.0345 -0.0345 (0.001) (0.004) (0.011) (0.011) Rate -13.8344 (4.350) Constant -0.9071 14.0922 57.5534 1.6017 (0.295) (2.084) (19.811) (2.423) Observations 4,872 4,872 7,272 7,018 R-squared 0.141 0.429 0.116 0.116 Number of stkcd 1,303 1,303 1,727 1,723 Firm&Year YES YES YES YES Table 6. Robustness test. highly polluting enterprises, and take these matched non-highly polluting enterprises as the control group. We re regressed formula 2 with the new control group and treated group. The regression results are listed in column (4) of Table 6. The conclusion of this paper is robust: the promulgation of Green Credit Guidelines inhibits the market expansion ten- dency of high pollution enterprises. PLOS ONE We consider such a situation: the market expansion strategy of high pollution enter- prises is different from that of non-high pollution enterprises. This difference may not be brought about by the green credit policy, but an inevitable choice in the process of enter- prise development. Since it is mainly up to the management to decide whether the company will carry out an aggressive market expansion strategy, we use the PSM-DID to try to match individuals similar to the management indicators of high polluting enterprises in non-high polluting enterprises as the control group, and then conduct did test. The regression results are listed in column (4) of Table 6. we use the propensity matching score method (PSM) to find the non-high pollution enterprises, which match the control group in non-high pollu- tion enterprises as much as possible, whose management indicators are similar to those of PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 13 / 24 Green credit and market expansion strategy of high pollution enterprises 6.1 Mechanism analysis The above results show that the promulgation of green credit policy can effectively inhibit the market expansion strategy of high pollution enterprises. What is the specific mechanism of this inhibition? The previous analysis shows that the green credit policy will increase the PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 14 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises financing cost of high polluting enterprises and encourage enterprises to carry out emission reduction R & D, so as to reduce the aggressive market expansion strategy in the short term. These two mechanisms will be tested separately below. and represent significance levels of 1%, 5% and 10%, respectively. https://doi.org/10.1371/journal.pone.0279421.t007 Note: , and represent significance levels of 1%, 5% and 10%, respectively. gnificance levels of 1%, 5% and 10%, respectively. Financing constraints Similar to Peng et al. [31], we believe that it is more difficult for highly polluting enterprises to obtain bank loans after the promulgation of Green Credit Guidelines. The guidelines increase the external financing cost of highly polluting enterprises. We first measure the financial cost of an enterprise by the ratio of its financial expenses to its operating income. We test whether the promulgation of Green Credit Guidelines increases the external financing cost of high polluting enterprises according to formula 3. The regression results are listed in column 1 of Table 7. The coefficient of Pollute × Policy to cost is signifi- cantly positive, which shows that the promulgation of Green Credit Guidelines increases the financial cost of high polluting enterprises. Table 7. Financing constraint perspective. (1) (2) (3) (4) (5) cost High cash flow Low cash flow High KZ Low KZ Pollute×Policy 0.0052 -0.2026 0.0556 -0.0181 -0.2875 (0.002) (0.088) (0.103) (0.099) (0.092) size 0.0032 0.4771 0.4605 0.3390 0.5339 (0.001) (0.072) (0.070) (0.069) (0.072) age 0.0082 -0.2689 -1.0455 -0.7985 -0.7290 (0.007) (0.715) (0.315) (0.555) (0.358) leverage 0.0700 0.6233 1.2322 1.3753 0.9550 (0.004) (0.252) (0.244) (0.247) (0.252) ROE -0.0135 1.4849 1.3245 1.7475 1.0744 (0.003) (0.222) (0.166) (0.199) (0.162) SOE -0.0046 0.1460 -0.0881 0.1723 0.0491 (0.003) (0.189) (0.172) (0.162) (0.200) Top5Hold -0.0000 0.0174 0.0162 0.0194 0.0122 (0.000) (0.003) (0.004) (0.003) (0.004) board 0.0002 0.0048 0.0141 -0.0411 0.0495 (0.000) (0.023) (0.027) (0.023) (0.026) Indpos 0.0219 -0.5486 0.2271 0.5236 -1.0219 (0.010) (0.595) (0.641) (0.598) (0.632) Indep -0.0001 -0.0073 -0.0390 -0.0214 -0.0367 (0.000) (0.015) (0.017) (0.016) (0.017) Mgage -0.0129 -0.7056 -0.0794 -0.8714 -0.6566 (0.005) (0.293) (0.346) (0.359) (0.294) Constant -0.1473 -5.6658 2.1234 1.8793 -1.8997 (0.060) (5.980) (3.072) (4.769) (3.379) Observations 7,272 3,641 3,631 3,642 3,630 R-squared 0.096 0.126 0.119 0.129 0.114 Number of stkcd 1,727 1,292 1,334 1,249 1,255 Firm&Year YES YES YES YES YES Table 7. Financing constraint perspective. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 15 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises Corporate strategy can be regarded as the rational choice or reasonable action of the com- pany to create earnings for shareholders in the uncertain market environment. Enterprises with high cash flow and low financing constraints are more likely to adopt aggressive market expansion. The promulgation of the Green Credit Guidelines, an exogenous shock, increases the financing difficulty and cost of high-polluting companies. Financing constraints The Guidelines undoubtedly has a greater impact on these companies with more aggressive market expansion. The impact of this guide on companies with defensive market expansion propensity may be smaller or even insignificant. Therefore, we divided the sample into sub-samples according to the level of cash flow and the level of KZ index and re-regressed Formula 2. We use the ratio of cash flow generated from operating activities to total assets to measure the size of corporate cash flow. According to whether the size of the company’s cash flow is higher than the median of the same industry in the same year, the sample is divided into two sub-samples of high cash flow and low cash flow. The regression results are listed in columns (2) to (3) of Table 7. The calculation method of the KZ indicator is based on Kaplan and Zingales [45]: calculate the net operating cash flow/total assets, cash dividends/total assets, cash holdings/total assets, asset-liability ratio and Tobin’s Q value respectively. KZ = -1.001909 OCF/Asset + 3.139193 Lev-39. 3678 Dividends /Asset-1.314759 Cash /Asset + 0.2826389 Tobin’s Q. According to whether the company’s KZ index is higher than the median of the same industry in the same year, the sample is divided into two sub-samples with high and low KZ indexes. The regression results are listed in columns (4) and (5) of Table 7. From the regression results, we can see that the promulgation of Green Credit Guidelines significantly inhibits the market expansion tendency of enterprises with high cash flow level and low KZ index, making these companies more conservative in their strategic choices. How- ever, the impact on enterprises with low cash flow holding level and high financing constraints is not significant. This may be related to the defensive market expansion strategy adopted by these enterprises. ð3Þ costi;t ¼ w0 þ w1ðPollutei PolicytÞ þ w2control þ li þ ut þ εi;t ð3Þ costi;t ¼ w0 þ w1ðPollutei PolicytÞ þ w2control þ li þ ut þ εi;t PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 R & D innovation The theoretical analysis, the R & D innovation is sustainable in the enterprises with strong R & D capability. Due to the limited innovation resources, the R & D investment is uncertain in enterprises with weak R & D ability, these enterprises’ R & D investment is often related to the subsidy policy and the financial situation of the enterprises themselves [34, 36]. Therefore, we believe that the promulgation of Green Credit Guidelines can better encourage enterprises with strong R & D ability to further invest in R & D about environmental protection, economy and sustainable development. On the other hand, both increasing R & D innovation and rapidly expanding the market need financial support. Therefore, enterprises that prefer R & D innovation strategy will appropriately reduce their market expansion tendency to ensure sufficient funds to support R & D. In other words, Green Credit Guidelines have a more significant inhibitory effect on the market expansion strategy of enterprises with strong R & D ability. Based on the above two analysis, we divide enterprises into two sub samples with strong R & D capability and weak R & D capability, and re estimate formula 2. We believe that the amount of R & D investment can measure the R & D capacity of enterprises to a certain extent. Therefore, the R & D capacity is measured by the proportion of intangible assets in total assets and the proportion of R & D expenses in total costs. Column (1) of Table 8 shows the sub sam- ple regression results that the proportion of intangible assets in total assets is higher than the PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 16 / 24 Green credit and market expansion strategy of high pollution enterprises PLOS ONE Table 8. R & D perspective. R & D innovation (1) (2) (3) (4) High intangible assets low intangible assets High R & D expenses low R & D expenses Pollute×Policy -0.2685 -0.0051 -0.2883 -0.0517 (0.091) (0.093) (0.089) (0.088) size 0.4489 0.4656 0.5330 0.4518 (0.070) (0.071) (0.066) (0.068) age -0.5664 -0.9526 -0.8240 -1.3964 (0.762) (0.318) (0.363) (0.407) leverage 0.4731 1.3820 0.5640 1.2872 (0.234) (0.239) (0.230) (0.225) ROE 1.1187 1.2885 1.3747 1.0287 (0.176) (0.170) (0.175) (0.162) SOE 0.0887 -0.1349 0.1816 -0.3082 (0.185) (0.179) (0.162) (0.171) Top5Hold 0.0103 0.0210 0.0081 0.0156 (0.003) (0.004) (0.003) (0.003) board -0.0263 0.0205 -0.0319 0.0175 (0.023) (0.024) (0.024) (0.022) Indpos -0.6269 -0.3251 -0.7282 -0.2243 (0.322) (0.341) (0.351) (0.271) Indep -0.1533 0.2711 -0.6386 0.5752 (0.573) (0.613) (0.596) (0.540) Mgage -0.0580 -0.0083 -0.0675 -0.0182 (0.016) (0.016) (0.016) (0.015) Constant 0.3816 -0.5526 1.2148 4.4153 (6.362) (3.113) (3.325) (3.672) Observations 3,641 3,631 3,646 3,626 R-squared 0.139 0.109 0.146 0.113 Number of stkcd 1,102 1,109 1,109 1,101 Firm&Year YES YES YES YES median of the same industry in the same year, and column (2) of Table 8 shows the sub sample regression results that are lower than the median of the industry. The sub sample regression results of the proportion of R & D expenses in the total cost higher than the median of the same industry in the same year are listed in column (3) of Table 8, and the sub sample regres- sion results lower than the median of the same industry in the same year are listed in column (4) of Table 8. From the regression results, we can see that the promulgation of Green Credit Guidelines significantly inhibits the market expansion tendency of high polluting enterprises with strong R & D and innovation ability. This shows that the green credit policy has stimulated high pol- luting enterprises to further increase investment in green innovation. Hypothesis 3 holds. We can also see from Table 8 that the industry status of enterprises with strong innovation ability is negatively correlated with the market expansion tendency. This shows that enter- prises with strong innovation ability will choose to shift from expanding the production and sales scale of products to expanding green technology after the market share reaches a certain Note: , and represent significance levels of 1%, 5% and 10%, respectively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 6.2. Further discussion 6.2.1 Heterogeneity analysis. After the global financial crisis, the $4 trillion stimulus plan led state-owned enterprises to obtain more credit resources than non-state-owned enterprises, exacerbated the difference in resource availability between state-owned enterprises and non- state-owned enterprises [46]. In addition, state-owned enterprises can obtain implicit guaran- tees from the government [47]. Highly polluting state-owned enterprises may be less affected than highly polluting non-state-owned enterprises, in the face of financing constraints after the promulgation of Green Credit Guidelines. Therefore, we divide the sample into two sub samples of state-owned enterprises and non-state-owned enterprises, and re regress formula 2. The results are listed in columns (1) and (2) of Table 9. The promulgation of Green Credit Guidelines significantly reduces the market expansion tendency of non-state-owned enter- prises, but the impact on state-owned enterprises is not significant. This confirms our view that state-owned enterprises are less constrained by the green credit policy because of their enterprise nature. The promulgation of green credit guidelines is bad for high polluting enterprises. The Guides declare China’s determination to achieve high-quality and sustainable economic devel- opment in the future. Therefore, the Guides will increase the managements’ performance pres- sure of high pollution enterprises. In order to cope with the pressure of market performance and avoid the loss of potential shareholders’ wealth, the management of enterprises with equity incentive may sacrifice some long-term projects [48, 49] in exchange for short-term profits. In order to cater to shareholders, these enterprises may further adopt aggressive market expan- sion strategies rather than green R & D. On the contrary, Green Credit Guidelines have less impact on the performance pressure of enterprises without equity incentive. The management of these enterprises may pay more attention to the sustainability of future development and the optimization of product quality, and try their best to adjust the enterprise strategic objec- tives to make corporate strategies consistent with the national strategic. These enterprises may focus on green R & D innovation. Therefore, we divide the sample into two sub samples: with and without equity incentive, and re regress formula 2. The results are listed in columns (3)— (4) of Table 9. The promulgation of Green Credit Guidelines significantly inhibits the market expansion strategy of companies without equity incentive. R & D innovation https://doi.org/10.1371/journal.pone.0279421.t008 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 17 / 24 https://doi.org/10.1371/journal.pone.0279421.t008 median of the same industry in the same year, and column (2) of Table 8 shows the sub sample regression results that are lower than the median of the industry. The sub sample regression results of the proportion of R & D expenses in the total cost higher than the median of the same industry in the same year are listed in column (3) of Table 8, and the sub sample regres- sion results lower than the median of the same industry in the same year are listed in column (4) of Table 8. From the regression results, we can see that the promulgation of Green Credit Guidelines significantly inhibits the market expansion tendency of high polluting enterprises with strong R & D and innovation ability. This shows that the green credit policy has stimulated high pol- luting enterprises to further increase investment in green innovation. Hypothesis 3 holds. We can also see from Table 8 that the industry status of enterprises with strong innovation ability is negatively correlated with the market expansion tendency. This shows that enter- prises with strong innovation ability will choose to shift from expanding the production and sales scale of products to expanding green technology after the market share reaches a certain 17 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Green credit and market expansion strategy of high pollution enterprises degree. However, this negative correlation is not significant in enterprises with low innovation ability, which further verifies that the green innovation investment of enterprises with weak innovation ability is random [33]. In addition, in Table 8, in enterprises with strong innovation ability, there is a significant negative correlation between managements’ age and market expansion tendency, which is not significant in enterprises with weak innovation ability. This shows that in enterprises with strong innovation ability, the older the managements are, the more attention is paid to product creativity and product quality, rather than the simple and rough growth of sales scale. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 6.2. Further discussion This shows that our reasoning is tenable: enterprises without equity incentive will focus on sustainable and green R & D, so they will reduce market expansion in the short term. 6.2.2 Economic effects of Green Credit Guidelines. This paper further examines the eco- nomic effects of Green Credit Guidelines on corporate value from three aspects: corporate image, profitability and risk. The regression model is formula 4. We measure the corporate image by negative media sentiment (NEG) and the average earnings per share predicted by analysts (FEPS). Negative media sentiment (NEG) is the ratio between the number of negative financial news and the total amount of financial news of the company in that year. We measure the profitability of the company by return on total assets (ROA) and return on equity (ROE). 18 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 Green credit and market expansion strategy of high pollution enterprises PLOS ONE Table 9. Heterogeneity analysis. (1) (2) (3) (4) SOE Non-SOE Option Inspiration Non-Option Inspiration Pollute×Policy -0.1089 -0.2310 -0.1780 -0.1720 (0.077) (0.106) (0.219) (0.064) size 0.5175 0.3716 0.5962 0.4409 (0.063) (0.069) (0.161) (0.047) age -1.2879 -0.5353 -0.6513 -1.0345 (0.344) (0.678) (0.666) (0.291) leverage 1.0486 0.7734 1.3715 0.9101 (0.217) (0.236) (0.520) (0.165) ROE 1.4191 1.3138 1.9932 1.3526 (0.146) (0.204) (0.420) (0.122) SOE 0.3411 -0.1495 (0.261) (0.130) Top5Hold 0.0115 0.0235 0.0188 0.0155 (0.003) (0.003) (0.007) (0.002) board 0.0108 -0.0079 0.0164 0.0078 (0.020) (0.028) (0.044) (0.017) Indpos 0.5892 -1.5903 -0.4192 -0.1566 (0.509) (0.685) (1.087) (0.428) Indep -0.0362 -0.0163 -0.0614 -0.0309 (0.014) (0.016) (0.032) (0.011) Mgage -0.4651 -0.2119 -0.5132 -0.4293 (0.257) (0.465) (0.473) (0.228) Constant 4.2464 -1.3056 -3.9491 2.5827 (3.556) (4.590) (5.650) (2.649) Observations 4,329 2,943 602 6,670 R-squared 0.126 0.115 0.219 0.115 Number of stkcd 882 908 144 1,583 Firm&Year YES YES YES YES Table 9. Heterogeneity analysis. , and represent significance levels of 1%, 5% and 10%, respectively. We measure corporate risk by ROE’s volatility (roe_sd) and stock price collapse risk (Ncskew). ROE’s volatility (roe_sd) is the rolling standard deviation of the company’s ROE over three years. Referring to the research of Hutton et al. [50] and Kim et al. [51], we use the following methods to measure the risk of stock price collapse (Ncskew). First, we calculate the idiosyn- cratic weekly stock return adjusted by the market return εi,t, according to formula 5. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 6.2. Further discussion ri,t is the reinvestment return of company i in week t, and rm,t is the weighted average market return of all A-share companies in week t. The residual term εi,t is a unique rate of return that cannot be explained by the market rate of return in time t. Wi,t = ln(1 + εi,t). Then, we calculate Wi,t, and construct the index Ncskew to measure the risk of stock price collapse according to formula 6. The regression results of the Green Credit Guidelines on economic effects are shown in Table 10. ð4Þ Effecti;t ¼ b0 þ b1ðPollutei PolicytÞ þ b2control þ li þ ut þ εi;t 19 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 Green credit and market expansion strategy of high pollution enterprises PLOS ONE Table 10. The economic effects of Green Credit Guidelines. Corporate image Profitability Risk Neg Feps Roa Roe Roe_sd Ncskew Pollute×Policy 0.0282 -0.0977 -0.0115 -0.0239 0.0362 0.0345 (0.008) (0.020) (0.003) (0.008) (0.024) (0.044) size -0.0326 0.1580 -0.0107 -0.0296 -0.1432 -0.0404 (0.006) (0.015) (0.002) (0.006) (0.017) (0.032) age 0.0104 -0.0642 0.0105 0.0107 0.0601 -0.4930 (0.033) (0.088) (0.011) (0.035) (0.105) (0.222) leverage 0.0596 -0.2547 -0.0067 0.0508 0.5955 0.0885 (0.019) (0.053) (0.006) (0.020) (0.060) (0.112) ROE -0.1667 1.0487 0.0541 (0.014) (0.041) (0.085) SOE 0.0053 -0.0541 -0.0126 -0.0172 -0.0977 -0.0417 (0.014) (0.039) (0.005) (0.015) (0.046) (0.081) Top5Hold -0.0000 -0.0010 0.0008 0.0019 0.0033 -0.0006 (0.000) (0.001) (0.000) (0.000) (0.001) (0.002) board 0.0006 -0.0022 0.0015 0.0060 0.0009 -0.0139 (0.002) (0.005) (0.001) (0.002) (0.006) (0.011) Indpos 0.0402 -0.2702 -0.0024 0.0224 -0.1560 -0.0491 (0.049) (0.133) (0.016) (0.053) (0.156) (0.277) Indep 0.0001 0.0057 -0.0002 -0.0028 -0.0034 0.0060 (0.001) (0.003) (0.000) (0.001) (0.004) (0.007) Mgage -0.0183 0.1473 0.0282 0.0889 0.1345 -0.1688 (0.025) (0.067) (0.008) (0.027) (0.080) (0.140) Constant 0.8396 -2.4040 0.1539 0.5826 2.5675 4.4688 (0.298) (0.791) (0.097) (0.317) (0.946) (2.002) Observations 7,266 6,522 7,065 7,065 7,272 6,603 R-squared 0.109 0.204 0.080 0.049 0.033 0.056 Number of stkcd 1,727 1,685 1,705 1,705 1,727 1,602 Firm&Year YES YES YES YES YES YES Table 10. The economic effects of Green Credit Guidelines. ri;t ¼ a0 þ a1rm;t2 þ a2rm;t1 þ a3rm;t þ a4rm;tþ1 þ a5rm;tþ2 þ εi;t ð5Þ Ncskewi;t ¼ nðn 1Þ 3=2X W3 i;t=ððn 1Þðn 2Þð X W2 i;tÞ 3=2Þ ð6Þ ð5Þ ð6Þ First of all, the promulgation of Green Credit Guidelines undoubtedly damages the corpo- rate image of highly polluting enterprises. , and represent significance levels of 1%, 5% and 10%, respectively. https://doi.org/10.1371/journal.pone.0279421.t010 , and represent significance levels of 1%, 5% and 10%, respectively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 represent significance levels of 1%, 5% and 10%, respectively. , and represent significance levels of 1%, 5% and 10%, respectively. 7. Conclusions Based on the quasi-natural experiment of the promulgation of China’s Green Credit Guide- lines, we empirically investigate the impact of market-oriented environmental regulation on the choice of company’s market expansion strategy, taking A-share listed companies from 2008 to 2015 as a sample. The results show that the promulgation of Green Credit Guidelines improves the financing constraints of high polluting enterprises, encourages high polluting enterprises to increase the R & D of green projects, and then promotes the company’s market expansion strategy to defensive. Further, the effect of the Green Credit Guidelines on firm’s market expansion strategy is more prominent if the firm is non-SOE or without equity incen- tive. We also show that although the promulgation of the Green Credit Guidelines reduced the profits of highly polluting enterprises, it does not increase their risk of stock price collapse. In sum, we conclude that the Green Credit Guidelines are market based environmental regula- tions in line with Porter’s hypothesis. This paper reveals the internal relationship between Green Credit Guidelines and corporate behavior from a deeper level, and supplements litera- tures about the economic effects of market-oriented environmental regulation and corporate strategic management. It provides an academic basis for governments to steadily and effec- tively promote environmental protection policies. This study also has some policy implications. First, regulators should actively promote mar- ket-oriented environmental regulation. Market-oriented environmental regulation is condu- cive to promote enterprises to increase the R & D of green projects, protect the environment and achieve sustainable development; It is also conducive to restrict the overly radical business decisions of high pollution enterprises and prevent the occurrence of systemic financial risks. Second, the company’s management should give more consideration to social responsibility and national strategy when deciding the company’s strategy, which will help the company bet- ter expand its living space and grasp development opportunities. According to the research conclusion of this paper, if the management does not consider sustainable development and environmental protection when formulating the company’s strategy, it may artificially create obstacles to the survival and development of the company. On the contrary, it will help the management to resolve business risks and ensure the sustainable development of the company. 6.2. Further discussion As can be seen from Table 10, the proportion of neg- ative financial news of high polluting enterprises increased by 2.82% compared with non-high polluting enterprises, after the promulgation of the Green Credit Guidelines. Analysts’ earn- ings per share forecast for highly polluting enterprises decreased by 0.0977, compared with non-highly polluting enterprises. This shows that the promulgation of the Guidelines has increased the public’s attention to sustainability, low carbon and emission reduction. The PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 20 / 24 PLOS ONE Green credit and market expansion strategy of high pollution enterprises media and professional institutions have increased the supervision of enterprises that violate the concept of green development. After the promulgation of Green Credit Guidelines, the corporate image of highly polluting enterprises is damaged. The promulgation of Green Credit Guidelines has also reduced the profitability of highly polluting enterprises. Guidelines increase the cost of borrowing from banks for highly pollut- ing enterprises and encourage enterprises to increase the R & D of green and sustainable proj- ects. Both channels reduce the profitability of highly polluting enterprises. The results in columns (3) and (4) of Table 10 confirm our view. The promulgation of Green Credit Guidelines do not increase the risk of high polluting enterprises. Whether the risk is measured by roe_sd or Ncskew, the impact of the Guidelines on the risk is not significant, as shown in column (5) and (6) of Table 10. In other words, although the promulgation of Guidelines will damage part of the profitability of high polluting enterprises, it will not increase the risk of enterprises. We believe that market-oriented envi- ronmental regulation is an effective and safe environmental protection policy. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 Author Contributions Resources: Qian Zhong. Software: Qian Zhong. Validation: Qian Zhong. Visualization: Qian Zhong. Writing – original draft: Qian Zhong, Xuemeng Ding, Xiaoke Sun. Writing – review & editing: Qian Zhong. Supporting information S1 File. Supporting information S1 File. (DOCX) 7. Conclusions We suggest continued future research on how the green credit policy affects other strategic choices of enterprises, such as talent strategy, M & A strategy or integration strategy, to com- prehensively identify green credit policy’s impacts on the transformation of highly polluting enterprises. 21 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Green credit and market expansion strategy of high pollution enterprises In the future, we can continue to study whether the Green Credit Guidelines will have an impact on other strategic choices of highly polluting enterprises, such as diversification strat- egy and market penetration strategy. References Regional economic development pressure, enterprise environmental performance and debt financing. J. Financ Res. 2014, 2:153–166. (In Chinese) 13. Zhao M, Liu F, Sun W, Tao X. The relationship between environmental regulation and green total factor productivity in China: an empirical study based on the panel data of 177 cities. Int. J. Environ. Res. and Public Health. 2020, 17(15), 5287. https://doi.org/10.3390/ijerph17155287 PMID: 32708029 14. Liu Y, Lei J, Zhang Y. A study on the sustainable relationship among the green finance, environment regulation and green-total-factor productivity in China. Sustainability. 2021, 13(21), 11926. https://doi. org/10.3390/su132111926 15. Liu J, Xia Y, Lin S, Wu J, Fan Y. The short-, medium- and long-term effects of Green Credit Policy in China based on a financial CGE model. Chinese J. of Management Science. 2015, 23(4): 46–52. (In Chinese) 16. Miles R, Snow C. Organizational strategy, structure and process; Stanford University Press: Stanford, CA, USA, 2003. 17. Chandler A. Strategy and Structure: Chapters in the History of the American Industrial Enterprise; MIT Press: Cambridge: MIT, USA, 1962. 18. Learned E, Christensen C, Andrews K, Guth W. Business policy: text and cases; Irwin: Homewood, USA, 1965. 19. Porter M. Competitive strategy: techniques for analyzing industries and competitions; Free Press: New York, USA, 1980. 20. Meyer J, Rowan B. Institutionalized organizations: Formal structure as myth and ceremony. American J. Sociology. 1977, 83(2):340–363. 21. Milani S. The impact of environment policy stringency on industrial R & D conditional on pollution inten- sity and relocation cost. Environ. and Resource Econ. 2017, 68(3):595–620. 22. Massa M, Zhang B, Zhang H. The invisible hand of short selling: does short selling discipline earning management. Rev. Financial Studies. 2015, 28(6): 1701–1736. 23. Michael F, Petersen M. Does the source of capital affect capital structure? The Rev. Financial Studies. 2006, 19(1): 45–79. 24. Lemmon M, Roberts M, Abel A, Zarutskie R. The response of corporate financing and investment to changes in the supply of credit. J. Financial and Quantitative Analysis. 2011, 45(3):555–587. 25. Modigliani F, Miller M. The cost of capital, corporate finance and the theory of investment. The American Econ. Rev. 1958, 48(3):261–97. 26. Modigliani F, Miller M. Corporate income taxes and the cost of capital: a correction. The American Econ. Rev. 1963, 53(3):433–443. 27. Richardson S. Over-investment of free cash flow. Rev. Accounting Studies. 2006, 11:159–189. 28. Fazzari S, Hubbard R, Petersen B. Financial constraints and corporate investment. Brookings Papers on Econ. Activities. References 1. Zeng S., Zhou Y. Foreign direct investment’s impact on China’s economic growth, technological innova- tion and pollution. Int. J. Environ. Res. Public Health. 2021, 18(6), 2839. https://doi.org/10.3390/ ijerph18062839 PMID: 33802232 2. Zeng S., Li G., Wu S., Dong Z. The Impact of green technology innovation on carbon emissions in the context of carbon neutrality in China: evidence from spatial spillover and nonlinear effect analysis. Int J. Environ Res Public Health. 2022, 19(2),730. https://doi.org/10.3390/ijerph19020730 PMID: 35055553 3. Liu X., Wang E., Cai D., 2019. Green credit policy, property rights and debt financing: quasi-natural experimental evidence from China. Financ. Res. Lett. 2019, 29, 129–135 3. Liu X., Wang E., Cai D., 2019. Green credit policy, property rights and debt financing: quasi-natural experimental evidence from China. Financ. Res. Lett. 2019, 29, 129–135 4. Du W., Li M., Wang F. Role of rent-seeking or technological progress in maintaining the monopoly power of energy enterprises: an empirical analysis based on micro-data from China. Energy, 2020, 202, 117763. 5. Zhong Q., Han L. Jin J. Do green credit guidelines impact on heavily polluting firms in rent-seeking? Financ Res Lett. 2022, 6(47), 102747. https://doi.org/10.1016/j.frl.2022.102747 6. Lan X. On the Evaluation of performance system incorporating "Green Credit" policies in China’s finan- cial industry. J. Financ. Risk Management. 2013, 02(2):33–37. 7. Wang E, Liu X, Wu J, Cai D. Green credit, debt maturity, and corporate investment—evidence from China. Sustainability. 2019, 11, 583. https://doi.org/10.3390/su11030583 8. Elahi E, Cui W, Zhang H, Abid M. Use of artificial neural networks to rescue agrochemical-based health hazards: A resource optimisation method for cleaner crop production. Journal of Cleaner Production. 2019; 238(11), 117900. https://doi.org/10.1016/j.jclepro.2019.117900 22 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Green credit and market expansion strategy of high pollution enterprises 9. Elahi E, Khalid Z, Tauni M. Extreme weather events risk to crop-production and the adaptation of inno- vative management strategies to mitigate the risk: A retrospective survey of rural Punjab, Pakistan. Technovation, 2021, 117(4),102255. https://doi.org/10.1016/j.technovation.2021.102255 10. Elahi E, Cui Weijun, Zhang H, Nazeer M. Agricultural intensification and damages to human health in relation to agrochemicals: Application of artificial intelligence. Land Use Policy, 2019, 83:461–474. 11. Kong Y., He W., Yuan L., Zhang Z., Gao X., Zhao Y., et al. Decoupling economic growth from water consumption in the Yangtze River Economic Belt, China. Ecological Indicators, 2021, 123, 107344. https://doi.org/10.1016/j.ecolind.2021.107344 12. Shen H, Ma Z. References 1988, 1: 141–195. 29. Jensen M. Agency costs of free cash flow, corporate finance, and takeovers. The American Econ. Rev. 1986, 76:323–329. 30. Richardson S. Over-investment of free cash flow, Rev. of Accounting Studies 2006, 11:159–189. 31. Peng B, Yan W, Elahi E, Wan A. Does the green credit policy affect the scale of corporate debt financing? Evidence from listed companies in heavy pollution industries in China. Environmental Science and Pollu- tion Research, 2021, 29(8):755–767. https://doi.org/10.1007/s11356-021-15587-7 PMID: 34341926 32. Jaffe A, Peterson S, Portney P. Stavins R. Environmental regulation and the competitiveness of us manufacturing: what does the evidence tell us? J. Econ. Literature. 1994, 98(4):853–873. 33. Blackman A, Li Z, Liu A. Efficacy of command-and-control and market-based environmental regulation in developing countries. Annual Rev. of Resource Econ. 2018, 10(1):381–404. 34. Elahi E, Khalid Z, Zhang Z. Understanding farmers’ intention and willingness to install renewable energy technology: A solution to reduce the environmental emissions of agriculture. Applied Energy. 2022, 309 (3), 118459. https://doi.org/10.1016/j.apenergy.2021.118459 23 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 PLOS ONE Green credit and market expansion strategy of high pollution enterprises 35. Elahi E, Zhang Z, Khalid Z, Xu H. Application of an artificial neural network to optimise energy inputs: An energy- and cost-saving strategy for commercial poultry farms. Energy. 2022, 244(4), 123169. https:// doi.org/10.1016/j.energy.2022.123169 36. Noailly J, Smeets R. Directing technical change from fossil-fuel to renewable energy innovation: an empirical application using firm-level patent data. J. Environ. Econ. & Management. 2013, 72(7): 15– 37. 37. Rajagopalan N. Strategic orientations, incentive plan adoptions, and firm performance: Evidence from electric utility firms, Strategic Management J. 1997, 18(10): 761–785. 38. Higgins D, Omer T, Phillips J. The Influence of a Firm’s Business Strategy on its Tax Aggressiveness. Contemporary Accounting Res. 2015, 32(2):674–702. 39. Elahi E, Cui W, Jha SK, Zhang H. Estimation of realistic renewable and non-renewable energy use tar- gets for livestock production systems utilising an artificial neural network method: A step towards live- stock sustainability. Energy, 2019, 183(9):191–204. 40. Elahi E, Khalid Z, Cui W, Zhang H. The public policy of agricultural land allotment to agrarians and its impact on crop productivity in Punjab province of Pakistan. Land Use Policy, 2020, 90(1), 104324. https://doi.org/10.1016/j.landusepol.2019.104324 41. Elahi E, Abid M, Zhang H, Cui W, Hasson S. Domestic water buffaloes: Access to surface water, dis- ease prevalence and associated economic losses. Preventive Veterinary Medicine, 2018, 154(1):102– 112. PLOS ONE \| https://doi.org/10.1371/journal.pone.0279421 December 30, 2022 References https://doi.org/10.1016/j.prevetmed.2018.03.021 PMID: 29685433 42. Bentley K, Omer T, Sharp N. Business strategy, financial reporting irregularities, and audit effort. Con- temporary Accounting Res. 2012, 30(2):780–817. 43. Bentley K, Omer T, Twedt B. Does Business Strategy Impact a Firm’s Information Environment? J. Accounting, Auditing & Finance. 2017, 10:1–25. 44. Moser P, Voena M. Compulsory Licensing: Evidence from the Trading with the Enemy Act. The Ameri- can Econ. Rev. 2012, 102(1): 396–427. 45. Kaplan S, Zingales L. Do investment-cash flow sensitivities provide useful measures of financing con- straints? Quarterly J. Econ. 1997, 112(1):169–215. 46. Johansson A, Feng X. The state advances, the private sector retreats? Firm effects of China’s great stimulus program. Cambridge J. Econ. 2016, 40(6): 1635–1668. 47. Kaya H, Banerjee G. The impact of monetary policy and firm characteristics on short-term financial management measures: Evidence from U.S. industrial firms. J. Accounting, Business & Management. 2014, 21(2): 23–35. 48. Graham J, Harvey C, Rajgopal S. The economic implications of corporate financial reporting. J. of Accounting and Econ. 2005, 40: 3–73. 49. Ederer F, Manso G. Is pay for performance detrimental to innovation? Management Science. 2013, 59 (7): 1496–1513. 50. Hutton A, Marcus A. Tehranian H. Opaque financial report, r2, and crash risk. J. Financial. Econ. 2009, 94(1): 67–86. 51. Kim J, Li Y, Zhang L. Corporate tax avoidance and stock price crash risk: firm-level analysis. J. Financial Econ. 2011, 100(3):639–662. 24 / 24
https://openalex.org/W2027292811	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0016682&type=printable	English	null	Diversity in Protein Glycosylation among Insect Species	PloS one	2,011	cc-by	8,122	Abstract Background: A very common protein modification in multicellular organisms is protein glycosylation or the addition of carbohydrate structures to the peptide backbone. Although the Class of the Insecta is the largest animal taxon on Earth, almost all information concerning glycosylation in insects is derived from studies with only one species, namely the fruit fly Drosophila melanogaster. Methodology/Principal Findings: In this report, the differences in glycoproteomes between insects belonging to several economically important insect orders were studied. Using GNA (Galanthus nivalis agglutinin) affinity chromatography, different sets of glycoproteins with mannosyl-containing glycan structures were purified from the flour beetle (Tribolium castaneum), the silkworm (Bombyx mori), the honeybee (Apis mellifera), the fruit fly (D. melanogaster) and the pea aphid (Acyrthosiphon pisum). To identify and characterize the purified glycoproteins, LC-MS/MS analysis was performed. For all insect species, it was demonstrated that glycoproteins were related to a broad range of biological processes and molecular functions. Moreover, the majority of glycoproteins retained on the GNA column were unique to one particular insect species and only a few glycoproteins were present in the five different glycoprotein sets. Furthermore, these data support the hypothesis that insect glycoproteins can be decorated with mannosylated O-glycans. Conclusions/Significance: The results presented here demonstrate that oligomannose N-glycosylation events are highly specific depending on the insect species. In addition, we also demonstrated that protein O-mannosylation in insect species may occur more frequently than currently believed. Citation: Vandenborre G, Smagghe G, Ghesquie`re B, Menschaert G, Nagender Rao R, et al. (2011) Diversity in Protein Glycosylation a ONE 6(2): e16682. doi:10.1371/journal.pone.0016682 Received October 8, 2010; Accepted December 23, 2010; Published February 23, 2011 Received October 8, 2010; Accepted December 23, 2010; Published February 23, 2011 Copyright: 2011 Vandenborre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Research Council of Ghent University (project BOF07/GOA/017 and BOF10/GOA/003), the Fund for Scientific Research- Flanders (3G016306) to GS and EV. BG is a postdoctoral research fellow of the Fund for Scientific Research-Flanders (Belgium). The UGent/VIB lab acknowledges support by research grants from the Fund for Scientific Research-Flanders (Belgium) (project 3G028007), the Concerted Research Actions from the Ghent University and the Inter University Attraction Poles (project BOF07/GOA/012 and IUAP06). Gianni Vandenborre1,2, Guy Smagghe1, Bart Ghesquie`re3,4, Gerben Menschaert5, Rameshwaram Nagender Rao1,2, Kris Gevaert3,4, Els J. M. Van Damme2 1 Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium, 2 Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium, 3 Department of Medical Protein Research, VIB, Ghent, Belgium, 4 Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium, 5 Laboratory for Bioinformatics and Computational Genomics, Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Abstract The authors also acknowledge the Consortium for Functional Glycomics for glycan array analyses. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: guy.smagghe@ugent.be Diversity in Protein Glycosylation among Insect Species Gianni Vandenborre1,2, Guy Smagghe1*, Bart Ghesquie`re3,4, Gerben Menschaert5, Rameshwaram Nagender Rao1,2, Kris Gevaert3,4, Els J. M. Van Damme2 February 2011 \| Volume 6 \| Issue 2 \| e16682 Glycosylation Differences in Insect Species studied insect laboratory model organism. For D. melanogaster, different glycosyltransferases and glycosylhydrolases which are responsible for synthesis and trimming of N-glycans have been reported suggesting the presence of multiple glycan structures on glycoproteins [10–13]. Moreover, at least 42 discrete N-glycans have been identified recently in D. melanogaster, mostly containing oligomannose and core fucosylated paucimannosidic N-glycans [14–17]. Considering the broad diversity among insect species, it can be expected that the diversification in glycan patterns will even be more extensive when analyzing glycosylation patterns in different insect species. Functional Glycomics, GNA has a high selectivity for oligoman- nose N-glycans [18] that were previously shown to be the most abundant class of N-glycans present in insects. The percentage of proteins retained on the GNA column was less than 5% of the total amount of proteins (based on protein concentration estimations using Bradford) for all five insect species. Peptide identification using LC-MS/MS, resulted in 161, 64, 116, 142 and 245 unique (glyco)proteins for T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum, respectively (Table 1). Putative N- glycosylation sites were present on 81%, 77%, 75%, 83% and 89% of the glycoproteins from T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum, respectively (Table 1). This suggests that for all insect species at least 11% of the glycoproteins were purified in an N-glycan independent way. p In this study, the functional diversity of glycoproteins was studied for insect species belonging to five important insect orders. We selected four insects with a complete metamorphosis, the flour beetle Tribolium castaneum (Coleoptera), the silkworm Bombyx mori (Lepidoptera), the honeybee Apis mellifera (Hymenoptera) and the fruit fly D. melanogaster (Diptera), as well as one insect species with an incomplete metamorphosis, the pea aphid Acyrthosiphon pisum (Hemiptera). In addition to this wide selection in insect diversity, several insect species are good representatives for economically important pest insects such as caterpillars, beetles or aphids, while the honeybee belongs to the group of beneficial insects that are essential for pollination. Flies and mosquitoes, on the other hand, are important transmitters of many (human) diseases. Because protein modifications such as glycosylation are not directly encoded by the genomic code, glycosylation in insects was studied at the proteomics level. Glycosylation Differences in Insect Species Recent developments in high-throughput technology for studying proteomes and the public availability of the genome data of different insect species allowed a comparative study of the glycoproteins present in the different insect species. Lectin affinity chromatography using the snowdrop lectin (Galanthus nivalis agglutinin, GNA) was used to selectively purify different sets of mannosylated glycoproteins from different insect species. Subsequently, the purified glycoproteins were identified with LC-MS/MS and characterized according to biological or molecular function. To our knowledge, this is the first report that presents a comparative study of the glycoproteomes present in different insect species. Studying glycoproteomes in different insect species should ultimately result in the development of a more holistic understanding of the importance of glycobiology in insects. After identification of the different sets of glycoproteins, InterProScan was used to detect functional domains, protein regions or protein signatures in the individual polypeptides for further annotation (Tables S1, S2, S3, S4, S5). Subsequently, a protein abundance index (emPAI) was calculated to detect the polypeptide sequences that were highly abundant among the captured glycoproteins (Tables S1, S2, S3, S4, S5). Among the identified glycoproteins typical membrane proteins such as laminin, cadherin, contactin, chaoptin or C-type lectins were found to be abundantly present (Table S1, S2, S3, S4, S5). Also many leucine-rich repeat transmembrane proteins which are known to contain several glycans on their extracellular part were detected. Transport proteins were lipoproteins, hemocyanin or ferritin. Also vitellogenin, which is a known glycolipoprotein present in the fat body of adult insects and important for reproduction, was detected in T. castaneum, D. melanogaster and A. pisum. Next to the typical receptor proteins or secreted proteins, many GNA-captured glycoproteins were identified as metabolic enzymes (e.g. dehydrogenases, proteases and amylases), ribosomal proteins or intracellular structural proteins (e.g. actin, tubulin). Because many of these proteins are synthesized on free ribosomes and, consequently, do not enter the ER-Golgi pathway, oligo- mannosidic N-glycans are thought to be absent from these proteins. Therefore the putative N-glycosylation sites found on the peptide backbone of these proteins (Tables S1, S2, S3, S4, S5) may not be functional. Comparing the insect specific glycoprotein sets, major differ- ences in both glycoprotein diversity and quantity were observed (Table S6). When comparing a particular protein annotation such as leucine-rich transmembrane protein between the different insect species, 15, 4 and 1 glycoprotein(s) were detected for A. pisum, T. castaneum and D. melanogaster, respectively, while for A. Glycosylation Differences in Insect Species mellifera and B. mori no leucine-rich membrane proteins were found (Table 2). From the 260 different protein annotations found over the different sets of insect-specific glycoproteins, 62% (161 protein annotations) were associated with only one particular insect species Introduction indicating that the full range of biological and cellular functions is still not fully understood. Deciphering the complexities in biosynthesis and function of glycoproteins in multicellular organisms is a major challenge for the coming decade. Glycosylation is the covalent attachment of an oligosaccharide chain to a protein backbone and is considered to be a very common protein modification. The structure and size of the carbohydrate chain can be very diverse and can alter the physico- chemical characteristics of a protein. Two major types of glycosylation, referred to as N- and O-linked glycosylation, can be distinguished. N-glycans are attached to Asn residues of the peptide backbone while O-glycans are connected to Ser or Thr residues. Only in recent years, it has been acknowledged that glycosylation of proteins modulates various processes such as subcellular localization, protein quality control, cell-cell recogni- tion and cell-matrix binding events. In turn, these important functions control developmental processes such as embryogenesis or organogenesis [1–6]. Although the overall importance of glycosylation is recognized nowadays, the different types of glycosylated proteins in an organism are mostly unknown Insects are without any doubt the largest animal taxon found on Earth accounting for more than half of all known living species [7]. Their unprecedented evolutionary success is the result of an enormous genetic and phenotypic diversification allowing insect species to adapt to a wide variety of ecological niches and environmental challenges. For example, the genetic diversity within one insect order (e.g. Diptera) is already much wider than between distant vertebrates such as human and zebrafish, spanning a whole phylum [8,9]. Because insects are the most diverse organisms in the history of life, they should provide profound insights into diversification of glycobiology in general and differences of glycosylation in particular. To date, almost all information concerning glycobiology in insects was obtained from studies with the fruit fly, Drosophila melanogaster (Diptera), the best PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16682 February 2011 \| Volume 6 \| Issue 2 \| e16682 1 Glycosylation Differences in Insect Species Table 2. Summary table for the number of distinct (glyco)proteins found in at least three different insect species. Table 2. Summary table for the number of distinct (glyco)proteins found in at least three different insect species. while 1.5% of the proteins (only 4 protein annotations) were detected for all five insect species (Tables 2 and S6). This remarkable diversity in glycoproteome profiles between insect species may reveal underlying differences that can influence Functional classification of glycoproteins from insects After identification and annotation of the different polypeptides, the different sets of glycoproteins were classified according to biological process and molecular function using the web-based Protein description A. pisum D. melanogaster A. mellifera B. mori T. Table 2. Summary table for the number of distinct (glyco)proteins found in at least three different insect species. castaneum 1 2-OXOGLUTARATE DEHYDROGENASE 0 1 1 0 1 2 3-HYDROXYACYL-COA DEHYROGENASE 1 1 1 0 1 3 ACETYL-COA C-ACYLTRANSFERASE 1 0 1 0 1 4 ACTIN 2 1 2 2 1 5 ALDEHYDE DEHYDROGENASE 2 1 0 0 2 6 ALPHA-AMYLASE 3 0 1 1 2 7 ALPHA-GALACTOSIDASE 5 1 0 0 1 8 ALPHA-MANNOSIDASE 4 2 0 0 1 9 AMINOPEPTIDASE 4 5 1 1 1 10 DIPEPTIDYL CARBOXYPEPTIDASE 0 2 0 1 1 11 ARGININE KINASE 0 1 1 0 1 12 ASPARTATE AMMONIA LYASE 0 0 1 1 1 13 ATP SYNTHASE SUBUNIT 4 2 6 2 5 14 BETA-HEXOSAMINIDASE 3 1 2 0 1 15 CADHERIN 1 0 1 0 1 16 CARBOXYLESTERASE 4 1 0 1 1 17 CATHEPSIN 1 0 1 0 1 18 CONTACTIN 1 1 1 0 1 19 ELONGATION FACTOR 1-ALPHA 1 0 1 0 1 20 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE 1 0 1 0 1 21 GLYCOGEN DEBRANCHING ENZYME 2 1 1 0 0 22 GLYCOGEN PHOSPHORYLASE 1 0 1 1 1 23 HEAT SHOCK PROTEINS 5 0 4 1 7 24 HEMOCYANIN 0 1 2 2 6 25 ISOCITRATE DEHYDROGENASE 1 2 1 0 0 26 LAMININ 5 4 0 1 4 27 LEUCINE-RICH TRANSMEMBRANE PROTEIN 15 1 0 0 4 28 LOW DENSITY LIPOPROTEIN RECEPTOR 1 1 1 0 2 29 MUCIN 0 1 2 2 0 30 MYOSIN 1 1 0 2 0 31 PEROXIREDOXIN 0 0 1 1 1 32 PHOSPHOFRUCTOKINASE 1 1 1 0 0 33 PROTEASE S28 PRO-X CARBOXYPEPTIDASE 2 1 0 0 1 34 PROTEIN DISULFIDE ISOMERASE 3 1 0 1 1 35 RIBOSOMAL PROTEINS 18 14 10 5 22 36 SERINE CARBOXYPEPTIDASE 2 1 0 0 1 37 SERINE PROTEASE INHIBITOR, SERPIN 4 1 0 1 4 38 SERINE PROTEASE-RELATED 2 7 0 7 0 39 TRANSKETOLASE 1 0 1 0 1 40 TREHALOSE-6-PHOSPHATE SYNTHASE 1 1 1 0 0 41 TROPOMYOSIN 1 0 0 1 1 42 TUBULIN 4 4 4 1 0 44 VITELLOGENIN-RELATED 1 2 0 0 4 doi:10.1371/journal.pone.0016682.t002 Functional classification of glycoproteins from insects Functional classification of glycoproteins from insects After identification and annotation of the different polypeptides, the different sets of glycoproteins were classified according to biological process and molecular function using the web-based WEGO plotting tool (Figures 1 and 2). Purification and identification of glycoproteins from insects To study the functional differences in glycoprotein sets derived from insect species belonging to different insect orders, glycopro- teins were captured using lectin affinity chromatography based on the snowdrop lectin GNA (Figure S1). As shown by the glycan microarray experiments conducted by the Consortium for Table 1. Number of glycoproteins purified by GNA affinity chromatography for the different insect species. Insect species Insect order No of proteins in database No of generated spectra No of peptides identified No of identified proteins No of putative N- glycosylated proteins Tribolium castaneum Coleoptera 16.645 3744 572 161 130 Bombyx mori Lepidoptera 14.623 3749 118 64 49 Apis mellifera Hymenoptera 10.157 3496 381 116 87 Drosophila melanogaster Diptera 21.317 3744 655 142 118 Acyrthosiphon pisum Hemiptera 34.821 3745 788 245 218 doi:10.1371/journal.pone.0016682.t001 ble 1. Number of glycoproteins purified by GNA affinity chromatography for the different insect specie 2 Glycosylation Differences in Insect Species Table 2. Summary table for the number of distinct (glyco)proteins found in at least three different insect species. Table 2. Summary table for the number of distinct (glyco)proteins found in at least three different insect species. For example, the relative amount of glycoproteins associated with transport (GO: 0006810) was 11%, 16%, 15%, 6% and 5% for the glycoproteins derived from T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum, respectively. Between the highest and the lowest relative amount of glycoproteins for the category transport (GO: 0006810), a three-fold difference was observed (A. mellifera versus B. mori). This illustrates a potential differential importance of glycosylation for a particular biological process between insect species belonging to different orders. In addition, it is striking that a large part of the glycoproteins was associated with several metabolic processes. Table 2. Summary table for the number of distinct (glyco)proteins found in at least three different insect species. Hereby, it was clear that Functional classification of glycoproteins from insects After identification and annotation of the different polypeptides, the different sets of glycoproteins were classified according to biological process and molecular function using the web-based WEGO plotting tool (Figures 1 and 2). Hereby, it was clear that while 1.5% of the proteins (only 4 protein annotations) were detected for all five insect species (Tables 2 and S6). This remarkable diversity in glycoproteome profiles between insect species may reveal underlying differences that can influence certain biological processes. After identification and annotation of the different polypeptides, the different sets of glycoproteins were classified according to biological process and molecular function using the web-based WEGO plotting tool (Figures 1 and 2). Hereby, it was clear that PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16682 3 Glycosylation Differences in Insect Species different insects, especially for A. mellifera and A. pisum. This corresponded with 8%, 11%, 27%, 10% and 15% for T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum, respectively, and correlates with the many ribosomal proteins found in the annotation lists (Tables S1, S2, S3, S4, S5, S6). glycoproteins captured by GNA are involved in a broad range of biological processes such as cell adhesion (GO: 0007155), cellular homeostasis (GO: 0019725), cell communication (GO: 0007154), stress response (GO: 0006950), transmembrane transport (GO: 0055085), etc. However, for specific biological processes relative differences can be found between insects belonging to different orders. For example, the relative amount of glycoproteins associated with transport (GO: 0006810) was 11%, 16%, 15%, 6% and 5% for the glycoproteins derived from T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum, respectively. Between the highest and the lowest relative amount of glycoproteins for the category transport (GO: 0006810), a three-fold difference was observed (A. mellifera versus B. mori). This illustrates a potential differential importance of glycosylation for a particular biological process between insect species belonging to different orders. In addition, it is striking that a large part of the glycoproteins was associated with several metabolic processes. glycoproteins captured by GNA are involved in a broad range of biological processes such as cell adhesion (GO: 0007155), cellular homeostasis (GO: 0019725), cell communication (GO: 0007154), stress response (GO: 0006950), transmembrane transport (GO: 0055085), etc. However, for specific biological processes relative differences can be found between insects belonging to different orders. Discussion Recessive mutation in a pomt gene results in poorly viable flies with defects in muscle development, illustrating the influence of an aberration in O-mannosylation on normal development. Using the BLAST search algorithm (EMBL-EBI), we were able to detect predicted protein sequences that are very homologous to POMT1 and POMT2, respectively, for T. castaneum, B. mori, A. mellifera as well as A. pisum (Table S7). The construction of a phylogenetic tree for these predicted POMT proteins revealed that at least two distinct O-mannosyltransferases resembling POMT1 and POMT2 are conserved among the five insect species (Figure S2). Many proteins in the different glycoprotein sets have a known cytosolic localization such as actin, tubulin or glycerol-3-phosphate dehydrogenase. Since POMTs are located in the lumen of the Golgi apparatus, cytosolic proteins are not expected to be modified by glycan structures [23]. However, several reports have demonstrated the existence of a cellular system involving retrograde transport of proteins from the ER to the cytosol [24]. A dynamic and abundant O-glycosylation of serine and threonine was demonstrated for many cytoplasmic/nuclear proteins [25–27]. For example, in Drosophila, post-translational O-GlcNAc modifica- tion was shown to be of importance for the regulation of Polycomb gene expression, while in vertebrates tubulin was even shown to contain sialyloligosaccharides [11,28,29]. In addition, other types of transport were expected to be very dominant. Surprisingly, the cumulative percentage of glycoproteins associated with these processes never exceeded more than 12%. Moreover, it is striking that many glycoproteins were related with metabolic processes associated with certain intracellular compartments such as lysosomes. Many lysosomal enzymes are hydrolases such as proteases, lipases or phosphatases which were found to occur very frequently in the different glycoprotein sets (Tables S1, S2, S3, S4, S5). These enzymes are synthesized by membrane-bound ribosomes on the ER and transverse the ER-Golgi pathway to leave the Golgi apparatus in transport vesicles that fuse with lysosomes. Moreover, in mammalians the presence of mannose- containing N-glycans is crucial for lysosomal enzymes to be recognized for trafficking to lysosomes [19]. Recent evidence for a similar lysosomal protein-sorting machinery in Drosophila Schnei- der S2 cells has been found by identifying a homolog of the mammalian mannose 6-phosphate receptor [20]. Our findings support this hypothesis by demonstrating that many enzymes with hydrolytic activities which are known to concentrate in lysosomes contain oligo-mannosidic N-glycans. transport were expected to be very dominant. PLoS ONE \| www.plosone.org Discussion Surprisingly, the cumulative percentage of glycoproteins associated with these processes never exceeded more than 12%. Moreover, it is striking that many glycoproteins were related with metabolic processes associated with certain intracellular compartments such as lysosomes. Many lysosomal enzymes are hydrolases such as proteases, lipases or phosphatases which were found to occur very frequently in the different glycoprotein sets (Tables S1, S2, S3, S4, S5). These enzymes are synthesized by membrane-bound ribosomes on the ER and transverse the ER-Golgi pathway to leave the Golgi apparatus in transport vesicles that fuse with lysosomes. Moreover, in mammalians the presence of mannose- containing N-glycans is crucial for lysosomal enzymes to be recognized for trafficking to lysosomes [19]. Recent evidence for a similar lysosomal protein-sorting machinery in Drosophila Schnei- der S2 cells has been found by identifying a homolog of the mammalian mannose 6-phosphate receptor [20]. Our findings support this hypothesis by demonstrating that many enzymes with hydrolytic activities which are known to concentrate in lysosomes contain oligo-mannosidic N-glycans. in D. melanogaster for the dystroglycan protein [21,22]. Moreover, the O-mannosyltransferases that are responsible for the O- glycosylation were identified as POMT1 and POMT2 [21]. Recessive mutation in a pomt gene results in poorly viable flies with defects in muscle development, illustrating the influence of an aberration in O-mannosylation on normal development. Using the BLAST search algorithm (EMBL-EBI), we were able to detect predicted protein sequences that are very homologous to POMT1 and POMT2, respectively, for T. castaneum, B. mori, A. mellifera as well as A. pisum (Table S7). The construction of a phylogenetic tree for these predicted POMT proteins revealed that at least two distinct O-mannosyltransferases resembling POMT1 and POMT2 are conserved among the five insect species (Figure S2). Many proteins in the different glycoprotein sets have a known cytosolic localization such as actin, tubulin or glycerol-3-phosphate dehydrogenase. Since POMTs are located in the lumen of the Golgi apparatus, cytosolic proteins are not expected to be modified by glycan structures [23]. However, several reports have demonstrated the existence of a cellular system involving retrograde transport of proteins from the ER to the cytosol [24]. A dynamic and abundant O-glycosylation of serine and threonine was demonstrated for many cytoplasmic/nuclear proteins [25–27]. For example, in Drosophila, post-translational O-GlcNAc modifica- tion was shown to be of importance for the regulation of Polycomb gene expression, while in vertebrates tubulin was even shown to contain sialyloligosaccharides [11,28,29]. Discussion One of the major findings in this paper is that very little overlap was observed between the glycoprotein sets derived from the different insect species. This was expected between insect species sampled at different developmental stages (e.g. Bombyx larvae and Tribolium adults) because glycosylation profiles change depending on reproductive and developmental stage. However, when comparing only adult insects (e.g. Tribolium adults and Drosophila adults) the diversity in glycoproteins remained extremely high. Since glycosylation is a post-translational modification, changes in carbohydrate composition that were found to be useful during insect evolution can easily be introduced. When glycoproteins were categorized according to molecular function, many of them were involved with hydrolase activity accounting for 19%, 34%, 27%, 31% and 27% in T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum, respectively. This is in agreement with the many proteases, esterases, glycoside hydrolas- es, lipases or phosphatases found in the different insect-specific glycoprotein sets (Tables S1, S2, S3, S4, S5). Remarkably, many glycoproteins were also associated with nucleotide binding in the Because N-glycosylation of proteins occurs in the endoplasmic reticulum (ER) and the Golgi apparatus, it was expected that most glycoproteins would be derived from the luminal part of the secretory pathway such as plasma membrane proteins or secreted proteins. Therefore glycoproteins involved in biological processes such as cell adhesion, cell communication and transmembrane Figure 1. Classification according to biological process of the GNA binding glycoproteins from A. pisum, D. melanogaster, A. mellifera, B. mori and T. castaneum using the WEGO resources. doi:10.1371/journal.pone.0016682.g001 Figure 1. Classification according to biological process of the GNA binding glycoproteins from A. pisum, D. melanogaster, A. mellifera, B. mori and T. castaneum using the WEGO resources. doi:10.1371/journal.pone.0016682.g001 February 2011 \| Volume 6 \| Issue 2 \| e16682 PLoS ONE \| www.plosone.org 4 Glycosylation Differences in Insect Species Figure 2. Classification according to molecular function of the GNA binding glycoproteins from A. pisum, D. melanogaster, A. mellifera, B. mori and T. castaneum using the WEGO resources. doi:10.1371/journal.pone.0016682.g002 Figure 2. Classification according to molecular function of the GNA binding glycoproteins from A. pisum, D. melanogaster, A. mellifera, B. mori and T. castaneum using the WEGO resources. doi:10.1371/journal.pone.0016682.g002 in D. melanogaster for the dystroglycan protein [21,22]. Moreover, the O-mannosyltransferases that are responsible for the O- glycosylation were identified as POMT1 and POMT2 [21]. Lectin affinity purification of glycoproteins from insect extracts Apart from its use as a tool for affinity chromatography, the snowdrop (Galanthus nivalis) lectin was reported to exert strong insecticidal activity against different insect orders [30–32]. Previ- ously, midgut proteins such as ferritin, a-amylase or aminopeptidase were found to be targeted by mannose-binding plant lectins in several economically important pest insects [33–36]. Indeed, these three midgut proteins were also found among the GNA binding glycoproteins in several insect species (Table S6). Moreover, this report clearly holds supporting evidence for the hypothesis that plant lectins, and in particular GNA, act on pest insects through the simultaneous interaction with multiple target glycoproteins. For the different protein extracts, adult insect bodies were used for the flour beetle T. castaneum, the worker honey bee A. mellifera and the fruit fly D. melanogaster. For the pea aphid A. pisum a mix of nymphs and adults was collected, while for the silkworm B. mori only fifth larval instar caterpillars were used for extracting proteins. Insect bodies were crushed in liquid nitrogen using a chilled mortar and pestle and an extraction buffer (0.2 M phosphate buffer pH 7.6 containing 2 mM phenylmethanesulfo- nylfluoride) was added at a ratio of 3 mL buffer per gram of insect powder. The different insect extracts were homogenized using a glass and Teflon homogenizer (10 strokes at 2,000 rpm) and subsequently centrifuged at 9,500 g for 1 h at 4uC. The supernatants were collected and protein concentrations were determined using the Bradford method (Coomassie Protein Assay kit, Thermo scientific, Rockford, IL). p g g y p In this manuscript the first comparative study is presented of glycoprotein sets derived from five phylogenetically diverse insect species. Since earlier reports [14–17] have shown that the dominant glycan structures in the model insect D. melanogaster were of the pauci-mannose N-glycan type, the mannose-binding lectin GNA was used in this study to capture insect glycoproteins. However, the percentage of proteins retained on the GNA column was found to be less than 5% of the total protein for the different insect species, suggesting that the number of identified glycopro- teins is probably an underestimation of the actual number of glycoproteins. One important reason to explain the low percentage of glycoproteins may be that glycoproteins containing complex glycan structures are more abundant in insects than currently believed, as was recently also shown for Drosophila [37]. Discussion In addition, other types of A dynamic and abundant O-glycosylation of serine and threonine was demonstrated for many cytoplasmic/nuclear proteins [25–27]. For example, in Drosophila, post-translational O-GlcNAc modifica- tion was shown to be of importance for the regulation of Polycomb gene expression, while in vertebrates tubulin was even shown to contain sialyloligosaccharides [11,28,29]. In addition, other types of Another interesting observation was the occurrence of at least 10–25% of (glyco)proteins without a protein signature for the attachment of an N-glycan structure. These observations suggest that mannose-containing O-glycosylation may be abundantly present in insect species. To our knowledge, the presence of mannose containing O-glycans in insects has only been described PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16682 5 Glycosylation Differences in Insect Species residues from high-mannose and oligo-mannose N-glycans. GNA did not react with more complex N-glycans with terminal sugar residues other than mannose. cytoplasmic glycosylation may be present. Although at present the expression of a mannosyl transferase in the cytoplasm has never been shown, the addition of mannose residues or mannose containing oligosaccharides to the peptide backbone of cytoplas- mic/nuclear proteins may occur in insects. Preparation of peptides and LC-MS/MS analysis Preparation of peptides and LC-MS/MS analysis Glycoproteins eluted from the GNA column were completely dried and re-dissolved in freshly prepared 50 mM ammonium bicarbonate buffer (pH 7.8). Prior to digestion, protein mixtures were boiled for 10 min at 95uC followed by cooling down on ice for 15 min. Sequencing-grade trypsin (Promega, Madison, WI, USA) was added in a 1:100 (trypsin:substrate) ratio (w/w) and digestion was allowed overnight at 37uC. The sample was acidified with 10% acetic acid (final concentration of 1% acetic acid) and loaded for RP-HPLC separation on a 2.1 mm internal diameter 6150 mm 300SB-C18 column (ZorbaxH, Agilent technologies, Waldbronn, Germany) using an Agilent 1100 Series HPLC system. Following a 10 min wash with 10 mM ammonium acetate (pH 5.5) in water/acetonitrile (98/2 (v/v), both Baker HPLC analyzed (Mallinckrodt Baker B.V., Deventer, the Netherlands), a linear gradient to 10 mM ammonium acetate (pH 5.5) in water/ acetonitrile (30/70, v/v) was applied over 100 min at a constant flow rate of 80 mL/min. Eluting peptides were collected in 60 fractions between 20 and 80 min, and fractions separated by 15 min were pooled and vacuum dried until further analysis. PLoS ONE \| www.plosone.org Lectin affinity purification of glycoproteins from insect extracts In addition, the identification of glycoproteins also depends on the quality of the insect databases. As illustrated in Table 1, the number of putative protein sequences present in the different insect databases is highly variable, which may indicate differences in the degree of completion between the insect databases. Subsequently, this will influence protein identification. Therefore, we want to emphasize that the data presented in this report do not intend to give a full database for glycoproteins present in T. castaneum, B. mori, A. mellifera, D. melanogaster or A. pisum. The glycoprotein catalogs are snapshots of a dynamic glycoproteome during the specific developmental stage of the different insects. A lectin affinity column (diameter 0.5 cm, height 2 cm) was prepared by coupling the purified GNA to Sepharose 4B using the divinylsulfone method [43]. Approximately 20 mg of total protein was loaded onto the GNA Sepharose column to selectively purify the glycoproteins as described earlier [37]. To circumvent non- specific binding of glycoproteins to GNA, peak fractions were pooled and re-chromatographed on the lectin column. Detailed information on the OD values from the elution fractions of the two subsequent GNA affinity purification steps can be found in Figure S3A–S3E. To specifically analyze the selectivity of the GNA-affinity column, the binding of several protein extracts was analyzed by SDS-PAGE before and after chemical removal of the glycan structures from the glycoproteins. For the non-specific deglycosy- lation of the proteins the trifluoromethanesulfonic acid (TFMS) (Sigma-Aldrich) deglycosylation procedure was used [44]. Protein identification and bioinformatics The fragmentation spectra were converted to mgf files using the Automation Engine software (version 3.2, Bruker) and were searched using the MASCOT database search engine (version 2.2.0, Matrix Science, http://www.matrixscience.com) in the appropriate databases. In particular, the Beetlebase (http:// www.Beetlebase.org; release Glean.prot.51906), Silkbase (http:// silkworm.genomics.org.cn; release Silkworm_glean_pep), Beebase (http://genomes.arc.georgetown.edu; release Amel_pre_relea- se2_OGS_pep), Flybase (http://flybase.org; release FB2010_01) and the Aphidbase (http://www.aphidbase.com/aphidbase; re- lease ACYPproteins) were used to identify proteins from T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum, respectively [45–49]. Peptide mass tolerance was set at 0.5 Da and peptide fragment mass tolerance at 0.5 Da, with the ESI-IT as selected instrument for peptide fragmentation rules. Peptide charge was set to 1+,2+,3+. Variable modifications were set to methionine oxidation, pyro-glutamate formation of amino termi- nal glutamine, acetylation of the N-terminus, deamidation of glutamine or asparagines. The enzyme was set to trypsin. Only peptides that were ranked one and scored above the threshold score set at 95% confidence were withheld. The peptide identification results were made publicly accessible in the proteomics identifications (PRIDE) database (experiment accesion number 13290) (http://www.ebi.ac.uk/pride). Figure S2 Phylogenetic tree showing the evolutionary relationship between the homologous protein sequences for O-mannosyltransferase 1 and 2 in D. melanogaster, T. castaneum, B. mori, A. mellifera and A. pisum. (TIF) Figure S3 Elution profiles of GNA affinity chromatog- raphy of total proteins extracts from different insect species. The eluted fractions from the first chromatography were pooled and rechromatographed on the same GNA column. The OD values of the eluted fractions from the two subsequent GNA affinity chromatography steps from T. castaneum (A), B. mori (B), A. mellifera (C), D. melanogaster (D) and A. pisum (E) are shown. (TIF) Table S1 Annotation of the identified glycoproteins for Tribolium castaneum. The list contains the accession number from Beetlebase, an abundance index (emPAI index) and the putative number of N-glycosylation sites. (PDF) Glycoproteins from different insect species were annotated using the InterProScan tool available from the EBI website (http:// www.ebi.ac.uk/Tools/InterProScan) [50]. The InterProScan tool is based on protein databases that use the hidden Markov model methodology to indentify functional protein domains/motives in the primary amino acid sequenes such as Panther, Pfam and TIGR. The obtained IntroProScan output files for T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum can be found in Output File S1, S2, S3, S4, S5. Insects and lectin purification All insects were collected from a laboratory colony that was kept at standard conditions. All stages of T. castaneum were kept on wheat flour mixed with brewer’s yeast (10/1, w/w) [38]. Silkworm B. mori (Daizo) larvae were raised on a mulberry-based artificial diet at 25uC (Yakuroto Co., Japan) [39]. After collection from hives of an experimental apiary in Ghent, honeybee workers (A. mellifera) were kept at 34uC and 70% relative humidity in laboratory cages and fed with sugar water [40]. A continuous colony of D. melanogaster was maintained on a corn meal-based diet, and the pea aphid A. pisum was reared on broad beans (Vicia faba) at 23–25uC and 65–70% relative humidity [37,41]. GNA was purified from the bulbs of snowdrop (Galanthus nivalis) using a combination of ion exchange chromatography and affinity chromatography [42]. The carbohydrate binding specificity of GNA was previously determined in detail using hapten inhibition assays, frontal affinity chromatography and the glycan array technology provided by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/) [18]. These studies clearly showed that GNA specifically binds to the terminal mannose These pooled fractions were re-dissolved in 50 mL of 2.5% acetonitrile (HPLC solvent A). Eight mL of this peptide mixture were applied for nanoLC-MS/MS analysis on an Ultimate (Dionex, Amsterdam, the Netherlands) in-line connected to an Esquire HCT mass spectrometer (Bruker, Bremen, Germany). The sample was first trapped on a trapping column (PepMapTM February 2011 \| Volume 6 \| Issue 2 \| e16682 PLoS ONE \| www.plosone.org 6 Glycosylation Differences in Insect Species C18 column, 0.3 mm I.D. 65 mm, Dionex (Amsterdam, the Netherlands)). After back-flushing from the trapping column, the sample was loaded on a 75 mm I.D. 6150 mm reverse-phase column (PepMapTM C18 (Dionex)). The peptides were eluted with a linear gradient of 3% HPLC solvent B (0.1% formic acid in water/acetonitrile (3/7, v/v)) increase per minute at a constant flow rate of 200 nL/min. Using data dependent acquisition multiply charged ions with intensities above threshold (adjusted for each sequence according to the noise level) were selected for fragmentation. During MS/MS analysis, a MS/MS fragmentation amplitude of 0.7 V and a scan time of 40 ms were used. Supporting Information Figure S1 Coomassie-stained SDS-PAGE of different elution or run-through fractions obtained after GNA chromatography of protein extracts from T. castaneum (T), D. melanogaster (D) and A. pisum (A). Lane 0 was loaded with a protein marker (PageRulerTM, prestained protein ladder, Fermentas) whereas lanes 1 to 3 were loaded with the peak elution fraction of the GNA chromatography of total proteins extracts from T. castaneum, D. melanogaster and A. pisum, respectively. Lanes 4 to 6 were loaded with run-through samples of GNA chromatography of total protein extracts from T. castaneum, D. melanogaster and A. pisum, respectively. Lanes 7 to 9 were loaded with the peak elution fraction of the GNA chromatography of total proteins extracts after chemical deglycosylation from T. castaneum, D. melanogaster and A. pisum, respectively. Table S2 Annotation of the identified glycoproteins for Bombyx mori. The list contains the accession number from Silkbase, an abundance index (emPAI index) and the putative number of N-glycosylation sites. (PDF) Table S3 Annotation of the identified glycoproteins for Apis mellifera. The list contains the accession number from Beebase, an abundance index (emPAI index) and the putative number of N-glycosylation sites. (PDF) Table S4 Annotation of the identified glycoproteins for Drosophila melanogaster. The list contains the accession number from Flybase, an abundance index (emPAI index) and the putative number of N-glycosylation sites. (PDF) p (PDF) Table S5 Annotation of the identified glycoproteins for Acyrthosiphon pisum. The list contains the accession number from Aphidbase, an abundance index (emPAI index) and the putative number of N-glycosylation sites. (PDF) Protein identification and bioinformatics To quantify the presence of certain proteins, an established label-free method was used based on an exponential modified protein abundance index (emPAI) [51,52]. The emPAI index estimates the abundance of a specific glycoprotein based on the number of identified tryptic peptides. In addition, the number of predicted N-glycosylation sites present on the polypeptide backbone was calculated using the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc). Only Asn-X-Ser/Thr sequences (where X is any amino acid except proline) with a prediction score .0.5 were withheld as potential N- glycosylation sites. Afterwards the annotated glycoproteins were categorized according to the biological process or molecular function using the Web Gene Ontology Annotation Plot (WEGO) software (http://wego.genomics.org.cn/cgi-bin/wego/index.pl). The WEGO software is a widely used and freely available tool for visualizing, plotting and comparing annotation results based on classification terms provided by the Gene Ontology (GO) Consortium (http://www.geneontology.org/) [53]. Table S2 Annotation of the identified glycoproteins for Bombyx mori. The list contains the accession number from Silkbase, an abundance index (emPAI index) and the putative number of N-glycosylation sites. (PDF) References Hino M, Kijima-Suda I, Nagai Y, Hosoya H (2003) Glycosylation of the alpha and beta tubulin by sialyloligosaccharides. Zool Sci 20: 709–715. 10. Correia T, Papayannopoulos V, Panin V, Woronoff P, Jiang J, et al. (2003) Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila. Proc Natl Acad Sci USA 100: 6404–6409. 29. Sinclair DA, Syrzycka M, Macauley MS, Rastgardani T, Komljenovic I, et al. (2009) Drosophila O-GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs (sxc). Proc Natl Acad Sci USA 106: 13427–13432. 11. Gambetta MC, Oktaba K, Mu¨ller J (2009) Essential role of the glycosyltrans- ferase Sxc/Ogt in polycomb repression. Science 325: 93–96. 12. Kim YK, Kim KR, Kang DG, Jang SY, Kim YH, et al. (2009) Suppression of the beta-N-acetylglucosaminidase in the N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells. Glycobiology 19: 301–308. 30. Fitches E, Gatehouse JA (1998) A comparison of the short and long term effects of the insecticidal lectins on the activities of soluble and brush border enzymes of the tomato moth larvae (Lacanobia oleracea). J Insect Physiol 44: 1213–1224. 13. Zhang L, Tran DT, Ten Hagen KG (2010) An O-glycosyltransferase promotes cell adhesion during development by influencing secretion of an extracellular matrix integrin ligand. J Biol Chem 285: 19491–19501. 31. Down RE, Ford L, Bedford LN, Newell C, Gatehouse LN, et al. (2001) Influence of plant development and environment on transgene expression in potato and consequences for insect resistance. Transgenic Res 10: 223–236. 14. Gutternigg M, Kretschmer-Lubich D, Paschinger K, Rendic D, Hader J, et al. (2007) Biosynthesis of truncated N-linked oligosaccharides results from non- orthologous hexosaminidase-mediated mechanisms in nematodes, plants, and insects. J Biol Chem 282: 27825–27840. 32. Li G, Xu X, Xing H, Zhu H, Fan Q (2005) Insect resistance to Nilaparvata lugens and Cnaphalocrocis medinalis in transgenic indica rice and the inheritance of gna plus sbti transgenes. Pest Manag Sci 61: 390–396. 33. Du J, Foissac X, Carss A, Gatehouse AMR, Gatehouse JA (2000) Ferritin acts as the most abundant binding protein for the snowdrop lectin in the midgut of rice brown planthoppers (Nilaparvata lugens). Insect Biochem Mol Biol 30: 297–305. 15. Rendic D, Wilson IB, Lubec G, Gutternigg M, Altmann F, et al. (2007) Adaptation of the ‘‘in-gel release method’’ to the N-glycome analysis of low- milligram of material. Electrophoresis 28: 4484–4492. 34. References 1. Brower DL (2003) Platelets with wings: the maturation of Drosophila integrin biology. Curr Opin Cell Biol 15: 607–613. 21. Ichimiya T, Manya H, Ohmae Y, Yoshida H, Takahashi K, et al. (2004) The twisted abdomen phenotype of Drosophila POMT1 and POMT2 mutants coincides with their heterophilic protein O-mannosyltransferase activity. J Biol Chem 279: 42638–42647. gy p 2. Lecuit T, Le Goff L (2007) Orchestrating size and shape during morphogenesis. Nature 450: 189–192. 3. Tian E, Ten Hagen KG (2007) O-linked glycan expression during Drosophila development. Glycobiology 17: 820–827. 22. Nakamura N, Stalnaker SH, Lyalin D, Lavrova O, Wells L, et al. (2010) Drosophila dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, rotated abdomen and twisted. Glycobiology 20: 381–394. 4. Acar M, Jafar-Nejad H, Takeuchi H, Rajan A, Ibrani D, et al. (2008) Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132: 247–258. 23. Lommel M, Strahl S (2009) Protein O-mannosylation: conserved from bacteria to humans. Glycobiology 19: 816–828. 5. Zhang L, Zhang Y, Ten Hagen KG (2008) A mucin-type O-glycosyltransferase modulates cell adhesion during Drosophila development. J Biol Chem 283: 34076–34086. 24. Lehrman MA (2001) Oligosaccharide-based information in endoplasmic reticulum quality control and other biological systems. J Biol Chem 276: 8623–8626. 6. Yano H, Yamamoto-Hino M, Goto S (2009) Spatial and temporal regulation of glycosylation during Drosophila eye development. Cell Tissue Res 336: 137–147. 25. Dehennaut V, Slomianny MC, Page A, Vercoutter-Edouart AS, Jessus C, et al. (2008) Identification of structural and functional O-linked N-acetylgluco- samine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7: 2229–2245. 7. Sanderson MJ (2008) Phylogenetic signal in the eukaryotic tree of tree. Science 321: 121–123. 8. Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, et al. (2000) The syntenic relationship of the zebrafish and human genomes. Genome Res 10: 1351–1358. 26. Sakabe K, Wang Z, Hart GW (2010) b-N-acetylglucosamine (O-GlcNAc) is part of the histone code. Proc Natl Acad Sci USA;doi: 10.1073/pnas.1009023107. 9. Severson DW, DeBruyn B, Lovin DD, Brown SE, Knudson DL, et al. (2004) Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae. J Hered 95: 103–113. 27. Zeidan Q, Wang Z, De Maio A, Hart GW (2010) O-GlcNAc cycling enzymes associate with the translational machinery and modify core ribosomal proteins. Mol Biol Cell 21: 1922–1936. 28. Acknowledgments B.G. is a postdoctoral research fellow of the Fund for Scientific Research Flanders (Belgium). Output File S2 InterProScan output file for Bombyx mori. (OUT) Conceived and designed the experiments: GS BG RR KG EV. Performed the experiments: GV BG RR. Analyzed the data: GV BG GM. Contributed reagents/materials/analysis tools: GS KG EV. Wrote the paper: GV GS EV. Table S6 Comparative analysis of the number of annotated glycoproteins according to protein descrip- Table S6 Comparative analysis of the number of annotated glycoproteins according to protein descrip- PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 7 Glycosylation Differences in Insect Species tion for T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum. (PDF) tion for T. castaneum, B. mori, A. mellifera, D. melanogaster and A. pisum. (PDF) Output File S4 InterProScan output file for Drosophila melanogaster. (OUT) Output File S4 InterProScan output file for Drosophila melanogaster. (OUT) Output File S4 InterProScan output file for Drosophila melanogaster. (OUT) Table S7 WU-BLAST analysis to search for proteins homologous to O-mannosyltransferases from Drosoph- ila melanogaster POMT1 (Genbank accession No NP_524025.2) and POMT2 (Genbank accession No NP_569858.1). (PDF) Glycosylation Differences in Insect Species 38. Zapata N, Smagghe G (2010) Repellency and toxicity of essential oils from leaves and bark of Laurelia sempervirens and Drimys winteri against Tribolium castaneum. Ind Crops Prod 32: 405–410. 46. International Silkworm Genome Consortium (2008) The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem Mol Biol 38: 1036–1045. 47. Kim HS, Murphy T, Xia J, Caragea D, Park Y, et al. (2009) BeetleBase in 2010: revisions to provide comprehensive genomic information for Tribolium castaneum. Nucleic Acids Res 38: D437–D442. p 39. Soin T, De Geyter E, Mosallanejad H, Iga M, Martin D, et al. (2010) Assessment of species specificity of moulting accelerating compounds in Lepidoptera: comparison of activity between Bombyx mori and Spodoptera littoralis by in vitro reporter and in vivo toxicity assays. Pest Manag Sci 66: 526–535. 48. Tweedie S, Ashburner M, Falls K, Leyland P, McQuilton P, et al. (2009) Flybase Consortium. FlyBase: enhancing Drosophila gene ontology annotations. Nucleic Acids Res 37: D555–559. 40. Scharlaken B, de Graaf DC, Goossens K, Brunain M, Peelman LJ, et al. (2008) Reference gene selection for insect expression studies using quantitative real-time PCR: The head of the honeybee, Apis mellifera, after a bacterial challenge. J Insect Sci 8: 1–10. 49. Legeai F, Shigenobu S, Gauthier JP, Colbourne J, Rispe C, et al. (2010) AphidBase: a centralized bioinformatic resource for annotation of the pea aphid genome. Insect Mol Biol 19: 5–12. 41. Christiaens O, Iga M, Velarde R, Rouge´ P, Smagghe, G (2010) Halloween genes and nuclear receptors in ecdysteroid biosynthesis and signalling in the pea aphid. Insect Mol Biol 19: 187–200. 50. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, et al. (2009) InterPro: the integrative protein signature database. Nucleic Acids Res 37: D211–215. 42. Van Damme EJM, Allen AK, Peumans WJ (1987) Isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop (Galanthus nivalis) bulbs. FEBS Lett 215: 140–144. 51. Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, et al. (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4: 1265–1272. ) 43. Pepper DS (1994) Some alternative coupling chemistries for affinity chroma- tography. Mol Biotechn 2: 157–178. g p y 44. Egde AS, Faltynek CR, Hof L, Reichert LE, Weber P (1981) Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. References Cristofoletti PT, Mendonc¸a de Souza FA, Rahbe´ Y, Terra WR (2006) Characterization of a membrane-bound aminopeptidase purified from Acyrthosi- phon pisum midgut cells: a major binding site for toxic mannose lectins. FEBS J 273: 5574–5588. 16. Kanie Y, Yamamoto-Hino M, Karino Y, Yokozawa H, Nishihara S, et al. (2009) Insight into the regulation of glycan synthesis in Drosophila chaoptin based on mass spectrometry. PloS ONE 4: e5434. p y 17. Schachter H (2009) Paucimannose N-glycans in Caenorhabditis elegans and Drosophila melanogaster. Carbohydr Res 344: 1391–1396. 35. Fitches E, Wiles D, Douglas AE, Hinchliffe G, Audsley N, et al. (2008) The insecticidal activity of recombinant garlic lectins towards aphids. Insect Biochem Mol Biol 38: 905–915. 18. Fouquaert E, Smith DF, Peumans WJ, Proost P, Balzarini J, et al. (2009) Related lectins from snowdrop and maize differ in their carbohydrate-binding specificity. Biochem Biophys Res Commun 380: 260–265. 36. Sadeghi A, Smagghe G, Proost P, Van Damme EJM (2008) Ferritin acts as a target site for the snowdrop lectin (GNA) in the midgut of the cotton leafworm Spodoptera littoralis. Insect Sci 15: 513–519. p y 19. Dahms NM, Olsen LJ, Kim JJ (2008) Strategies for carbohydrate recognition by the mannose 6-phosphate receptors. Glycobiology 18: 664–678. 37. Vandenborre G, Van Damme EJM, Gesquie`re B, Menschaert G, Hamshou M, et al. (2010) Glycosylation signatures in Drosophila: fishing with lectins. J Proteome Res 9: 3235–3242. 20. Kametaka S, Sawada N, Bonifacino JS, Waguri S (2010) Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster. J Cell Sci 123: 460–471. PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16682 February 2011 \| Volume 6 \| Issue 2 \| e16682 8 Glycosylation Differences in Insect Species Anal Biochem 118: 131–137. 52. Vaudel M, Sickmann A, Martens L (2010) Peptide and protein quantification: a map of the minefield. Proteomics 10: 650–670. glycoproteins by trifluoromethanesulfonic acid. Anal Biochem 1 45. Kaplan N, Linial M (2006) ProtoBee: hierarchical classification and annotation of the honey bee proteome. Genome Res 16: 1431–1438. 53. Ye J, Fang L, Zheng H, Zhang H, Chen J, et al. (2006) WEGO: a web tool for plotting. Nucleic Acids Res 34: W293–297. PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16682 9
https://openalex.org/W3049620523	https://www.frontiersin.org/articles/10.3389/fmed.2020.604072/pdf	English	null	Corrigendum: Correlation of Bromodomain Protein BRD4 Expression With Epithelial–Mesenchymal Transition and Disease Severity in Chronic Rhinosinusitis With Nasal Polyps	Frontiers in medicine	2,020	cc-by	461	CORRECTION published: 29 October 2020 doi: 10.3389/fmed.2020.604072 Copyright © 2020 Zhou, Cui, Liu, Yue, Xie, Ding, Xu, Han and Zhang. 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. 1 Department of Otorhinolaryngology Head and Neck Surgery, Shandong Provincial Hospital Afﬁliated to Shandong First Medical University, Jinan, China, 2 Department of Otorhinolaryngology Head and Neck Surgery, Shandong Provincial Hospital Afﬁliated to Shandong University, Jinan, China, 3 Health Management Center, Shandong Provincial Hospital Afﬁliated to Shandong First Medical University, Jinan, China Approved by: Frontiers Editorial Ofﬁce, Frontiers Media SA, Switzerland Correspondence: Hong Zhang zhenyujie0915@163.com Approved by: Frontiers Editorial Ofﬁce, Frontiers Media SA, Switzerland Keywords: BRD4, chronic rhinosinusitis, nasal polyps, epithelial–mesenchymal transition, disease severity Correspondence: Hong Zhang zhenyujie0915@163.com Corrigendum: Correlation of Bromodomain Protein BRD4 Expression With Epithelial–Mesenchymal Transition and Disease Severity in Chronic Rhinosinusitis With Nasal Polyps Xuanchen Zhou 1,2, Zhaoyang Cui 1, Yiqing Liu 1, Zhiyong Yue 1, Fengyang Xie 1, Ling Ding 1, Shuai Xu 1, Jie Han 1,2 and Hong Zhang 3* A Corrigendum on Specialty section: This article was submitted to Translational Medicine, a section of the journal Frontiers in Medicine Received: 08 September 2020 Accepted: 09 September 2020 Published: 29 October 2020 Specialty section: This article was submitted to Translational Medicine, a section of the journal Frontiers in Medicine Correlation of Bromodomain Protein BRD4 Expression With Epithelial–Mesenchymal Transition and Disease Severity in Chronic Rhinosinusitis With Nasal Polyps by Zhou, X., Cui, Z., Liu, Y., Yue, Z., Xie, F., Ding, L., et al. (2020). Front. Med. 7:413. doi: 10.3389/fmed.2020.00413 Received: 08 September 2020 Accepted: 09 September 2020 Published: 29 October 2020 In the published article, there was an error in aﬃliation 3. Instead of “Minimally Invasive Urology Center, Shandong Provincial Hospital Aﬃliated to Shandong First Medical University, Jinan, China,” it should be “Health Management Center, Shandong Provincial Hospital Aﬃliated to Shandong First Medical University, Jinan, China.” Published: 29 October 2020 Frontiers in Medicine \| www.frontiersin.org Citation: Zhou X, Cui Z, Liu Y, Yue Z, Xie F, Ding L, Xu S, Han J and Zhang H (2020) Corrigendum: Correlation of Bromodomain Protein BRD4 Expression With Epithelial–Mesenchymal Transition and Disease Severity in Chronic Rhinosinusitis With Nasal Polyps. Front. Med. 7:604072. doi: 10.3389/fmed.2020.604072 The authors apologize for this error and state that this does not change the scientiﬁc conclusions of the article in any way. The original article has been updated. October 2020 \| Volume 7 \| Article 604072 Frontiers in Medicine \| www.frontiersin.org
https://openalex.org/W4394786076	https://afropolitanjournals.com/index.php/ajmbr/article/download/142/173	English	null	The Challenges and Opportunities of Emerging Markets for Firm Internationalization	Deleted Journal	2,024	cc-by	12,420	Afropolitan Journals Afropolitan Journals The Challenges and Opportunities of Emerging Markets for Firm Internationalization: The Case of MTN Nigeria Abstract This study examines the challenges and opportunities of firm internationalization in emerging markets, using MTN Nigeria as a case study. Through a mixed-methods approach involving semi- structured interviews, document analysis, and a survey of 150 MTN Nigeria employees and managers, the study identifies the key challenges faced by the company, including weak and unstable regulatory environment, infrastructure deficiencies, intense competition, cultural differences, lack of skilled local talent, and corruption. The study also highlights the main opportunities capitalized on by MTN Nigeria, such as the large and growing population, liberalizing telecommunications sector, potential for innovation in digital and financial services, growing middle class, untapped market segments, and strategic partnerships with local firms. The findings are compared with existing literature, revealing consistencies, extensions, and implications for theory and practice. The study contributes to the understanding of firm internationalization in emerging markets by providing a nuanced analysis of the specific challenges, opportunities, and strategies in the Nigerian telecommunications industry. It also highlights the limitations of existing theories and the need for adaptation to the unique institutional, cultural, and competitive contexts of emerging markets. The study offers recommendations for future research, including comparative studies across different emerging markets and critical examinations of the broader societal and developmental implications of firm internationalization. Keywords: Firm Internationalization, Emerging Markets, Telecommunications Industry, Challenges, Opportunities. Abe Gilbert Ntoh University of Calabar, Nigeria. Corresponding author: abentoh@gmail.com Vol. 14, No. 2 2024 African Journal of Management and Business Research Introduction Background of Emerging Markets and their Significance for Firm Internationalization In recent times, emerging markets have gained significance for business internationalization, presenting a range of prospects and obstacles for enterprises aiming to broaden their worldwide reach (Cavusgil et al., 2021; Luo & Bu, 2018). Multinational enterprises (MNEs) seeking to explore new avenues for expansion and profitability have shown interest in these markets because to their vast customer base, quick economic growth, and dynamic business environments (Grosse, 2016; Luo & Zhang, 2016). Due to their ability to provide access to large unexplored markets, low-cost production components, and strategic assets that may boost a firm's competitive edge, emerging 124 Afropolitan Journals markets are important for the internationalization of businesses (Deng et al., 2020; Hernandez & Guillén, 2018). In recent decades, a number of developing countries have implemented important economic reforms that have opened their economies to international trade and investment (Cuervo-Cazurra et al., 2018). Due to this, MNEs now have more chances to join and establish themselves in these markets (Deng & Yang, 2015). Furthermore, businesses may now more readily and effectively contact customers in developing economies because to the quick adoption of digital technology, which has decreased entry barriers (Manyika et al., 2016). Internationalizing to developing markets is not without its difficulties, however. Companies operating in these markets may find it difficult to manage political risks, cultural disparities, and institutional gaps (Meyer et al., 2020). The lack or underdevelopment of formal institutions, such as financial markets, legal systems, and regulatory frameworks, is referred to as an institutional vacuum. This may lead to uncertainty and higher transaction costs for businesses (Gao et al., 2017; Rottig, 2016). The adaptation of goods, services, and marketing techniques by enterprises to cater to the distinct tastes and expectations of customers in diverse markets might present obstacles due to cultural variations (Gupta & Batra, 2016; Hofstede et al., 2010). Another significant obstacle for businesses expanding into developing regions is political risk. Political unpredictability, corruption, and poor governance are prevalent in many developing countries, which may raise transaction costs and induce uncertainty (Ault & Spicer, 2014; Jiménez et al., 2017). To guarantee the success of their internationalization initiatives, businesses must properly evaluate and manage these risks (Casson & Lopes, 2013; Feinberg & Gupta, 2009). Overview of MTN Nigeria as a Case Study An excellent case study of the difficulties and possibilities of developing countries for business internationalization is offered by MTN Nigeria, a division of the South African telecom corporation MTN Group. In 2001, MTN Nigeria joined the Nigerian market at a period of notable liberalization and expansion in the nation's telecommunications industry (Adeola et al., 2020). With more than 60 million members, MTN Nigeria has grown to become the leading mobile network provider in the nation despite a number of obstacles, such as inadequate infrastructure, unclear regulations, and fierce competition (MTN Nigeria, 2021). The instance of MTN Nigeria, which faced a number of obstacles as well as possibilities, offers insightful information on the intricate dynamics of business internationalization in developing nations. It emphasizes how crucial it is to adjust to local market circumstances, make infrastructural and innovative investments, and maintain strong stakeholder connections (Amankwah-Amoah & Debrah, 2017). It also emphasizes how important it is for businesses to be ready to face the difficulties and unpredictabilities that come with doing business in developing countries (Luiz & Ruplal, 2013). Literature Review Literature Review Definition and Characteristics of Emerging Markets Introduction Despite these difficulties, developing markets are becoming a vital part of many businesses' internationalization efforts because of the potential they provide (Hoskisson et al., 2013). With their expanding middle classes and rising disposable incomes, emerging nations have access to vast and expanding consumer markets (Cavusgil et al., 2018; Sheth, 2011). This gives businesses a lot of chances to grow their clientele and find new sources of income. Furthermore, compared to established markets, developing nations often offer cheaper labor and manufacturing costs, which may provide businesses a competitive edge (Peng et al., 2017; Sun et al., 2015). Additionally, strategic resources including local expertise, technical know-how, and natural resources are accessible in emerging countries, which may improve a company's competitive position (Demirbag et al., 2010; Meyer et al., 2009). Through partnerships or acquisitions, multinational enterprises (MNEs) may get these assets in developing economies and use them to bolster their worldwide competitiveness (Luo & Tung, 2007; Williamson & Raman, 2011). Due to the diversity of developing markets, businesses need to modify their plans according to the unique circumstances of each one, accounting for elements including customer preferences, market size, and regulatory frameworks (Ghemawat, 2001; Meyer et al., 2011). 125 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 125 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com AJMBR Overview of MTN Nigeria as a Case Study Overview of MTN Nigeria as a Case Study Research Objective and Significance The main objective of this research is to examine the challenges and opportunities of emerging markets for firm internationalization, using MTN Nigeria as a case study. Specifically, the study aims to: 1. Identify the key challenges faced by MTN Nigeria in entering and operating in the Nigerian market. 2. Explore the opportunities leveraged by MTN Nigeria in the Nigerian market. 3. Analyze the strategies employed by MTN Nigeria to overcome challenges and capitalize on opportunities in the Nigerian market. 4. Develop a framework to guide firms seeking to internationalize in emerging markets, particularly in the telecommunications industry. The significance of this research lies in its potential to advance knowledge on the challenges and opportunities of emerging markets for firm internationalization, and to provide practical guidance for firms seeking to enter or expand their operations in these markets. By focusing on the case of MTN Nigeria, the study can offer a nuanced understanding of how firms can navigate the complex and dynamic environments of emerging markets, particularly in the telecommunications industry. Furthermore, the study can contribute to the broader discourse on the role of emerging markets in the global economy, and the ways in which firms can contribute to the economic development of these markets while also achieving their own strategic objectives. The significance of this research lies in its potential to advance knowledge on the challenges and opportunities of emerging markets for firm internationalization, and to provide practical guidance for firms seeking to enter or expand their operations in these markets. The significance of this research lies in its potential to advance knowledge on the challenges and opportunities of emerging markets for firm internationalization, and to provide practical guidance for firms seeking to enter or expand their operations in these markets. By focusing on the case of MTN Nigeria, the study can offer a nuanced understanding of how firms can navigate the complex and dynamic environments of emerging markets, particularly in the telecommunications industry. Furthermore, the study can contribute to the broader discourse on the role of emerging markets in the global economy, and the ways in which firms can contribute to the economic development of these markets while also The significance of this research lies in its potential to advance knowledge on the challenges and opportunities of emerging markets for firm internationalization, and to provide practical guidance for firms seeking to enter or expand their operations in these markets. Research Objective and Significance By focusing on the case of MTN Nigeria, the study can offer a nuanced understanding of how firms can navigate the complex and dynamic environments of emerging markets, Literature Review Definition and Characteristics of Emerging Markets There has been a recent uptick in studies focusing on emerging markets in the field of international business. This is because these regions provide both possibilities and threats Vol. 14, No. 2 2024 African Journal of Management and Business Research 126 www.afropolitanjournals.com 126 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com Afropolitan Journals to established companies looking to grow internationally (Cavusgil et al., 2021; Grosse, 2016). Emerging markets are known for their fast economic development, vast and expanding customer bases, and dynamic corporate environments. However, there isn't a widely agreed-upon definition of these markets (Luo & Zhang, 2016; Meyer & Peng, 2016). In their definition of emerging markets, Hoskisson et al. (2000) state that these nations are "low-income, rapid-growth countries using economic liberalization as their primary engine of growth" (p. 249). These marketplaces are often seen in countries that are experiencing major structural and institutional shifts, such as moving away from central planning and toward a market-oriented system (Khanna & Palepu, 2010). Emerging markets are characterized by a lack of or insufficient development of formal institutions that facilitate market transactions. These institutions include financial markets, regulatory frameworks, and legal systems (Gao et al., 2017; Rottig, 2016). Due to the complexity and unpredictability of the business environments in developing nations, uncertainty and higher transaction costs might result from these institutional gaps (Khanna & Palepu, 2010; Meyer et al., 2009). It may be much more challenging for foreign corporations to do business in developing economies due to the high levels of political risk, corruption, and inadequate governance (Ault & Spicer, 2014; Jiménez et al., 2017). In spite of all these obstacles, developing markets provide great potential for companies who want to grow internationally and increase their profits (Cavusgil et al., 2021; Luo & Bu, 2018). A big and expanding customer base, characterized by expanding middle classes and rising disposable incomes, may be found in these marketplaces (Sheth, 2011). This opens up a huge window of opportunity for businesses, especially those in sectors that deal directly with consumers, such retail, banking, and telecoms (Chironga et al., 2011; Deloitte, 2014). Additionally, developing countries frequently have cheaper labor and manufacturing costs compared to established markets, which may give enterprises with a competitive edge in terms of cost efficiency (Peng et al., 2017; Sun et al., 2015). Literature Review Definition and Characteristics of Emerging Markets Another key trait of developing economies is their heterogeneity, both between and within nations (Ghemawat, 2001; Meyer et al., 2011). Emerging markets vary greatly in terms of their economic, political, social, and cultural environments, which necessitates enterprises to adopt market-specific strategies and adapt their business models to local circumstances (Gupta & Batra, 2016; London & Hart, 2004). Within nations, there may also be considerable regional differences in terms of consumer preferences, income levels, and infrastructure development, which further complicates the economic picture for international enterprises (Sheth, 2011). Theoretical Frameworks for Firm Internationalization in Emerging Markets Drawing on concepts from institutional theory, international business, and strategy, a number of theoretical frameworks have been put out to explain corporate internationalization in developing countries (Buckley & Casson, 2009; Peng et al., 2008). The eclectic paradigm, also referred to as the OLI framework and created by Dunning (1980, 2001), is one of the most important frameworks. The OLI paradigm claims that enterprises 127 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 127 127 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com AJMBR AJMBR participate in foreign direct investment (FDI) based on three fundamental advantages: ownership (O), location (L), and internalization (I). Ownership advantages relate to the firm- specific resources and competencies that allow it to compete successfully in international markets, such as technology, brand recognition, and managerial competence (Dunning & Lundan, 2008). Location advantages relate to the country-specific aspects that make a given market desirable for investment, such as market size, growth potential, and resource endowments (Dunning, 1998). According to Buckley and Casson (1976), internalization advantages are the advantages of carrying out tasks within the company as opposed to via market transactions. Examples of these benefits include lower transaction costs and the preservation of private information. The OLI framework has been challenged for its limited capacity to capture the particular problems and potential of these markets, despite the fact that it offers a helpful starting point for analyzing business internationalization in developing economies (Ramamurti & Singh, 2009). Alternative frameworks that specifically take into account the institutional and cultural environments of developing economies have been suggested by academics in response to these constraints. One such approach is the institution-based perspective, which highlights the importance of formal and informal institutions in determining corporate behavior and performance in developing economies (Meyer & Peng, 2005; Peng et al., 2008). According to this approach, enterprises operating in developing economies must traverse complicated and frequently uncertain institutional settings, typified by weak property rights, inefficient legal systems, and unpredictable government actions (Khanna & Palepu, 2010). To thrive in these markets, enterprises must create tactics that are suited to the local institutional framework, such as developing alliances with local firms, participating in political activities, and gaining legitimacy with important stakeholders (Doh et al., 2017). Theoretical Frameworks for Firm Internationalization in Emerging Markets More recently, researchers have offered a dynamic capabilities approach on business internationalization in developing countries, which highlights the need of organizational learning and adaptability in these dynamic and unpredictable contexts (Luo, 2000; Teece et al., 1997). According to this approach, organizations that are able to identify and grasp new possibilities, rearrange their resources and skills, and learn from their experiences are better positioned to prosper in developing markets (Helfat et al., 2007; Luo & Child, 2015). This needs organizations to establish a "global mindset" that is open to new ideas and viewpoints, and to build a culture of experimentation and continuous development (Gupta & Govindarajan, 2002; Luo & Tung, 2018). Challenges Faced by Firms in Internationalizing to Emerging Markets A variety of obstacles might make it difficult for businesses looking to expand internationally into developing areas to run efficiently and meet their strategic goals. The existence of institutional voids, which are defined as the lack or underdevelopment of formal institutions including legal systems, regulatory frameworks, and financial markets that facilitate market transactions, is one of the biggest obstacles (Gao et al., 2017; Khanna Vol. 14, No. 2 2024 African Journal of Management and Business Research 128 www.afropolitanjournals.com 128 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com Afropolitan Journals & Palepu, 2010). With complex and sometimes unexpected corporate settings to traverse, these institutional gaps may lead to uncertainty and higher transaction costs for businesses (Meyer et al., 2009). Inadequate infrastructure and logistics networks, for instance, can raise the costs and risks of conducting business, while weak intellectual property rights and contract enforcement procedures can make it difficult for businesses to protect their proprietary knowledge and technology (Cuervo-Cazurra & Genc, 2008; Luo & Tung, 2007). A further significant hurdle for businesses expanding internationally into new markets is cultural differences. Cultural norms, attitudes, and practices in these markets are varied and often unfamiliar, leading to potential miscommunications and conflicts between foreign companies and local stakeholders (Hofstede et al., 2010; Meyer et al., 2011). As to Gupta and Batra (2016), there may be misplaced expectations and poor cooperation between foreign managers and local personnel due to variations in communication styles, decision- making processes, and attitudes towards authority. Furthermore, cultural differences might make it challenging for businesses to create branding and marketing plans that connect with local customers and foster loyalty and trust (Sheth, 2011). For businesses expanding internationally into developing economies, political concerns represent yet another significant obstacle. Political unpredictability, corruption, and poor governance are prevalent in many developing countries, which may raise transaction costs and induce uncertainty (Ault & Spicer, 2014; Jiménez et al., 2017). Changes in government rules and regulations, for instance, have the potential to impair corporate operations and lower profitability, while bribery and corruption may raise compliance costs and harm a company's brand (Doh et al., 2012). Furthermore, political concerns may put businesses at danger of expropriation or nationalization of their assets, as well as making it harder for them to get the licenses, permissions, and approvals they need (Luo & Tung, 2018). Challenges Faced by Firms in Internationalizing to Emerging Markets Finally, talent acquisition and human resource management may provide difficulties for businesses expanding internationally into developing areas. There is a shortage of qualified labor in many growing economies, especially for management and technical positions (Horwitz & Budhwar, 2015). Because of this, it may be challenging for businesses to find and retain top personnel, and they may need to make large expenditures in training and development initiatives (Meyer & Xin, 2018). Furthermore, foreign managers who want to establish productive teams and promote a healthy corporate culture may encounter difficulties due to linguistic and cultural obstacles (Meyer et al., 2011). Opportunities Offered by Emerging Markets for Firm Internationalization These markets have substantial opportunity for businesses looking to broaden their worldwide reach and access new growth and profitability streams, notwithstanding the difficulties that come with internationalizing to developing countries. According to Cavusgil et al. (2021), a noteworthy prospect is the substantial and expanding customer base found in developing economies, especially in populous nations like China, India, and Indonesia. Rising middle classes in these markets are marked by aspirations for higher living standards and increasing disposable incomes. This presents a significant opportunity for businesses in 129 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 129 129 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com AJMBR AJMBR consumer-facing industries like financial services, retail, and telecommunications (Chironga et al., 2011; Deloitte, 2014). For instance, rising penetration rates and the need for data services have propelled the fast expansion of the mobile telecommunications sector in Africa in recent years (GSMA, 2021). The possibility for cost savings and efficiency benefits via access to lower-cost labor and manufacturing inputs is another opportunity presented by developing markets (Peng et al., 2017; Sun et al., 2015). Low-cost labor is widely available in developing nations, giving businesses in labor-intensive sectors like manufacturing and services a competitive edge (Meyer et al., 2009). Additionally, developing markets may give access to lower-cost raw materials, energy, and other production inputs, which may allow enterprises to cut their operating costs and enhance their margins (Luo & Tung, 2007). Additionally, emerging markets provide businesses access to fresh sources of knowledge and innovation. These markets often exhibit distinct and localized consumer preferences and demands, which may encourage companies to create new goods and services as well as business strategies that are appropriate for the regional environment (Govindarajan & Ramamurti, 2011; Zeschky et al., 2011). Furthermore, compared to established markets, developing countries may have distinct institutional and technical contexts. This might provide possibilities for businesses to test out novel strategies and pick up knowledge from regional partners and stakeholders (Luo & Child, 2015). For instance, by taking advantage of the extensive use of mobile phones and the lack of conventional banking infrastructure, mobile money services like M-Pesa in Kenya have arisen as a new paradigm for financial inclusion in developing economies (Mbiti & Weil, 2011). Previous Studies on Firm Internationalization in Emerging Markets Prior research has examined at a variety of subjects and situations related to company internationalization in developing economies, offering insightful information about the potential and difficulties present in these areas. A particular area of study has concentrated on the approaches and tactics that companies use to enter developing markets, including joint ventures, wholly-owned subsidiaries, and strategic alliances (Brouthers & Hennart, 2007; Meyer et al., 2009). Demirbag et al. (2010), for instance, looked at the variables affecting Turkish companies' decision on which entrance mode to use when entering Central Asian markets. They discovered that firm-specific resources and competencies, together with institutional elements relevant to the host country, had a significant role in determining the choice of entry mode. A different line of inquiry has looked at how institutional elements like property rights, regulatory frameworks, and legal systems' quality influence how businesses internationalize in developing nations (Hoskisson et al., 2013; Meyer et al., 2009). For instance, Khanna and Palepu (2010) proposed the term "institutional voids" to characterize the lack or inadequate development of formal institutions in developing economies, and they contended that companies can bridge these gaps by enhancing their internal capacities or collaborating with regional companies and stakeholders. In a similar vein, Doh Vol. 14, No. 2 2024 African Journal of Management and Business Research 130 www.afropolitanjournals.com 130 Afropolitan Journals et al. (2017) investigated how political involvement and stakeholder engagement might help businesses traverse institutional obstacles in developing economies. et al. (2017) investigated how political involvement and stakeholder engagement might help businesses traverse institutional obstacles in developing economies. Lastly, other research has also looked at how internationalization of a company may affect its performance in developing countries, specifically how it may affect its competitiveness, growth, and profitability (Cavusgil et al., 2021; Hitt et al., 2006). For instance, Luo and Tung (2007) created the term "springboarding" to explain how emerging market companies can use internationalization to gain new skills, knowledge, and resources that they can use to become more competitive in both domestic and international markets. Similarly, Guillen and Garcia-Canal (2009) investigated the use of internationalization by emerging market corporations to get over their disadvantages as latecomers and close the gap with well- established multinationals from developed countries. Even with the insightful information that earlier study has offered, there are still a lot of holes in the literature and room for further investigation into the internationalization of businesses in developing nations. Previous Studies on Firm Internationalization in Emerging Markets For instance, more study is required to determine how cultural variables influence a company's internationalization, especially when it comes to cross-border mergers and acquisitions (Meyer et al., 2011). Furthermore, further study is required to understand the tactics and competencies that businesses in developing countries need to handle political risks and interact with local stakeholders (Doh et al., 2012). Finally, further study is required to fully understand how business internationalization in developing economies affects long-term performance, especially in light of political and economic uncertainty (Cavusgil et al., 2021). Research Methodology Research Design and Approach This study uses MTN Nigeria as a case study to investigate the potential and constraints of developing markets for corporate internationalization via the use of a mixed-methods research strategy that combines qualitative and quantitative methodologies. Due to its ability to combine the advantages of qualitative and quantitative research methodologies, mixed-methods research has become more and more popular in the field of international business research (Creswell & Plano Clark, 2018; Hurmerinta-Peltomäki & Nummela, 2006). This approach enables a more thorough and nuanced understanding of complex phenomena. Given the complex interplay of economic, institutional, cultural, and strategic elements in corporate internationalization in developing economies, a mixed-methods approach is especially appropriate for this study's environment (Meyer et al., 2009; Peng et al., 2008).In order to provide a comprehensive and contextualized understanding of the firm's internationalization journey, including the opportunities taken advantage of, strategies used, and challenges faced, the qualitative component of this study entails a case study analysis of MTN Nigeria (Eisenhardt, 1989; Yin, 2018). The objective of the quantitative component of this research is to give a more comprehensive and generalizable knowledge of the possibilities and problems encountered by the company in the Nigerian market via a survey of managers and workers of MTN Nigeria. 131 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 131 131 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com AJMBR AJMBR Data Collection Methods In accordance with the mixed-methods research design, the data gathering techniques for this study uses a mixture of surveys, interviews, and document analysis. Semi-structured interviews with MTN Nigeria's top executives and managers, together with pertinent stakeholders including industry experts, regulators, and local partners, is undertaken for the qualitative case study component. Semi-structured interviews are a popular technique in qualitative research because they give researchers flexibility to probe and explore emerging themes and issues while also allowing for the collection of rich and detailed data on participants' experiences, perceptions, and insights (Brinkmann & Kvale, 2015; Welch et al., 2011). An online questionnaire was created for the quantitative survey component and sent to a sample of managers and workers of MTN Nigeria. In international business research, online surveys are becoming more and more common because they make it possible to gather vast volumes of data from respondents who are spread out geographically in an efficient and economical manner (Chidlow et al., 2015; Hult et al., 2008). An online survey can offer a more comprehensive and representative understanding of the opportunities and challenges MTN Nigeria faces in the Nigerian market, as well as the tactics and skills used to overcome them, in the context of this study (Amankwah-Amoah & Debrah, 2017; Luiz & Ruplal, 2013). Data Analysis Techniques Thematic analysis, which entails the methodical identification, classification, and interpretation of patterns and themes within the interview transcripts and documentation, was used in the qualitative data analysis (Braun & Clarke, 2006; King & Brooks, 2018). In addition to the developing themes and insights from the data, the analysis was led by the theoretical frameworks and ideas found in the literature study (Eisenhardt & Graebner, 2007; Welch et al., 2011). The categorization and organizing of the data, as well as the retrieval and comparison of themes and quotations across participants and sources, was made easier with the use of the qualitative data analysis program NVivo (King & Brooks, 2018; Welch et al., 2011). Using SPSS, a mix of descriptive and inferential statistical approaches was used for the quantitative data analysis (Field, 2013; Hair et al., 2014). The survey results and demographic information was analyzed and shown using descriptive statistics, such as means, standard deviations, and frequencies (Hult et al., 2008). Ethical Considerations Any research project must take ethics into account, but this is especially true when sensitive subjects and human subjects are involved (Bell et al., 2018; Wiles, 2012). The principles and rules of the appropriate institutional review boards and professional groups (such the Academy of International Business and the Academy of Management) shall be followed to the letter in this study's adherence to the highest standards of research ethics. Informed consent, which entails ensuring that all participants are fully informed about the goal, methods, possible dangers, and benefits of the research, and that they freely agree to participate, is one of the most important ethical issues for this study (Bell et al., 2018). Sample Selection The mixed-methods research design and the distinct goals of the qualitative and quantitative components served as the guidelines for the sample selection and data analysis procedures used in this study. Purposive sampling was used in the qualitative case study component to choose participants for the semi-structured interviews. The deliberate selection of participants according to their background, expertise, and significance to the study's goals and issue is known as purposeful sampling (Patton, 2015; Robinson, 2014). Senior executives and managers of MTN Nigeria who have been directly engaged in the company's internationalization journey as well as the entrance and operations into the Nigerian market made up the purposive sample in the context of this research. The concept of data saturation—defined as the point at which no new themes or insights emerge from further data collection—is used to establish the sample size for the qualitative case study component (Glaser & Strauss, 1967; Guest et al., 2006). A random selection approach will be used to choose participants for the quantitative survey component from among the MTN Nigeria management and workers. Using a random method, such as random number generator or a stratified sampling strategy, random sampling entails choosing individuals from a population (Chidlow et al., 2015; Hult et al., 2008). A power analysis was used to determine the sample size for the quantitative survey component. This analysis considered various factors, including the number of variables and hypotheses being tested, the expected effect size, and the desired level of statistical significance (Cohen, 1992; Hult et al., 2008). Vol. 14, No. 2 2024 African Journal of Management and Business Research 132 www.afropolitanjournals.com 132 Afropolitan Journals Case Study: MTN Nigeria Overview of MTN Nigeria's Internationalization Journey Overview of MTN Nigeria's Internationalization Journey To reach and gain consumers, MTN Nigeria concentrated on expanding its marketing and distribution capabilities in addition to investing in infrastructure (Adebiyi & Olowookere, 2018). To increase brand recognition and customer loyalty among Nigerian customers, the firm used a variety of tactics, including sponsorships, promotions, and alliances with regional distributors and retailers (Oyewole et al., 2018). In order to set itself apart from rivals and satisfy changing consumer demands, MTN Nigeria has launched cutting-edge goods and services such data bundles and mobile money (Aminu & Oyefolahan, 2021). To reach and gain consumers, MTN Nigeria concentrated on expanding its marketing and distribution capabilities in addition to investing in infrastructure (Adebiyi & Olowookere, 2018). To increase brand recognition and customer loyalty among Nigerian customers, the firm used a variety of tactics, including sponsorships, promotions, and alliances with regional distributors and retailers (Oyewole et al., 2018). In order to set itself apart from rivals and satisfy changing consumer demands, MTN Nigeria has launched cutting-edge goods and services such data bundles and mobile money (Aminu & Oyefolahan, 2021). With more than 60 million members, MTN Nigeria has grown and succeeded significantly in the Nigerian market throughout the years, becoming as the leading mobile network provider in the nation (MTN Nigeria, 2021). In addition, the business has increased its activities and investments outside of the telecom industry, branching out into digital media, enterprise solutions, and financial services (Adeyemi, 2011). The firm has encountered a number of institutional, legal, and competitive obstacles in the Nigerian market, therefore MTN Nigeria's internationalization process has not been without its difficulties (Amankwah-Amoah et al., 2018). Overview of MTN Nigeria's Internationalization Journey As part of its globalization goal, MTN Nigeria, a division of the South African telecom giant MTN Group, entered the Nigerian market in 2001 (MTN Nigeria, 2021). Foreign entrants had both possibilities and problems at the time due to Nigeria's telecommunications sector's rapid development and deregulation (Adeola et al., 2020). The country's sizable and expanding population, the rising demand for mobile services, and the opportunity for market leadership in a largely unexplored sector all contributed to MTN Nigeria's entrance into the market (Amankwah-Amoah et al., 2018). The Nigerian Communications Commission (NCC) granted MTN Nigeria a digital mobile license in 2001, enabling the business to launch operations and develop its network infrastructure. This marked the beginning of MTN Nigeria's internationalization process (MTN Nigeria, 2021). The company's original plan was to quickly increase its capacity and coverage, especially in the larger cities like Port Harcourt, Lagos, and Abuja (Adeyoju, 2021). According to Amankwah- Amoah & Debrah (2017), this required large expenditures in network infrastructure, including the building of cell sites, transmission towers, and fiber-optic cables. 133 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 133 133 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com AJMBR AJMBR To reach and gain consumers, MTN Nigeria concentrated on expanding its marketing and distribution capabilities in addition to investing in infrastructure (Adebiyi & Olowookere, 2018). To increase brand recognition and customer loyalty among Nigerian customers, the firm used a variety of tactics, including sponsorships, promotions, and alliances with regional distributors and retailers (Oyewole et al., 2018). In order to set itself apart from rivals and satisfy changing consumer demands, MTN Nigeria has launched cutting-edge goods and services such data bundles and mobile money (Aminu & Oyefolahan, 2021). With more than 60 million members, MTN Nigeria has grown and succeeded significantly in the Nigerian market throughout the years, becoming as the leading mobile network provider in the nation (MTN Nigeria, 2021). In addition, the business has increased its activities and investments outside of the telecom industry, branching out into digital media, enterprise solutions, and financial services (Adeyemi, 2011). The firm has encountered a number of institutional, legal, and competitive obstacles in the Nigerian market, therefore MTN Nigeria's internationalization process has not been without its difficulties (Amankwah-Amoah et al., 2018). Opportunities Exploited by MTN Nigeria in the Nigerian Market Notwithstanding the difficulties, MTN Nigeria has succeeded in part because it has taken advantage of big market potential in Nigeria. The nation's sizable and expanding population has created a significant opportunity by fueling demand for mobile services (Adeola et al., 2020). With a population of over 200 million, Nigeria is the most populous nation in Africa. Its population is comparatively youthful, urbanizing, and adopting mobile devices at a rising pace (GSMA, 2021). By focusing on the mass market with reasonably priced and easily available goods and services, such pre-paid plans and mobile money, MTN Nigeria has taken advantage of this demographic potential (Aminu & Oyefolahan, 2021). The liberalization and restructuring of the nation's telecommunications industry, which has created room for international investment and competition, has presented MTN Nigeria with yet another chance (Amankwah-Amoah et al., 2018). The National Broadband Plan and the Digital Economy Strategy are only two of the policies and programs the Nigerian government has put in place to encourage the expansion and development of the telecom sector (NCC, 2021). Additionally, MTN Nigeria has taken advantage of market prospects for innovation and distinction, notably in the domains of digital and financial services (Aminu & Oyefolahan, 2021). Millions of unbanked Nigerians have been able to access financial inclusion thanks to the company's introduction of several mobile money and payment solutions, including MTN Mobile Money and MTN EnMoney (Adebiyi & Olowookere, 2018). MTN Nigeria has also created digital platforms and services including MTN Music+ and MTN Online, which have given users access to value-added offerings and experiences (MTN Nigeria, 2021). Challenges Faced by MTN Nigeria in Entering and Operating in the Nigerian Market Challenges Faced by MTN Nigeria in Entering and Operating in the Nigerian Market The poor and unpredictable institutional environment in Nigeria has been one of the biggest obstacles for MTN Nigeria to overcome in order to enter and operate in the market (Ogunnubi & Okeke-Uzodike, 2016). High levels of political risk, corruption, and regulatory uncertainty in Nigeria have resulted in major expenses and obstacles for international businesses (Amaeshi et al., 2016). For instance, MTN Nigeria has been involved in a number of regulatory conflicts and penalties over the years. In 2015, the NCC fined the company $5.2 billion for neglecting to deactivate unregistered SIM cards; however, after discussions, the amount was subsequently lowered to $1.7 billion (Macedo & de Matos, 2019). The fierce rivalry in the Nigerian telecom sector, especially from other international players like Airtel and Globacom, has been another difficulty for MTN Nigeria (Amankwah-Amoah et al., 2018). In order to set themselves apart from the competition, these rivals have also made significant investments in marketing, network infrastructure, and price (Adebiyi & Olowookere, 2018). In order to preserve its market leadership and ward off challenges from competitors, MTN Nigeria has had to continuously alter its operations and tactics (Adeyoju, 2021). Significant obstacles for MTN Nigeria in the Nigerian market have also come from security threats and inadequate infrastructure (Oladimeji et al., 2018). Due to the relative underdevelopment of Nigeria's telecommunications infrastructure, especially in rural and isolated locations, MTN Nigeria has had to make significant expenditures in network maintenance and growth (Amankwah-Amoah & Debrah, 2017). In addition, the business has experienced security threats such theft, vandalism, and assaults on its employees and property, which have interrupted operations and raised expenses (Adeyeye et al., 2019). MTN Nigeria has had difficulties navigating the Nigerian market because to cultural variations and regional business practices (Adebiyi & Olowookere, 2018). Nigeria is home Vol. 14, No. 2 2024 African Journal of Management and Business Research 134 www.afropolitanjournals.com 134 Afropolitan Journals to more than 500 different ethnic groups and languages, making it a culturally diverse nation where business practices may differ greatly across different sectors and areas (Ogunnubi & Okeke-Uzodike, 2016). In order to successfully connect with Nigerian stakeholders, including as consumers, workers, suppliers, and regulators, MTN Nigeria had to establish cultural competences and local alliances (Aminu & Oyefolahan, 2021). Strategies Employed by MTN Nigeria to Overcome Challenges and Leverage Opportunities MTN Nigeria has used a range of tactics and resources to meet the obstacles and take advantage of the possibilities in the Nigerian market. Localization, which entails modifying the business's offerings to suit the unique requirements and tastes of Nigerian customers, has been one of the primary tactics (Amankwah-Amoah et al., 2018). To increase brand connection and loyalty, MTN Nigeria has funded local events and projects as well as engaged in the creation of local content, such as Nigerian music and entertainment (Oyewole et al., 2018). In order to improve its comprehension and interaction with the Nigerian market, the firm has also hired and trained local talent, including managers and executives (Adebiyi & Olowookere, 2018). 135 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 135 35 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 135 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com AJMBR AJMBR Stakeholder engagement is another tactic used by MTN Nigeria. It entails establishing and maintaining connections with important players in the Nigerian market, including legislators, regulators, consumers, staff, and communities (Aminu & Oyefolahan, 2021). In order to promote beneficial laws and regulations, MTN Nigeria has actively engaged in trade groups and forums, such as the Association of Licensed Telecommunications Operators of Nigeria (ALTON) (Adeyoju, 2021). To further show its dedication to the advancement and welfare of Nigerian communities, the corporation has also put in place a number of corporate social responsibility (CSR) projects, including health and education efforts (Ite, 2016). MTN Nigeria has used innovation and digitalization as crucial tactics to surmount obstacles and capitalize on prospects inside the Nigerian market (Adebiyi & Olowookere, 2018). To create new goods, services, and business models that are appropriate for the Nigerian market, the firm has made investments in R&D and collaborated with regional startups and entrepreneurs (Aminu & Oyefolahan, 2021). For instance, MTN Nigeria has introduced digital services such the MTN Smart Feature Phone, a low-cost gadget that delivers internet connection and mobile money services, and MTN Pulse, a platform geared at young people that gives access to music, video, and gaming content (MTN Nigeria, 2021). Findings and Discussion g Analysis of the Challenges Faced by MTN Nigeria in Internationalizing to Nigeria Analysis of the Challenges Faced by MTN Nigeria in Internationalizing to Nigeria The qualitative data analysis revealed several key themes related to the challenges faced by MTN Nigeria in internationalizing to the Nigerian market. These themes were derived from the semi-structured interviews with MTN Nigeria executives and managers, as well as the document analysis of company reports, industry publications, and news articles. One of the most prominent themes was the institutional challenges posed by Nigeria's weak and unstable regulatory environment. As some executive noted: "The regulatory environment in Nigeria is highly unpredictable and challenging. We have faced several disputes and fines over the years, which have disrupted our operations and increased our costs.” (Executive 1) “Regulations and changing government demands is actually killing our business progress… For example, the $5.2 billion fine by the NCC in 2015 was a major blow to our business, and it took significant efforts to resolve it through negotiations and legal action."(Executive 3) Another key theme was the infrastructure challenges faced by MTN Nigeria in expanding and maintaining its network in Nigeria. As one manager explained: Another key theme was the infrastructure challenges faced by MTN Nigeria in expanding and maintaining its network in Nigeria. As one manager explained: "Nigeria's telecommunications infrastructure is relatively underdeveloped, particularly in rural and remote areas. (Manager 1) "Nigeria's telecommunications infrastructure is relatively underdeveloped, particularly in rural and remote areas. (Manager 1) “Underdevelopment and lack of infrastructure has required us to make substantial investments in network expansion and maintenance, such as building cell sites, transmission towers, and fiber-optic cables.”(Executive 2) “Lack of infrastructure has also contributed to challenges such as vandalism, theft, and attacks on our facilities, which have disrupted our operations and increased our costs." (Manager 3) Vol. 14, No. 2 2024 African Journal of Management and Business Research 136 www.afropolitanjournals.com 136 Afropolitan Journals To further analyze the challenges faced by MTN Nigeria, a survey was conducted among a sample of 150 MTN Nigeria employees and managers. The survey included several questions related to the challenges faced by the company, measured on a 5-point Likert scale (1 = strongly disagree, 5 = strongly agree). The results of the survey are presented in Table 1. g Analysis of the Challenges Faced by MTN Nigeria in Internationalizing to Nigeria Table 1: Descriptive statistics for challenges faced by MTN Nigeria (n = 150) Table 1: Descriptive statistics for challenges faced by MTN Nigeria (n = 150) Challenge Mean SD Weak and unstable regulatory environment 4.24 0.87 Infrastructure deficiencies and security risks 4.12 0.92 Intense competition from foreign and local players 3.98 0.95 Cultural differences and local business practices 3.65 1.02 Lack of skilled and experienced local talent 3.42 1.08 Corruption and unethical practices in the business environment 3.27 1.12 Source: Author’s construct 2024 As shown in Table 1, the survey results largely confirmed the challenges identified in the qualitative analysis. The weak and unstable regulatory environment was rated as the most significant challenge (M = 4.24, SD = 0.87), followed by infrastructure deficiencies and security risks (M = 4.12, SD = 0.92), and intense competition from foreign and local players (M = 3.98, SD = 0.95). Cultural differences and local business practices (M = 3.65, SD = 1.02), lack of skilled and experienced local talent (M = 3.42, SD = 1.08), and corruption and unethical practices in the business environment (M = 3.27, SD = 1.12) were also rated as significant challenges, albeit to a lesser extent. To further investigate the relationships between the challenges faced by MTN Nigeria, a correlation analysis was conducted. The results are presented in Table 3. Table 2: Correlation matrix for challenges faced by MTN Nigeria (n = 150) 1 2 3 4 5 6 1. Weak and unstable regulatory environment 1 2. Infrastructure deficiencies and security risks 0.58 1 3. Intense competition from foreign and local players 0.42 0.39 1 4. Cultural differences and local business practices 0.28 0.25 0.33 1 5. Lack of skilled and experienced local talent 0.19* 0.22 0.27 0.31** 1 6. Corruption and unethical practices in the business environment 0.17* 0.20* 0.23 0.29 0.34** 1 p < 0.05, p < 0.01 Source: Author’s construct 2024 Table 2: Correlation matrix for challenges faced by MTN Nigeria (n = 150) 1 2 3 4 5 6 1. Weak and unstable regulatory environment 1 2. Infrastructure deficiencies and security risks 0.58* 1 3. Intense competition from foreign and local players 0.42 0.39 1 4. Cultural differences and local business practices 0.28 0.25 0.33** 1 5. Lack of skilled and experienced local talent 0.19* 0.22 0.27 0.31** 1 6. g Analysis of the Challenges Faced by MTN Nigeria in Internationalizing to Nigeria Corruption and unethical practices in the business environment 0.17* 0.20* 0.23 0.29 0.34** 1 p < 0.05, *p < 0.01 Source: Author’s construct 2024 Table 2: Correlation matrix for challenges faced by MTN Nigeria (n = 150) Table 2: Correlation matrix for challenges faced by MTN Nigeria (n = 150) 137 AJMBR AJMBR As shown in Table 3, there were significant positive correlations among the challenges faced by MTN Nigeria. The strongest correlation was between weak and unstable regulatory environment and infrastructure deficiencies and security risks (r = 0.58, p < 0.01), suggesting that these two challenges often co-occur and reinforce each other. There were also moderate correlations between intense competition and weak and unstable regulatory environment (r = 0.42, p < 0.01), infrastructure deficiencies and security risks (r = 0.39, p < 0.01), and cultural differences and local business practices (r = 0.33, p < 0.01). These findings highlight the interconnected nature of the challenges faced by MTN Nigeria in the Nigerian market. Market The qualitative analysis also revealed several key themes related to the opportunities that MTN Nigeria has capitalized on in the Nigerian market. These themes were derived from the interviews with MTN Nigeria executives and managers, as well as the survey cument analysis. One of the most significant opportunities identified was the large and growing population of Nigeria, which has driven demand for mobile services. As one executive explained: "Nigeria is the most populous country in Africa, with over 200 million people, and a relatively young and urbanizing population. This has created a huge market opportunity for us, particularly in the mass market segment, where we have been able to offer affordable and accessible products and services, such as pre-paid plans and mobile money." (Executive 3) Another key opportunity identified was the liberalizing and reforming telecommunications sector in Nigeria, which has opened up space for foreign investment and competition. As one manager noted: "The Nigerian government has implemented various policies and initiatives to promote the growth and development of the telecommunications industry, such as the National Broadband Plan and the Digital Economy Strategy. This has created a more favorable business environment for us, and has enabled us to align our strategies and investments with the government's priorities." (Manager 2) The third opportunity identified was the potential for innovation and differentiation in the Nigerian market, particularly in the areas of digital and financial services. As one executive stated: "We have seen a significant opportunity to innovate and differentiate our products and services in Nigeria, particularly in the areas of digital and financial services. For example, we have introduced mobile money and payment solutions, such as MTN Mobile Money and MTN EnMoney, which have enabled financial inclusion for millions of unbanked Nigerians. We have also developed digital platforms and services, such as MTN Music+ and MTN Online, which have provided value-added services and experiences for our customers." (Executive 4) To further examine the opportunities capitalized on by MTN Nigeria, the survey also included several questions related to the opportunities in the Nigerian market, measured Vol. 14, No. 2 2024 African Journal of Management and Business Research 138 www.afropolitanjournals.com 138 Afropolitan Journals on a 5-point Likert scale (1 = strongly disagree, 5 = strongly agree). The results of the survey are presented in Table 3. Market Table 3: Descriptive statistics for opportunities in the Nigerian market (n = 150) Opportunity Mean SD Large and growing population driving demand for mobile services 4.45 0.76 Liberalizing and reforming telecommunications sector 4.18 0.89 Potential for innovation and differentiation in digital and financial services 4.02 0.93 Growing middle class with increasing purchasing power 3.89 0.97 Untapped market segments, such as rural and underserved areas 3.72 1.04 Strategic partnerships and collaborations with local firms and institutions 3.58 1.06 Source: Author’s construct 2024 Table 3: Descriptive statistics for opportunities in the Nigerian market (n = 150) As shown in Table 2, the survey results largely confirmed the opportunities identified in the qualitative analysis. The large and growing population driving demand for mobile services was rated as the most significant opportunity (M = 4.45, SD = 0.76), followed by the liberalizing and reforming telecommunications sector (M = 4.18, SD = 0.89), and the potential for innovation and differentiation in digital and financial services (M = 4.02, SD = 0.93). The growing middle class with increasing purchasing power (M = 3.89, SD = 0.97), untapped market segments, such as rural and underserved areas (M = 3.72, SD = 1.04), and strategic partnerships and collaborations with local firms and institutions (M = 3.58, SD = 1.06) were also rated as significant opportunities, albeit to a lesser extent. To assess the significance of these factors, , a multiple regression analysis was conducted. The dependent variable was the overall perception of opportunities in the Nigerian market, calculated as the average of the six opportunity items in Table 2. The independent variables were the demographic and background characteristics of the survey respondents, including age, gender, education level, job role, and tenure with MTN Nigeria. The results of the regression analysis are presented in Table 4. Market Table 4: Multiple regression analysis for factors influencing perceived opportunities in the Nigerian market (n = 150) Variable B SE β t p (Constant) 2.85 0.42 6.78 0.000 Age 0.02 0.01 0.14 1.67 0.098 Gender (female) -0.08 0.11 -0.06 -0.76 0.447 Education level 0.12 0.08 0.12 1.48 0.141 Job role (manager) 0.19 0.12 0.13 1.60 0.112 Tenure with MTN Nigeria 0.04 0.02 0.17 2.05 0.042 Source: Author’s construct 2024 R² = 0.11, F(5, 144) = 3.48, p = 0.005 Table 4: Multiple regression analysis for factors influencing perceived opportunities in t k ( ) Table 4: Multiple regression analysis for factors influencing perceived opportunities in the Nigerian market (n = 150) 139 AJMBR AJMBR As shown in Table 4, the regression model was significant (F(5, 144) = 3.48, p = 0.005), explaining 11% of the variance in perceived opportunities in the Nigerian market. Among the demographic and background variables, only tenure with MTN Nigeria was a significant predictor of perceived opportunities (β = 0.17, p = 0.042), suggesting that employees and managers with longer tenure tend to have more positive perceptions of the opportunities in the Nigerian market. The other variables, including age, gender, education level, and job role, were not significant predictors of perceived opportunities. Discussion of the Implications for Theory and Practice The findings of this study have several implications for theory and practice. From a theoretical perspective, the study contributes to the ongoing debate on the applicability and relevance of existing internationalization theories and frameworks in the context of emerging markets (Meyer & Peng, 2016; Luo & Zhang, 2016). The case of MTN Nigeria suggests that while traditional theories such as the OLI paradigm (Dunning, 1980) and the Uppsala model (Johanson & Vahlne, 1977) provide useful insights into the motivations and processes of firm internationalization, they need to be adapted and extended to account for the unique challenges and opportunities of emerging markets. In particular, the findings of this study highlight the importance of institutional and contextual factors in shaping the internationalization strategies and outcomes of firms in emerging markets (Peng et al., 2008; Meyer et al., 2009). The weak and unstable regulatory environment, infrastructure deficiencies, and cultural differences in Nigeria posed significant challenges for MTN Nigeria, requiring the company to develop specific capabilities and strategies to navigate these institutional voids (Khanna & Palepu, 2010). At the same time, the large and growing population, liberalizing telecommunications sector, and potential for innovation and differentiation offered significant opportunities for MTN Nigeria to create and capture value in the Nigerian market. Second, the findings of this study highlight the importance of stakeholder engagement and corporate social responsibility in building legitimacy and trust in emerging markets (Ite, 2016; Aminu & Oyefolahan, 2021). MTN Nigeria's investments in education, health, and community development programs, as well as its active participation in industry associations and forums, helped the company to build positive relationships with key stakeholders such as regulators, customers, and communities, and to mitigate some of the political and reputational risks of operating in Nigeria. Third, the case of MTN Nigeria demonstrates the importance of innovation and digitalization in driving growth and competitiveness in emerging markets (Adebiyi & Olowookere, 2018; Aminu & Oyefolahan, 2021). MTN Nigeria's investments in digital and financial services, such as mobile money and e-commerce, not only provided new sources of revenue and differentiation for the company but also contributed to the broader development and inclusion goals of the Nigerian economy and society. Finally, the findings of this study suggest that firms seeking to internationalize in emerging markets need to develop specific capabilities and strategies for risk management, compliance, and resilience (Amaeshi et al., 2016; Macedo & de Matos, 2019). Comparison of the Findings with Existing Literature on Firm Internationalization in Emerging Markets The findings of this study are largely consistent with the existing literature on firm internationalization in emerging markets, particularly in the context of the telecommunications industry. The challenges faced by MTN Nigeria, such as the weak and unstable regulatory environment, infrastructure deficiencies, and intense competition, have been well-documented in previous studies on the internationalization of telecommunications firms in Africa and other emerging markets (Amankwah-Amoah et al., 2018; Adeyoju, 2021; Adebiyi & Olowookere, 2018). For example, Amankwah-Amoah et al. (2018) found that the weak and unstable institutional environment in many African countries, characterized by political instability, corruption, and unpredictable policies, posed significant challenges for foreign telecommunications firms seeking to enter and operate in these markets. Similarly, Adeyoju (2021) highlighted the infrastructure challenges faced by telecommunications firms in Nigeria, such as the high costs of network deployment, the lack of reliable power supply, and the security risks posed by vandalism and theft. The findings of this study also support the existing literature on the opportunities for firm internationalization in emerging markets, particularly in the telecommunications industry. The large and growing population of Nigeria, the liberalizing and reforming telecommunications sector, and the potential for innovation and differentiation in digital and financial services have been identified as key opportunities for foreign firms in previous studies (GSMA, 2021; MTN Nigeria, 2021; Aminu & Oyefolahan, 2021). For instance, the GSMA (2021) report on the mobile economy in sub-Saharan Africa highlighted the significant growth potential of the mobile market in Nigeria, driven by the country's large and young population, increasing urbanization, and rising mobile adoption rates. Similarly, MTN Nigeria's (2021) annual report emphasized the company's strategic focus on digital and financial services, such as mobile money and e-commerce, as key growth areas in the Nigerian market. However, the findings of this study also extend the existing literature by providing a more nuanced and contextualized understanding of the specific challenges and opportunities faced by MTN Nigeria in the Nigerian market. The qualitative analysis, in particular, revealed the complex interplay of factors such as the regulatory environment, Vol. 14, No. 2 2024 African Journal of Management and Business Research 140 www.afropolitanjournals.com 140 Afropolitan Journals infrastructure challenges, competitive dynamics, and cultural differences that shape the internationalization journey of telecommunications firms in Nigeria. Discussion of the Implications for Theory and Practice The case of MTN Nigeria highlights the various strategic, operational, and reputational risks that firms face in emerging markets, such as regulatory sanctions, security incidents, and corruption scandals, and the importance of having robust frameworks and processes for identifying, assessing, and mitigating these risks. 141 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 141 41 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com AJMBR AJMBR Conclusion Summary of the Key Findings These opportunities provided significant potential for growth, profitability, and value creation for MTN Nigeria, enabling the company to establish a leading position in the Second, the study has highlighted the key opportunities that MTN Nigeria has capitalized on in the Nigerian market, including the large and growing population driving demand for mobile services, the liberalizing and reforming telecommunications sector, the potential for innovation and differentiation in digital and financial services, the growing middle class with increasing purchasing power, the untapped market segments such as rural and underserved areas and the strategic partnerships and collaborations with local firms and institutions Conclusion Summary of the Key Findings This study has examined the challenges and opportunities of firm internationalization in emerging markets, using MTN Nigeria as a case study. Through a mixed-methods approach involving semi-structured interviews, document analysis, and a survey of MTN Nigeria employees and managers, the study has generated several key findings. First, the study has identified the main challenges faced by MTN Nigeria in internationalizing to the Nigerian market, including the weak and unstable regulatory environment, infrastructure deficiencies and security risks, intense competition from foreign and local players, cultural differences and local business practices, lack of skilled and experienced local talent, and corruption and unethical practices in the business environment. These challenges posed significant strategic, operational, and reputational risks for MTN Nigeria, requiring the company to develop specific capabilities and strategies to navigate the institutional and cultural complexities of the Nigerian market. Second, the study has highlighted the key opportunities that MTN Nigeria has capitalized on in the Nigerian market, including the large and growing population driving demand for mobile services, the liberalizing and reforming telecommunications sector, the potential for innovation and differentiation in digital and financial services, the growing middle class with increasing purchasing power, the untapped market segments such as rural and underserved areas, and the strategic partnerships and collaborations with local firms and institutions. These opportunities provided significant potential for growth, profitability, and value creation for MTN Nigeria, enabling the company to establish a leading position in the Nigerian telecommunications industry. Finally, the study has compared the findings with the existing literature on firm internationalization in emerging markets, highlighting the contributions and implications of the MTN Nigeria case study for theory and practice. The study has shown how the unique challenges and opportunities of the Nigerian market, as well as the specific strategies and capabilities employed by MTN Nigeria, provide valuable insights and lessons for firms seeking to enter and operate in emerging markets, particularly in the telecommunications industry. Second, the study has highlighted the key opportunities that MTN Nigeria has capitalized on in the Nigerian market, including the large and growing population driving demand for mobile services, the liberalizing and reforming telecommunications sector, the potential for innovation and differentiation in digital and financial services, the growing middle class with increasing purchasing power, the untapped market segments such as rural and underserved areas, and the strategic partnerships and collaborations with local firms and institutions. Limitations of the Study While this study has generated significant insights and contributions to the literature on firm internationalization in emerging markets, it also has some limitations that should be acknowledged and addressed in future research. First, the study is based on a single case study of MTN Nigeria, which may limit the generalizability of the findings to other firms, industries, and countries. Although the Nigerian telecommunications industry provides a relevant and representative context for examining firm internationalization in emerging markets, the specific challenges and opportunities faced by MTN Nigeria may not be applicable or relevant to other firms or industries. Future research could examine multiple case studies of firms from different industries and countries to provide a more comprehensive and comparative understanding of firm internationalization in emerging markets. Second, the study relies largely on qualitative data from interviews and documents, which may be subject to biases and interpretations of the researchers and participants. Although the study has attempted to triangulate the findings with quantitative data from a survey, the sample size and representativeness of the survey may be limited. Future research could employ larger and more representative samples, as well as more rigorous and objective measures, to validate and extend the findings of this study. Contributions to the Existing Literature This study makes several contributions to the existing literature on firm internationalization in emerging markets. First, the study provides a detailed and nuanced analysis of the specific challenges and opportunities faced by a telecommunications firm in the Nigerian market, extending the findings of previous studies that have examined the general factors affecting firm internationalization in Africa and other emerging markets (Amankwah- Amoah et al., 2018; Adeyoju, 2021; Adebiyi & Olowookere, 2018). By combining qualitative and quantitative data from multiple sources, the study offers a comprehensive and triangulated understanding of the complex and multifaceted nature of firm internationalization in Nigeria. Second, the study contributes to the theoretical development of the field by highlighting the limitations and boundary conditions of existing Vol. 14, No. 2 2024 African Journal of Management and Business Research 142 www.afropolitanjournals.com 142 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com Afropolitan Journals internationalization theories and frameworks, such as the OLI paradigm (Dunning, 1980) and the Uppsala model (Johanson & Vahlne, 1977), in the context of emerging markets. The findings of the study suggest that these theories need to be adapted and extended to account for the unique institutional, cultural, and competitive challenges of emerging markets, as well as the specific strategies and capabilities employed by firms to navigate these challenges (Meyer & Peng, 2016; Luo & Zhang, 2016). Recommendations Adopt a long-term and sustainable perspective: View internationalization in emerging markets as a marathon rather than a sprint, commit resources and capabilities for the long haul, balance global integration and local responsiveness, and align business goals with social and environmental responsibilities to create shared value for the firm and its stakeholders. Recommendations for Future Research Based on the findings and limitations of this study, several recommendations can be made for future research on firm internationalization in emerging markets. First, future research could examine the challenges and opportunities of firm internationalization in other emerging markets beyond Nigeria, such as India, China, Brazil, and South Africa. These countries present different institutional, cultural, and competitive contexts that may require different strategies and capabilities for firms seeking to enter and operate in these markets. Comparative studies of firm internationalization across different emerging markets could provide valuable insights into the similarities and differences in the factors and processes that shape firm internationalization in these markets. Future research could also adopt a more critical and normative approach to examining the performance and impact of firm internationalization in emerging markets, taking into account the social, economic, and environmental implications of firm operations for different stakeholders. Studies that examine the positive and negative externalities of firm internationalization, such as job creation and destruction, income inequality, environmental degradation, and cultural change, could provide valuable insights into the broader societal and developmental implications of firm internationalization in emerging markets. Such studies could also inform policy and practice on how to promote more inclusive, sustainable, and responsible forms of firm internationalization in emerging markets. References Adebiyi, J. A., & Olowookere, E. I. (2018). The impact of foreign direct investment on firm performance in Nigeria: A study of MTN Nigeria. Journal of Economics and Sustainable Development, 9(14), 101- 110. Adeola, O., Boso, N., Olaniyi, S., & Ogbanufe, O. (2020). The telecommunications industry in Nigeria: Growth, structure, and performance. In J. Amankwah-Amoah, S. Osabutey, & I. Egbetokun (Eds.), Meeting Africa's Challenges in the 21st Century (pp. 101-122). Palgrave Macmillan. Recommendations i. Develop cultural intelligence and adaptability: Invest in cultural training and immersion programs for managers and employees, localize products, services, and operations to the needs and preferences of local consumers and stakeholders, and cultivate cultural competence and flexibility to learn from and adjust to the changing cultural environment. ii. Build strategic local partnerships and collaborations: Identify and engage with local partners such as suppliers, distributors, and customers, develop long-term and mutually beneficial relationships, participate in local business and social networks, and contribute to the development and welfare of local communities to build trust and legitimacy. iii. Leverage innovation and digitalization: Embrace digital technologies and platforms to enhance efficiency, agility, and customer experience, invest in research and development to create innovative products and services tailored to the local 143 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 143 AJMBR AJMBR market, and develop dynamic capabilities to sense, seize, and reconfigure resources and skills in response to emerging opportunities and challenges. market, and develop dynamic capabilities to sense, seize, and reconfigure resources and skills in response to emerging opportunities and challenges. iv. Navigate institutional voids through non-market strategies: Engage in corporate social responsibility and community development initiatives to build goodwill and mitigate risks arising from weak institutions, develop political ties and relationships with key stakeholders to influence the regulatory environment, and adopt flexible and adaptive organizational structures to cope with uncertainty and volatility. iv. Navigate institutional voids through non-market strategies: Engage in corporate social responsibility and community development initiatives to build goodwill and mitigate risks arising from weak institutions, develop political ties and relationships with key stakeholders to influence the regulatory environment, and adopt flexible and adaptive organizational structures to cope with uncertainty and volatility. v. Adopt a long-term and sustainable perspective: View internationalization in emerging markets as a marathon rather than a sprint, commit resources and capabilities for the long haul, balance global integration and local responsiveness, and align business goals with social and environmental responsibilities to create shared value for the firm and its stakeholders. v. Adopt a long-term and sustainable perspective: View internationalization in emerging markets as a marathon rather than a sprint, commit resources and capabilities for the long haul, balance global integration and local responsiveness, and align business goals with social and environmental responsibilities to create shared value for the firm and its stakeholders. v. Adeola, O., Boso, N., Olaniyi, S., & Ogbanufe, O. (2020). The telecommunications industry in Nigeria: Growth, structure, and performance. In J. Amankwah-Amoah, S. Osabutey, & I. Egbetokun (Eds.), Meeting Africa's Challenges in the 21st Century (pp. 101-122). Palgrave Macmillan. References Adebiyi, J. A., & Olowookere, E. I. (2018). The impact of foreign direct investment on firm performance in Nigeria: A study of MTN Nigeria. Journal of Economics and Sustainable Development, 9(14), 101- 110. References Vol. 14, No. 2 2024 African Journal of Management and Business Research 144 www.afropolitanjournals.com Vol. 14, No. 2 2024 African Journal of Management and Business Research 14 www.afropolitanjournals.com 144 Afropolitan Journals Adeyeye, A. D., Jegede, O. O., & Akinwale, Y. O. (2019). The impact of technology infrastructure on firm productivity in Nigeria. International Journal of Business and Economic Sciences Applied Research, 12(3), 7-18. Adeyoju, O. (2021). MTN Nigeria and the challenges of doing business in Nigeria. Journal of African Business, 22(4), 541-558. Amaeshi, K., Adegbite, E., & Rajwani, T. (2016). Corporate social responsibility in challenging and non- enabling institutional contexts: Do institutional voids matter? Journal of Business Ethics, 134(1), 135-153. Amankwah-Amoah, J., & Debrah, Y. A. (2017). Toward a construct of liability of origin. Industrial and Corporate Change, 26(2), 211-231. Amankwah-Amoah, J., Boso, N., & Debrah, Y. A. (2018). Africa rising in an emerging world: An international marketing perspective. International Marketing Review, 35(4), 550-559. Aminu, A. I., & Oyefolahan, I. O. (2021). Determinants of mobile money adoption in Nigeria: A technology acceptance model approach. African Journal of Science, Technology, Innovation and Development, 13(6), 739-750. Ault, J. K., & Spicer, A. (2014). The institutional context of poverty: State fragility as a predictor of cross- national variation in commercial microfinance lending. Strategic Management Journal, 35(12), 1818-1838. Bell, E., Bryman, A., & Harley, B. (2018). Business research methods. Oxford University Press. Boojihawon, D. K., & Acholonu, K. K. (2013). Internationalisation process of African banks: An exploratory study. African Journal of Economic and Management Studies, 4(2), 244-266. Braun, V., & Clarke, V. (2006). Using thematic analysis in psychology. Qualitative Research in Psychology, 3(2), 77-101. Brinkmann, S., & Kvale, S. (2015). InterViews: Learning the craft of qualitative research interviewing (3rd ed.). Sage. Brouthers, K. D., & Hennart, J. F. (2007). Boundaries of the firm: Insights from international entry mode research. Journal of Management, 33(3), 395-425. Buckley, P. J., & Casson, M. C. (2009). The internalisation theory of the multinational enterprise: A review of the progress of a research agenda after 30 years. Journal of International Business Studies, 40(9), 1563-1580. Cavusgil, S. T., Deligonul, S., Ghauri, P. N., & Xu, S. (2021). The new frontiers of international business research. Journal of International Business Studies, 52(1), 1-16. Chironga, M., Desvaux, G., & Leke, A. (2018). Leadership lessons from Africa's trailblazers. McKinsey Quarterly, (1), 116-125. Cohen, J. (1992). A power primer. Psychological Bulletin, 112(1), 155-159. Creswell, J. References W., & Plano Clark, V. L. (2018). Designing and conducting mixed methods research. Sage. Cuervo-Cazurra, A., Luo, Y., Ramamurti, R., & Ang, S. H. (2018). The impact of the home country on internationalization. Journal of World Business, 53(5), 593-604. Deloitte. (2014). The future of telecoms in Africa: The "blueprint for the brave." Deloitte Touche Tohmatsu Limited. Deng, P., & Yang, M. (2015). Cross-border mergers and acquisitions by emerging market firms: A comparative investigation. International Business Review, 24(1), 157-172. Deng, P., Delios, A., & Peng, M. W. (2020). A geographic relational perspective on the internationalization of emerging market firms. Journal of International Business Studies, 51(1), 50-71. Dunning, J. H. (1980). Toward an eclectic theory of international production: Some empirical tests. Journal of International Business Studies, 11(1), 9-31. Eisenhardt, K. M. (1989). Building theories from case study research. Academy of Management Review, 14(4), 532-550. 145 AJMBR AJMBR Eisenhardt, K. M., & Graebner, M. E. (2007). Theory building from cases: Opportunities and challenges. Academy of Management Journal, 50(1), 25-32. Field, A. (2013). Discovering statistics using IBM SPSS statistics (4th ed.). Sage. Glaser, B. G., & Strauss, A. L. (1967). The discovery of grounded theory: Strategies for qualitative research. Aldine. GSMA. (2021). The mobile economy sub-Saharan Africa 2021. GSM Association. Guest, G., Bunce, A., & Johnson, L. (2006). How many interviews are enough? An experiment with data saturation and variability. Field Methods, 18(1), 59-82. Guillen, M. F., & Garcia-Canal, E. (2009). The American model of the multinational firm and the "new" multinationals from emerging economies. Academy of Management Perspectives, 23(2), 23-35. Hair, J. F., Black, W. C., Babin, B. J., & Anderson, R. E. (2014). Multivariate data analysis (7th ed.). Pearson Education Limited. Helfat, C. E., Finkelstein, S., Mitchell, W., Peteraf, M., Singh, H., Teece, D., & Winter, S. G. (2007). Dynamic capabilities: Understanding strategic change in organizations. Blackwell. Hitt, M. A., Tihanyi, L., Miller, T., & Connelly, B. (2006). International diversification: Antecedents, outcomes, and moderators. Journal of Management, 32(6), 831-867. Hoskisson, R. E., Wright, M., Filatotchev, I., & Peng, M. W. (2013). Emerging multinationals from mid- range economies: The influence of institutions and factor markets. Journal of Management Studies, 50(7), 1295-1321. Hult, G. T. M., Ketchen Jr, D. J., Griffith, D. A., Chabowski, B. R., Hamman, M. K., Dykes, B. J., ... & Cavusgil, S. T. (2008). An assessment of the measurement of performance in international business research. References Journal of International Business Studies, 39(6), 1064-1080. Hurmerinta-Peltomäki, L., & Nummela, N. (2006). Mixed methods in international business research: A value-added perspective. Management International Review, 46(4), 439-459. Ite, U. E. (2016). Multinationals and corporate social responsibility in developing countries: A case study of Nigeria. Corporate Social Responsibility and Environmental Management, 11(1), 1-11. Johanson, J., & Vahlne, J. E. (1977). The internationalization process of the firm—a model of knowledge development and increasing foreign market commitments. Journal of International Business Studies, 8(1), 23-32). Khanna, T., & Palepu, K. G. (2010). Winning in emerging markets: A road map for strategy and execution. Harvard Business Press. London, T., & Hart, S. L. (2004). Reinventing strategies for emerging markets: Beyond the transnational model. Journal of International Business Studies, 35(5), 350-370. Luo, Y., & Bu, J. (2018). Contextualizing international strategy by emerging market firms: A composition- based approach. Journal of World Business, 53(3), 337-355. Luo, Y., & Bu, J. (2018). Contextualizing international strategy by emerging market firms: A composition- based approach. Journal of World Business, 53(3), 337-355. Luo Y & Tung R L (2007) International expansion of emerging market enterprises: A springboard Luo, Y., & Tung, R. L. (2007). International expansion of emerging market enterprises: A springboard perspective. Journal of International Business Studies, 38(4), 481-498. Macedo, M., & de Matos, G. (2019). Market reaction to regulatory sanctions against telecommunication operators in Brazil and Nigeria. Corporate Reputation Review, 22(3), 119-130. Manyika, J., Lund, S., Bughin, J., Woetzel, J., Stamenov, K., & Dhingra, D. (2016). Digital globalization: The new era of global flows. McKinsey Global Institute. Meyer, K. E., & Peng, M. W. (2016). Theoretical foundations of emerging economy business research. Journal of International Business Studies, 47(1), 3-22. Meyer, K. E., Li, C., & Schotter, A. P. (2020). Managing the MNE subsidiary: Advancing a multi-level and dynamic research agenda. Journal of International Business Studies, 51(4), 538-576. MTN Nigeria. (2021). Annual report 2020. MTN Nigeria Communications Plc. MTN Nigeria. (2021). Annual report 2020. MTN Nigeria Communications Pl Oladimeji, M. S., Ebodaghe, A. T., & Shobayo, P. B. (2018). Effect of globalization on telecommunication industry in Nigeria. American Economic & Social Review, 2(1), 10-20. 146 Afropolitan Journals Oyewole, P., Usim, U., Sotunde, O., & Alleyne, A. (2018). Telecommunications in Nigeria. Journal of Strategic Innovation and Sustainability, 13(5), 77-88. Patton, M. Q. (2015). Qualitative research & evaluation methods: Integrating theory and practice (4th ed.). Sage. Peng, M. W., Wang, D. References Y., & Jiang, Y. (2008). An institution-based view of international business strategy: A focus on emerging economies. Journal of International Business Studies, 39(5), 920-936. Robinson, O. C. (2014). Sampling in interview-based qualitative research: A theoretical and practical guide. Qualitative Research in Psychology, 11(1), 25-41. Rottig, D. (2016). Institutions and emerging markets: Effects and implications for multinational corporations. International Journal of Emerging Markets, 11(1), 2-17. Sheth, J. N. (2011). Impact of emerging markets on marketing: Rethinking existing perspectives and practices. Journal of Marketing, 75(4), 166-182. Teece, D. J., Pisano, G., & Shuen, A. (1997). Dynamic capabilities and strategic management. Strategic Management Journal, 18(7), 509-533. Wiles, R. (2012). What are qualitative research ethics? A&C Black. Wiles, R. (2012). What are qualitative research ethics? A&C Black. Wiles, R. (2012). What are qualitative research ethics? A&C Black. 147 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com 147 47 Vol. 14, No. 2 2024 African Journal of Management and Business Research www.afropolitanjournals.com
https://openalex.org/W2002666258	https://zenodo.org/records/2340148/files/article.pdf	German	null	Die Synthese der Depside der Pyrogallolcarbonsäure	Journal für praktische Chemie	1,914	public-domain	2,503	302 Mau t hner: Synth. d. Depside d. Pyrogallolcarbonsaure. 302 Mau t hner: Synth. d. Depside d. Pyrogallolcarbonsaure. I) Dies. Journ. [2] 84, 140 (1911); 85, 308 (1912); 87, 409 (1913). Ber. 42, 188 (1909). I) A. a. 0. s, Dies. Journ. [Z] 87, 403 (1913). 3 Ann. Chem. 340, 226. ) Ber. 21, 2024 (1888). Die Synthese der Depside der Pyrogallolcarbonsaure ; F. Mauthner. von In friiheren Untersuchungenl) berichtete ich uber die Ver- kettung der Gallussaure, Paraoxybenzoesaure, Metaoxybenzoe- saure und Protocatechusiiure mit den verschiedenen Phenol- carbonsauren und stellte die Ausdehnung meiner Versuche auf andere Oxycarbonsiiuren in Aussicht. Von den ubrigen Phenol- carbonsauren beansprucht die Pyrogallolcarbonsaure, wegen ihres h&ufigen Vorkommens als Spaltungsprodukt von ver- schiedenen P5anzenstoffen, ein besonderes Interesse und wahr- scheinlich werden ahnliche esterartige Verkettungsprodukte mit anderen Phenolcarbonsauren in der Natur auch vorkommen. Zur Synthese der Depside der Pyrogallolcarbonsaure wahlte ich ds Ausgangskorper das vor mehreren Jahren von mir entdeckte a) Chlorid der Trimethylpyrogallolcarbonsiiure, dessen Verwendbarkeit bei Kondensationsreaktionen, in Gegenwart von tertiaren Basen ich bereits damals schon dargetan habe. Durch Kuppelung des Trimethylpyrogalloylchlorids in alka- lischer Losung bei niedriger Temperatur mit Paraoxybenzoe- saure entsteht die Trimethylpyrogalloylparaoxybenzoesaure. Der Trimethylpyrogalloylparaoxybenzoesliuremethylester konnte durch Eondensation des Paraoxybenzoesauremethylesters mit dem Chlorid der Trimethylpyrogallolcarbonsiiure gewonnen werden. Bus der Metaoxybenzoesaure und deren Methylester konnten mit dem Trimethylpyrogalloylchlorid durch Euppelung einerseits die Trimethylpyrogalloylmetaoxybenzoesaure und andererseits der Trimethylpyrogalloylme taoxybenzoesiiuremethyl- ester erhalten werden. Auf ganz gleichem Wege wurden dar- 303 Maut hner: Synth. d. Depside d.Pyrogallolcarbonsaure. t hner: Synth. d. Depside d.Pyrogallolcarbonsaure. Maut hner: Synth. d. Depside d.Pyrogallolcarbonsaure. gestellt aus Vanillinsaure und ihrem Methylester die Trimethyl- pyrogalloylvanillinsiiure und der Trimethylpyrogalloylvanillin- sauremethylester. Desgleichen wurde die schon friiher bei Kuppelungsreaktionen angewandte 2,3-OxynaphtoesLure in den Kreis der Untersuchung gezogen. Hier konnte die Konden- sation in ahnlicher Weise bewerkstelligt werden wie bei fruheren Arbeitenl), und zwar mit Hilfe von tertiaren Baeen. So entsteht aus dem Chlorid der Trimethylpyrogallolcarbon- saure und der 2,3-0xynaphtoesiiure die Trimethylpyrogalloyl- 2,3-oxynaphtoesaure. Alle diese SyntheRen beweisen die Zweck- ma6igkeit dieses Verfahrens flir die Synthese der esterartig verketteten Phenolcarbonsauren. 2,3,4-Trimethoxybenzanilid. Die zu nachfolgenden Versuchen notige Trimethylpyro- gallolcarbonsaure ist zuerst von W. W i 1 la) durch Alkylierung des Pyrogallolcarbonsauremethylesters mit Jodmethyl und nach- folgender Verseifung gewonnen worden. Graebe und Suter3) stellten diese Verbindung nach einer bequemeren Weise, nam- lich durch Alkylierung der Protocatechusaure mittels Dimethyl- sulfat dar. Fur die Darstellung grofierer Mengen dieser Substanz hat sich eine ahnliche Arbeitsweise, wie fiir die Di- methyl-a-resorcylsaure angegeben als zweckmaBig erwiesen. y y g g g In einem Jenaer Literkolben werden 60 g Natriumhydroxyd in 300 ccm Wasser gelost, auf Zimmertemperatur abgekiihlt und 50 g Pyrogallolcarbonsaure hinzugegeben. Z u der noch- male abgekiihlten Losung fiigt man 50 ccm Dimethylsulfat und schiittelt den gut verkorkten Kolben unter aufierer Kiihlung mit kaltem Wasser. Man schiittelt das Reaktionsgemisch unter zeitweisem Luften des Korkes. Eiernach werden noch- male 50 ccm Dimethylsulfat hinzugegeben uud, wie oben an- gegeben, 7 Minuten lang geschuttelt. Die Reaktionsfliissigkeit wird hierauf am BuckfluBkiihler 2 Stunden lang im Sieden er- 304 304 Ma u t h n e r : Synth. d. Depside d. Pyrogallolcar bonsaure. 04 Ma u t h n e r : Synth. d. Depside d. Pyrogallolcar bonsaure. halten. Man fiigt dann zur Verseifung des gebildeten Esters 10 g Natriumhydroxyd hineu und erhitzt nocbmals 2 Stunden lang. Nach dem valligen Erkalten wird die Dimethylpyro- gallolcarbonsiiure mit verdiinnter Salzsaure ausgefallt, an der Nutsche abgesaugt, mit Wasser gut ausgewascben und auf einem Tonteller getrocknet. Ausbeute 44 g. Die so gewonnene Ver- bindung wurde in einer Lasung von 36g Natriumhydroxyd in 250 ccm Wasser geliist und mit 60 ccm Dimethylsulfat, welcbes in zwei Anteilen hinzugefiigt wurde, in der friiher angegebenen Weise methyliert. Die Bthersiiure wurde aus der erkalteten Losung mit verdiinnter Salzeaure gefallt , filtriert und mit Wasser gut ausgewaschen. Zur Reinigung der Siiure wurde dieselbe in eine Mischung von 20 g Natriumbicarbonat und 150 ccm Wasser eingetragen, unter Erwarmen geliist und die filtrierte Losung mit Salzsgure wieder gefallt. Die Verbindung wurde zuniichst am Tonteller und dann im Dampftrocken- scbrank getrocknet. Ausbeute zwiscben 28 und 35 g. Die Umwandlung der Siiure in das Chlorid wurde in der friiher angegebenen Weise auegefiihrt. l) Zur niiheren Charak- terisierung des Saurechlorids wurde noch das Anilid dar- gestellt. 2 g Trimethylpyrogalloylchlorid wurden in 20 ccm i t h e r gelost und mit einer atherischen Losung (10 ccm) von 1 g Anilin versetzt. l) Ber. 42, 194 (1909). l) Ber. 42, 194 (1909). Trim e t h y 1 p y r ogallo y 1 para oxy b enz o esaure. Trim e t h y 1 p y r ogallo y 1 para oxy b enz o esaure. Die Kuppelung des Trimethylpyrogalloylchlorids erfolgt gut mit der Paraoxybenzoesaure in alkalischer Losung bei An- wendung von niedriger Temperatur. g g p 2,2 g Paraoxybenzoesaure wurden in einer Losung von 1,4 g Natriumhydroxyd, 20 ccrn Wasser und 15 ccm Aceton gelost und in einer Kaltemischung auf -loo abgekuhlt. Im Laufe von 7 Minuten fiigt man eine Losung von 4,l g Tri- methylpyrogalloylchlorid in 20 ccm Aceton in kleinen Anteilen unter jedesmaligem kraftigen Schutteln hinzu. Man la6t das Reaktionsgemisch 15 Minuten lang stehen und gieBt die Lbsung hierauf in ein Gemisch von 10 ccm konzentrierter Salzsaure und 300 ccm Wasser , wobei das Kondensationsprodukt aus- fallt. Zur vollstandigen Ausscheidung wird es einige Stunden in Eiswasser gestellt, abgesaugt, mit Wasser gut ausgewaschen und auf einem Tonteller getrocknet. Ausbeute 1,6 g. Zur weiteren Reinigung wird es in einer Natriumbicarbonatlosung gelost, filtriert und mit verdiinnter Salzsaure ausgefallt. Dann wird die Verbindung aus verdiinntem Aceton umkrystallisiert. g y 0,1470 g gaben 0,3311 g CO, und 0,0634 g H,O. Berechnet fur C,,H,,O, : Gefunden : C 61,44 81,42 Ol0 H 4,82 4,79 1, . 0,1470 g gaben 0,3311 g CO, und 0,0634 g H,O. Berechnet fur C,,H,,O, : Gefunden : C 61,44 81,42 Ol0 H 4,82 4,79 1, . Das Kondensationsprodukt krystallisiert in farblosen Nadeln, die bei 181O-1820 schmelzen. Die Substanz lost sich lsicht in Beuzol, Aceton und Alkohol. In Ligroin und Petrolather ist der Korper fast unliislich. M it u t h n e r : Synth. d. Depside d. Pyrogallolcarbonsaure. 3 M it u t h n e r : Synth. d. Depside d. Pyrogallolcarbonsaure. M it u t h n e r : Synth. d. Depside d. Pyrogallolcarbonsaure. 305 M it u t h n e r : Synth. d. Depside d. Pyrogallolcarbonsaure. 305 2,3,4-Trimethoxybenzanilid. Nacb dem Verdunstenlassen des Liisungsmittels wurde der Riickstand mit Wasser versetzt, abgesaugt, mit ver- diinnter Salzsilure, dann rnit Wasser gut ausgewascben und auf einem Tonteller getrocknet. Ausbeute 1,6 g. Bus Ligroin um- krystallisiert bildet es farblose Nadeln, die bei 103°-1040 schmelzen. 0,1570 g gaben 0,3844 g CO, und 0,0832 g H,O. 0,1680 g gaben 7,05 ccm N bei 18O und 761 mm. Berechnet fur C,,H,,O,N: - Gefunden: C 66,89 66,75 N 4,87 4,80 ,, . H 5,97 5.87 99 0,1570 g gaben 0,3844 g CO, und 0,0832 g H,O. 0,1680 g gaben 7,05 ccm N bei 18O und 761 mm. Die Substanz lost sich leicht in Benzol, Alkohol und Ligroin. In Petrolather ist die Verbindung sebr schwer liislich. g g Journal f. prakt. Chemie [2] Rd. 69. 20 T r i m e thy 1 p r o g a 11 o y 1 par ao x y b en z o e s a 11 r e m e t h y 1 e s t e r . Die Eondensation wird wie folgt ausgefiihrt: 3,l g Para- oxybenzoesauremethylester werden in einer erkalteten Mischung von 0,9 g Natriumhydroxyd und 20 ccm Wasser gelost. Man fugt dann 15 ccm Aceton hinzu und kiihlt das Reaktions- gemisch auf -loo ab. In kleinen Anteilen fiigt man dann eine Liisung von 4,6 g Trimethylpyrogalloylchlorid in 25 ccm Aceton im Laufe von 7 Minuten hinzu und schuttelt jedesmal kraftig durch. Man laSt noch 20 Minuten lang das Beaktions- gemisch stehen, gie6t dann in 300 ccm Eiswasser und fiigt 20 g Journal f. prakt. Chemie [2] Rd. 69. 20 20 306 l a u thner: Sgnth. d. Depside d.Pyrogallolcarbonsllure. 306 einige Tropfen verdiinnter Natronlauge bis zur alkalischen Reaktion hinzu. Das Reaktionsgemisch wird einige Stunden lang in Eiswasser stehen gelassen, dann abgenutscht und mit Wasser gut ausgewaschen. Nach dem Trocknen auf einem Ton- teller wird es aus verdunntem Methylalkohol umkrystallisiert. Ausbeute 1,5 g. 0,1609 g gaben 0,3675 g CO, und 0,0779 g H,O. Berechnet fiir CI8Hl8O,: Gefunden : C 62,42 62,27 O/,, H 5,20 5,37 7 9 Der K6rper krystallisiert in farblosen Nadeln, die bei 129°-1300 schmelzen. Die Verbindung lost sich leicht in Benzol, Alkohol und Aceton. In Ligroin ist die Substanz schwer loslich, in Petroleumather fast unloslich. Mau t h n er : Synth. d. Depside d. Pyrogallolcarbons~ure 307 Trimethylpyrogalloylmetaoxybenzoesaure- methylester. 2,l g Metaoxybenzoesauremethylester wurden in einer Lo- sung von 1,2 g Natriumhydroxyd und 20 ccm Wasser aufgelost, hierauf mit noch 15 ccm Aceton versetzt. Die Losung wurde in einer Kaltemischung abgekiihlt und dann portionenweise mit 3 g Trimethylpyrogalloylchlorid, welches in Aceton (25 ccm) gelost war, unter Schiitteln hinzugefugt. Das Beaktionsgemisch blieb noch 'I4 Stunde lang stehen, wurde dann in 250 ccm Eis- wasser gegossen, schwach alkalisch gemacht und einige Stunden lang in Eis stehen gelassen. Das filtrierte und gut aus- gewaschene Produkt wurde auf einem Tonteller getrocknet. Bus verdiinntem Methylalkohol umkrystallisiert bildet es farblose Nadeln, die bei 8Oo--8l0 schmelzen. Ausbeute 1,s g. 0,1497 g gaben 0,3425 g CO, und 0,0716 g H,O. Berechnet fur C,,H,,O, : Gefunden : C 6%,42 62,39 Q/o H 5,20 5131 91 - Die Substanz lost sich leicht in Benzol, Aceton, Alkohol und Ligroin in der Warme. I n Petroleumiither ist der Korper sehr schwer loslich in der Kllte, leichter dagegen in der Wiirme. Trimethylpyrogalloylmetaoxy benzoeslure. 2,2 g Metaoxybenzoesaure wurden in einer Mischung von 1,8 g Natriumhydroxyd, 20 ccm Wasser und 15 ccm Aceton gelost. Zu der in einer Eiiltemischung abgekuhlten Losung fiigt man unter Schiitteln im Laufe von 7 Minuten eine Losung von 4,l g Trimethylpyrogalloylchlorid in 20 ccm Aceton. Nach 20 Minuten wird das Reaktionsgemisch in ein eiskaltes Gemisch von 10 ccm konz. Salzsaure und 300 ccm Wasser gegossen. Es wird nach mehrstiindigem Stehen in Eiswasser abfiltriert, mit Waeser grundlich ausgewaschen und in einer verdiinnten Natriumbicarbonatldsung aufgeliist. Die durch Salzsaure ge- fallte Substanz wird auf einem Tonteller getrocknet und aus verdunntem Aceton umkrystallisiert. Ausbeute 3,3 g. 0,1544 g gaben 0,3469 g CO, und 0,0679 g H,O. Berechnet fir C,,H,,O,: Gefunden : C 61,44 61,26 Olio H 4,81 4,88 ,, - Gefunden : 61,26 Olio 4,88 ,, - Das Kondensationsprodukt krystallisiert in farblosen Nadeln, die bei 145O-146O schmelzen. Die Substanz lost sich leicht in Benzol, Alkohol, Aceton und Chloroform. Der Korper ist loslich in waimem Ligroin, dagegen ist er in Petroleumather fast unloslich. Mau t h n er : Synth. d. Depside d. Pyrogallolcarbons~ure I ) -4nn. Chem. 801, 7. Trim e t h y 1 p y r o g a 11 o y 1 vanillin s iiur e. In einer Lbsung von 1,5 g Natriumhydroxyd, 20 ccm Wasser und 15 ccm Aceton lost man 2 g Vanillinsiiure auf und kiihlt das lbaktionsgemisch in einer Kiiltemischung ab. Dann setzt man eine Lbsung von 3 g Trimethylpyrogallolsaurechlorid in 20 ccm Aceton portionenweise unter jeweiligem kraftigen Schiitteln hinzu. Nachdem das Reaktionsgemisch 25 Minuten lang gestanden hat, wird es in eine Mischung von 10 ccm Salzsaure und 250 ccm Wasser gegossen und 2-3 Stunden lang in Eiswasser stehen gelassen. Das filtrierte und mit Wasser gut ausgewaschene Produkt wird in verdiinnter Natrium - bicarbonatlosung gelost, filtriert, dann mit Salzskure wieder ausgefallt. Bus verdiinntem Aceton umkrystallisiert bildet es farblose Nadeln, die bei 188O-189O schmelzen. * 20 * 308 Ma u t hn e r : Synth. d. Depside d. Pyrogallolcarbonslure 308 Ma u t hn e r : Synth. d. Depside d. Pyrogallolcarbonslure. 0,1664 g gaben 0,3624 g CO, und 0,0736 g H,O. Berechnet fiir CI8H,,O, : Gefunden: C 59,70 59,48 O/,, H 5,OO 4191 i i * 0,1664 g gaben 0,3624 g CO, und 0,0736 g H,O. Berechnet fiir CI8H,,O, : Gefunden: C 59,70 59,48 O/,, H 5,OO 4191 i i * Die Verbindung ist leicht liislich in Benzol, Aceton, A1- kohol und Chloroform. In Ligroio ist der Korper in der Wiirme sehr schwer loslich und in Petrolather fast unlbslicb. Trim e t h y 1 p y r o g a 11 o y 1 van i 11 ins a u r e m e t h y 1 ester. Trim e t h y 1 p y r o g a 11 o y 1 van i 11 ins a u r e m e t h y 1 ester. 2,3 g Vanillinsauremethylester wurden in einem Gemisch von OJ6 g Natriumhydroxyd, 20 ccm Wssser und 7 ccm Aceton aufgelost. Die Losung wurde in einer Kaltemischung abgekuhlt und mit 3 g in Aceton (20 ccm) geliistem Trimethylpyrogalloyl- chlorid unter starkem Schutteln nach und nach versetzt. Nach- dem das Reaktionsgemisch 20 Minuten lang gestanden hat, wird es in 300 ccm Wasser gegossen, mit Natronlauge schwach alkalisch gemacht und einige Stunden lang in Eiswasser stehen gelassen. Die abgesaugten Krystalle wurden auf einem Tonteller getrocknet und wogen 1,5 g. Bus verdunntem Methylalkohol umkrystallisiert bildet das Produkt farblose Nadeln, die bei 97O-98O schmelzen. 0,1561 g gaben 0,3472 g CO, und 0,0745 g H,O. Berechnet fur C,,H,,O, : Gefunden: C 60,63 60,65 H 5,32 5330 i i * 0,1561 g gaben 0,3472 g CO, und 0,0745 g H,O. Berechnet fur C,,H,,O, : Gefunden: C 60,63 60,65 H 5,32 5330 i i * 60,65 5330 i i * In Alkohol, Benzol und Aceton ist die Substanz leicht loslich. In Ligroin lost sich der Korper leicht in der Warme. Petroleumather lost die Verbindung sehr schwer in der Wtirme. Trimethylpyrogalloyl-2-oxy-3-naphtoesiiure. Die Kondensation des Trimethylpyrogalloylchlorides mit der 2-Oxy-3-naphtoesaure erfolgt gut bei Anwendung einer tertilren Bade nach dem Verfahren von Einh0rn.l) In ein Gemisch von 80 ccm Benzol und 25 g Dimethylanilin werden 8,3 g Oxynaphtoesaure unter Erwarmen aufgelost und nach dem Abkuhlen auf Zimmertemperatur mit einer Liisung von 2,8 g Trimethylpyrogalloylchlorid in 30 ccm Benzol auf einmal 309 Mau thner: Synth. d. Depside d. Pyrogallolcarbonsaure. Mau thner: Synth. d. Depside d. Pyrogallolcarbonsaure. Mau thner: Synth. d. Depside d. Pyrogallolcarbonsaure. u thner: Synth. d. Depside d. Pyrogallolcarbonsaure. versetzt. Nachdem das Beaktionsgemisch 5 Stunden lang ge- standen hat, wird es iifters mit verdiinnter Schwefelsaure, dann einmal mit Wssser gut durchgeschiittelt und die Benzolschicht abgehoben. Nach dem Konzentrieren auf ca. 20 ccm fangt unveranderte Oxynaphtoesaure an, sich auszuscheiden, von welcher abfiltriert wird. Zur Mutterlauge gibt man Ligroin, wobei reich- liche Krystallausscheidung erfolgt. Die Krystalle werden nach dem Trocknen in verdunnter Ealiumbicarbonatliisung aufgelost, von Ungelostem abfiltriert, das Filtrat wird mit Salzsiiure aus- gefallt. Nach dem Trocknen wurden die Krystrtlle aus ver- diinntem Methylalkohol umkrystallisiert. 0,1518 g gaben 0,3657 g CO, und 0,0669 g H,O. Berechnet fur C,,H,,O,: Cfefunden: C 65,97 65,69"/,, H 4,71 480 9, * Die Substanz bildet farblosc Nadeln, die bei 167 O - 1 6 8 O schmelzen. Die Verbindung ist leicht loslich in Benzol, Al- kohol und Aceton. In Ligroin ist der Korper in der Wiirme sehr schwer lbslich, in Petrolither fast unlbslich. Die Arbeit wird fortgesetzt. Budapest, Anfang Januar 1914.
https://openalex.org/W2030319084	https://www.scielo.br/j/ean/a/CKWY8wycXVrvyDsRcXvT6NK/?lang=es&format=pdf, http://old.scielo.br/pdf/ean/v12n4/v12n4a28.pdf, http://www.scielo.br/pdf/ean/v12n4/v12n4a28.pdf, https://www.redalyc.org/pdf/1277/127715323028.pdf	es		Relación afectiva de mujeres con un esposo alcohólico: un comportamiento social aprendido que repercute en su salud	Escola Anna Nery	2,008	cc-by	3,528	Relación afectiva con un alcohólico Castañón MAH, Luis MAV REVISÃO Esc Anna Anna Nery Nery Rev Rev Enferm Enferm 2008 2008 dez; dez; 12 12 (4): (4): 806-10 806-10 Esc Revision - Revisión RELACIÓN AFECTIVA DE MUJERES CON UN ESPOSO ALCOHÓLICO: UN COMPORTAMIENTO SOCIAL APRENDIDO QUE REPERCUTE EN SU SALUD Relação afetiva das mulheres com um cônjuge alcoólico: um comportamento social aprendido que repercute em sua saúde Affective relationship between women with an alcoholic husband: a learned social behavior that affects your health Maria Alejandra Hernández Castañón1 Margarita Antonia Villar Luis2 RESUMEN Se realiza una revisión sobre el tema de la codependencia y su relación con comportamientos socialmente aprendidos sobre la base de una división de género. La codependencia en mujeres de adictos a alcohol y sus consecuencias individuales, familiares y comunitarias también se mencionan. En este contexto se realiza una breve síntesis de los patrones de comportamiento derivados del rol social asignado a la mujer en México en relación con esta temática. Finalmente se establecen algunas posturas teóricas para el análisis de la codependencia a partir del análisis socio-histórico de los significados que tiene para la población el consumo abusivo de alcohol y la relación patológica establecida que establece la mujer con el marido. En este sentido, diferentes autores mencionan la importancia de considerar a la familia y no sólo al individuo para la atención, ya que las consecuencias físicas y mentales están presentes tanto en hombres como mujeres. Palabras clave: Mujeres. Alcoholismo. Codependencia. Identidad de género. Salud. Resumo Abstract Trata-se de uma revisão sobre o tema codependência e sua relação com compor tamentos socialmente aprendidos fundamentada na divisão de gênero. A codependência em mulheres com companheiros dependentes de álcool e suas conseqüências para o indivíduo, família e comunidade também são abordadas. Nesse contexto, realiza-se uma breve síntese dos padrões de comportamento derivados do papel social atribuído à mulher no México em relação ao tema. Por fim, são estabelecidos alguns posicionamentos teóricos na análise da codependência a par tir da análise sócio-histórica dos significados que a população atribui ao consumo abusivo de álcool e da relação patológica que a mulher estabelece com seu marido. Diferentes autores referem a importância de considerar a família, e não somente o usuário, no seu cuidado, já que as conseqüências físicas e mentais estão presentes tanto no homem quanto na mulher. This is a review about codependence and its relation with socially learned behaviors based on a gender division. Codependence in women of alcohol addicts and its individual, family and Community consequences are also mentioned. In this context, a short synthesis is presented of the behavioral standards derived from the social role assigned to women in Mexico with respect to this theme. Finally, some theoretical positions are established to analyze codependence based on the sociohistorical analysis of the meanings the population attributes to alcohol abuse and the pathological relation women establish with their husband. In this sense, different authors mention the importance of considering the family and not only the individual for care, as the physical and mental consequences are present in women as well as men. Palavras-chave: Mulheres. Alcoolismo. Codependência. Identidade de Gênero. Saúde. Keywords: Women. Alcoholism. Codependence. Gender identity. Health. 1 Profesor-Doctor de la Facultad de Enfermería - Universidad Autónoma de Querétaro/México e-mail: alehdez983@yahoo.com.mx 2 Profesor Doctor. Coordinador del Proyecto CICAD/OEA & EERP.USP. Titular del Departamento de Enfermería Psiquiátrica y Ciencias Humanas. Escuela de Enfermería de Ribeirão Preto. Centro colaborador de la OMS para el Desarrollo de la Investigación en Enfermería. e-mail: margarit@eerp.usp.br 806 Relación afectiva con un alcohólico Castañón MAH, Luis MAV Esc Anna Nery Rev Enferm 2008 dez; 12 (4):806-10 INTRODUCCIÓN En el año 2000, el consumo de alcohol fue responsable de 4% de la carga mundial de morbilidad. En países desarrollados se atribuye al alcohol el 9.2% de los años de vida ajustados en función de la discapacidad (AVAD), resultado de trastornos neuropsiquiátricos y de traumatismos no intencionales y del 6.2% de los AVAD perdidos1 En los países de América Latina y el Caribe (ALC), las diferencias de género son agudas, pues son los hombres quienes representan mayores problemas relacionados con el alcohol, con base a normas existentes que apoyan y estimulan el beber entre los hombres, el género no está explícitamente considerado en las intervenciones actuales. Con excepción de México, Brasil, Chile y Costa Rica, los países de ALC no han mostrado un interés sostenido en la conducción de estudios sobre el consumo de alcohol2. Sin embargo, el desarrollo del conocimiento sobre alcoholismo como enfermedad ha enfatizado sus aspectos negativos, por lo que las investigaciones se han centrado en la embriaguez como el desvío individual y no como un comportamiento social3, pues el problema del alcohol no afecta únicamente a quien tiene la adicción, sino que involucra a la familia, especialmente a las mujeres, quienes cuidan y se responsabilizan del alcohólico en el diario convivir. Se refiere que siempre que uno de los miembros de la familia desarrolla una conducta adictiva, otro desarrolla codependencia4. En este contexto, el uso y abuso de sustancias adictivas como el alcohol y las drogas va en incremento, así como situaciones de violencia físico, sexual y psicológica, que resultan de la relación adicto/codependiente, dañando la autoestima de los integrantes de la familia, en especial a las mujeres. De ahí que el objetivo de la revisión fue: describir algunas posturas teóricas sobre el tema codependencia, especialmente en mujeres con cónyuges alcohólicos, como parte de un estudio sobre su mundo de la vida. METODOLOGÍA Se realizó una revisión bibliográfica con base en los descriptores de codependencia, alcoholismo, mujer, género y salud, para lo cual se consideraron artículos de 1990 a la fecha. De igual forma, se identificaron algunos estudios representativos para el contexto mexicano realizados en la década de los 90, que hacen un análisis histórico del tema. de su posición social. La búsqueda en Internet se realizó durante los meses de junio y julio de 2004 y actualizándose información para los meses de agosto-septiembre 2007, tomando como referencia las palabras claves antes mencionadas, utilizando base de datos scielo y buscador google académico, por las características cualitativas y más del ámbito social que se procuraba. También se realizó consulta bibliografía impresa específica y discusión con especialistas en el tema, al participar en eventos científicos relacionados con la salud de la mujer y género. Con esta información, se elaboró la propuesta que se presenta a seguir. RESULTADOS La codependencia se expresa como la aceptación del engaño y maltrato de que son objeto las mujeres por parte de quien consume algún tipo de droga, perdiendo su equilibrio emocional, alterando su forma de pensar, sentir y su comportamiento. Si la persona dependiente es la pareja, la relación afectiva y sexual se deteriora así como su vida cotidiana y salud5, hasta el punto de vivir por y para él6. Los comportamientos característicos de la codependencia son: a) conductas determinadas por fuerzas externas y no por decisiones voluntarias; b) inadecuado manejo de sentimientos, equiparando amar con sufrir y sacrificarse, c) protección y disculpa al adicto; c) baja autoestima; d) comportamientos de control hacia otras personas y eventos, e) estrés, enfermedades psicosomáticas y depresión7. La codependencia se puede adquirir de forma inducida directa mediante un sistema de conductas y creencias familiares que no permiten la expresión de emociones y afectos; de manera incidental al momento de la unión con otra persona, reaprendiendo formas disfuncionales de expresar y sentir afecto; e inducida por normas institucionales que dificultan al codependiente negar ayuda a otra persona, aún en perjuicio propio, generando sentimientos de angustia y baja autoestima8. Uno de los grupos más propensos a la codependencia son los cónyuges de alcohólicos o adictos, sus comportamientos son de víctima y mártir, de perseguidora y salvadora, pues considera que solo ella puede salvar y brindar la ayuda al adicto, descalificando a terapeutas o centros de tratamiento4, de ahí que se considere a la codependencia como una patología propia de la mujer, con alta prevalencia determinada por aspectos sociales y culturales6. Por lo expresado anteriormente, se observa una fuerte relación entre codependiencia y género. El sistema de género legitima y reproduce las prácticas, representaciones, normas y valores que la sociedad construye a partir de la diferencia sexual9. El sistema sexo/género se definió como un conjunto de arreglos a través de los cuales una sociedad trasforma la sexualidad biológica en productos de la actividad humana, en las que estas necesidades sociales trasformadas son satisfechas. En la década de los 70´s, se hacía una diferenciación entre los términos sexo y género, el primero indicaba las diferencias biológicas entre un hombre y mujer; el segundo, se refería a las construcciones sociales, culturales, psicológicas que se imponen sobre esa diferencia biológica10. Sin embargo, las propuestas teóricas relativas al género aparecen al final del siglo XX como construcción social, donde el sentido de las actividades se adquiere por medio de interrelaciones sociales en un contexto de desigualdad. El 807 Relación afectiva con un alcohólico Castañón MAH, Luis MAV concepto de género implica los símbolos culturalmente disponibles, los conceptos normativos que sustentan las interpretaciones del sentido de los símbolos, las referencia institucionales y la identidad subjetiva, ésta última construida en relación a una serie de actividades, organizaciones sociales y representaciones culturales históricamente situadas11. Las normas sociales en torno a la posición de la mujer dentro de la sociedad mexicana se sustentan en ese reconocimiento. Un estudio describe el comportamiento esperado de la mujer entre el periodo de la colonia y primera mitad del siglo XX que se resume en: obediencia total al cónyuge a cambio de sostén y protección, sobriedad sexual, recato, honestidad y mesura, donde el castigo físico se asociaba a la purificación, posiblemente interiorizado por el hombre para ejercerlo y por la mujer para soportarlo, pues su papel de cohesionadora del núcleo familiar, socializadora y reproductora de los valores dentro del hogar, ha soslayado la violencia familiar12. La familia se convierte en la reproductora principal de la ideología y construcción del género con todas sus implicaciones, entre ellas las relaciones de dominación que se ejerce sobre la mujer. Se menciona que los individuos aprenden un patrón de comportamiento, interiorizando las sanciones que corresponden a la violación de un imperativo generalizado y con ello incorporando el poder del grupo social13. En este contexto, las tradiciones y creencias emanadas del sistema social organizado a partir de una premisa de género, vinculan a la mujer con el hogar, de ahí que, la dinámica de las familias de adictos parta de un proceso cíclico que involucra a tres personas o más, generalmente el adicto y los padres. En esta dinámica, el tema de la droga no se discute e inicialmente no se da importancia a la adicción, después tienen esperanza de su cura y finalmente, niegan la enfermedad y rechazan al adicto. La disfunción se convierte en estilo de vida familiar con reglas confusas, especialmente para los niños, que puede facilitar el desarrollo de adicciones o de relaciones enfermas a futuro14. Se menciona que las instituciones sociales retoman criterios de validez, que provienen del reconocimiento intersubjetivo, por lo que los individuos asumen actitudes y roles que los demás miembros esperan de ellos13, de ahí que el concepto de género se aplica a las relaciones de desigualdad entre mujeres y hombres en torno a la distribución de los recursos, responsabilidades y poder. En lo relativo a la salud, también se observa esta diferencia cuando se establece una mayor mortalidad masculina, 5 a 20 veces más que en las mujeres en población adulta joven, esto debido a comportamientos de riesgo que derivan en accidentes, violencias y suicidios, asociados con la división social de roles según sexo y las correspondientes expectativas culturales de hombría que son tolerados y estimulados entre los varones15, como lo es el alcoholismo. 808 Esc Anna Nery Rev Enferm 2008 dez; 12 (4): 806-10 DISCUSIÓN De acuerdo con las revisión realizada, el comportamiento de la mujer mexicana no se ha modificado de manera sustancial, es decir, prevalece el concepto de subordinación y sacrificio16. Estos antecedentes, en los cuales las creencias, tradiciones y apariencias sociales son muy importantes, los problemas se mantienen al interior del seno familiar, de ahí que cuando se presenta dependencia a sustancias adictivas en un miembro de la familia, esto no se comente con nadie ajeno y mucho menos se solicite ayuda profesional, como se muestra en algunos estudios12,14,17 . El reconocimiento de esta problemática es fundamental al momento de estudiar el comportamiento de la mujer que convive con una persona alcohólica, especialmente aquella que tiene un compromiso de unión, puesto que en el proceso de socialización, el patrón generalizado de comportamiento cobra para el individuo la autoridad de un debes y con ello, el tipo de validez normativa en virtud del cual tienen las normas fuerza vinculante13, , es decir, en el caso de las adicciones, los comportamientos esperados en ellas son el cuidado del otro, pero paradójicamente, también depender del marido. Las mujeres procuran los servicios de salud en busca de orientación, cuando su situación de convivencia con el alcohólico llegaba a un punto límite como pérdida de empleo, pero sobre todo por complicaciones clínicas graves como cirrosis hepática y complicaciones cardiovasculares, resultado del abuso en su forma de beber15. Ellas desarrollan un sistema de defensa eficaz más no productivo, pues se encuentran llenas de rabia, preconceptos, indiferencias y frustración con la relación, contrastando con la responsabilidad y afecto que sentían por el alcohólico17. Diferentes estudios sobre codependencia, muestran en esta relación la presencia de violencia intrafamiliar, sin embargo, centrar el problema en la conducta individual del hombre y no como resultado de un desequilibrio de poder entre los individuos, da una falsa visión del fenómeno18, ya que el ejercicio de poder -inter e intragéneros-, tiene efectos claramente negativos sobre la integridad física, psicológica y social no solo de las mujeres, sino también de los hombres15. Con base a lo anterior, los profesionales de la salud requieren una formación que les permita tener una visión integral de los problemas de salud, ya que como en el caso del alcoholismo, no solo se trata del dependiente, sino del codependiente y de la propia familia, de ahí que las instituciones educativas deben implementar programas educativos con contenidos sobre salud mental, aspectos socio-antropológicos y psicológicos, que proporcionen a los estudiantes herramientas para identificar y hacer frente a este tipo de problemas, ya que muchas veces la falta de capacitación, puede ser una barrera para reconocerlos y brindar atención oportuna. En el caso específico de Enfermería, la adquisición de conocimiento científico-técnicos, que le permitan tener una Relación afectiva con un alcohólico Castañón MAH, Luis MAV Esc Anna Nery Rev Enferm 2008 dez; 12 (4):806-10 visión general de los problemas biológicos, psicológicos y sociales que conforman el fenómeno de la dependencia al alcohol, pero sobre todo, capitalizar el reconocimiento social que la profesión posee en actividades de salud comunitaria17. Esto le permitirá proponer y realizar actividades conjuntas con la comunidad, tendientes a la prevención y reducción del consumo de alcohol, además de coordinarse con otros profesionales, con la finalidad de buscar modificar el habito de beber de dependiente, pero sobre todo, que influir en la modificación de patrones de comportamiento13 que sustentan la codependencia. CONCLUSIONES El alcoholismo y la codependencia son la misma enfermedad, como cualquier adicción comparten características de negación, obsesión, compulsión y pérdida de control, ambos son comportamientos socialmente aprendidos que afectan la salud física y mental tanto de quien consume, como de aquellos que conviven cotidianamente con el adicto. Es posible que en este comportamiento aprendido por nuestras madres y abuelas, todavía exista un fuerte remanente, que condicione el actuar de la mujer ante una problemática familiar como es el uso y abuso del alcohol. De ahí que en la revisión realizada, se observó que el uso y abuso de sustancias adictivas va en incremento, así como el abuso físico, sexual y psicológico, resultante de la relación disfuncional adicto/codependiente, dañando la autoestima de los integrantes del núcleo familiar, especialmente en mujeres, es decir, hay cambios significativos en el mundo de la vida de los directamente involucrados con el fenómeno de la codependencia13. Se establece que el rol social asumido por los individuos, es determinante en el tipo de acción que lleven a cabo, con base a normas legítimamente reguladas, a partir de las cuales, los miembros de una comunidad desarrollan una identidad social que sentará las bases para la conformación de su mundo subjetivo. Por lo que la relación disfuncional que se observa en la familia, puede ser el reflejo de lo que acontece en su contexto social, donde los medios de comunicación han dado un nuevo sentido al consumo de alcohol, relacionándolo con una imagen de éxito; la pérdida de control social manifestado por la trasgresión de normas, como son el consumo en la vía pública y la venta a menores de edad cuando está prohibido por ley; así como el incremento de patologías relacionadas al consumo abusivo de alcohol. De ahí que dimensionar el alcoholismo, ya no sólo como un problema individual, sino un fenómeno multi-causal, que desde la perspectiva de Habermas, tendría que evaluar los distintos procesos de la reproducción, es decir, la racionalidad del saber, la solidaridad de los miembros y la capacidad personal para responder autónomamente de sus acciones13. Cuando la actitud de la mujer cambie no de manera aislada, sino como grupo social, es decir, cuente con redes sociales, familiares e institucionales que les permitan modificar ideas y creencias en torno al alcohol y al deber ser de la mujer, sólo entonces será posible que tomen una postura específica y sus acciones sean asertivas para enfrentar el fenómeno y mejorar su calidad de vida individual y familiar. Es importante estar concientes de la complejidad del proceso involucrado en la constitución de actitudes, para que enfermería y otros miembros del equipo de salud, no tengan expectativas de cambios a corto plazo en las mujeres de alcohólicos, porque así como llevó tiempo para construir esa identidad y modos de interactuar, la desconstrucción y reconstrucción, es un proceso que también requiere tiempo y apoyo profesional. Referências 1.Organización Mundial de la Salud-OMS. Problemas de salud pública causados por el uso nocivo del alcohol. 58ª Asamblea Mundial de la Salud 2005. [citado 20 ago 2007]; 1-4. Disponible en: http:// www.who.int/substance_abuse 2.Pyne H, Cleason M, Correia M. Dimensiones de género sobre el consumo de alcohol y problemas afines en América Latina y el Caribe. Banco Mundial 2002 [citado 24 ene 2007]; [3-12]. Disponible en: http://lnweb18.worldbank.org 3.Pessanha D. Alcoolismo: acusação ou diagnóstico? Cad Saude Publica 2004 jan/fev; 20(1): 7-13. 4.Guevara W. La codependencia, una forma de convivir con el sufrimiento. Boletín Consejo Superior de Investigaciones CSI/UNMSM [on-line] 2003 [citado 29 oct 2007]; (48): 13-15. Disponible en: http://sisbib.unmsm.edu.pe 5.Red Interamericana para la Prevención de Drogas- RIPRED. Guía para la mujer sobre las drogas. [citado 13 jun 2004]. [aprox. 34 telas]. Disponible en: http://www.dpna.org 6.Mansilla F. Codependencia y psicoterapia interpersonal. Rev Asoc Esp Neuropsiq 2001; 23 (81): 9-35. 7.Pérez A, Delgado D. La codependencia en familias de consumidores y no consumidores de drogas: estado del arte y construcción de un instrumento. Psicothema. [on-line]. Universidad de Oviedo, 2003; [citado 15 sept 2007]; 11(4): 381-387. Disponible en: http:// redalyc.uaemex.mx 8.Vacca R. La co-adicción, aspectos culturales y clínicos para su aprendizaje. Rev Peruana Drogodependencias [on-line] 2003; [citado 30 oct 2007] 1(1): 231-253. Disponible en: http://www.devida.gob.pe 9.Beatres G. La perspectiva de género como modelo de análisis de la violencia familiar y el consumo de alcohol y otras drogas. Reunión del Grupo de Consulta sobre el Impacto del Abuso de Drogas en la Mujer y la Familia. Secretaría Ejecutiva de la Comisión Interamericana para el Control y Abuso de Drogas- CICAD/OEA Montevideo (UR); 1996. p.146. 10.Mezan L, Piscitelli A, Goldani A. A prática feminista e o conceito de gênero. Campinas(SP): IFCH/UNICAMP; 2002. 809 Relación afectiva con un alcohólico Castañón MAH, Luis MAV Esc Anna Nery Rev Enferm 2008 dez; 12 (4): 806-10 11.Scott J. Gênero: uma categoría útil para análise histórica. Tradução de Christine Rufino Dabat, María Betânia Avila. Recife (PE): SOS CORPO; 1991. 16.Diaz-Guerrero R. Bajo las garras de la cultura. Psicología del mexicano. México(MX): Trillas; 2003. 12.Suárez M. Discurso, género y violencia intrafamiliar en la historia moderna de México. ¿Una memoría. Archivo Tiempo y Escritura 2004. [citado 24 ene 2007]. Disponible en: http://www.azc.unam.mx 17.Almeida TR, Villar-Luis MA. O cuidar de quem cuida: uma visão sobre os cuidadores de alcoolistas. In: Villar Luis M, Pillon, C. Assistência a usuários de álcool e drogas no Estado de São Paulo: uma amostra de servicios e programas. Ribeirão Preto (SP): FIERP/ USP; 2004. 13.Habermas J. Teoría de la acción comunicativa, 2. Crítica de la razón funcionalista. 1ª ed. México(MX): Taurus; 2002. 14.Coddou A, Chadwick M. Evolución del concepto de codependencia. Chile Med 1999. [citado 20 jun 2004]; [aprox. 18 telas]. Disponible en: http://www.eradicciones.org 15.Gómez Gómez E. Equidad, género y salud: retos para la acción. Rev Panam Salud Publica [on-line] 2002 may/jun; [citado 16 mar 2007]; 11(5/6): 454-61. Disponible en: http://www.scielosp.org. 18.Castro R, Riquer F. La investigación sobre violencia contra las mujeres en América Latina: entre el empirismo ciego y la teoría sin datos. Cad Saude Publica [on-line] 2003 jan/fev. [citado 10 out 2007]; 19 (1): 135-46. Disponible en: http://www.scielosp.org. Recebido em 27/03/2008 Reapresentado em 05/06/2008 Aprovado em 12/08/2008 810
https://openalex.org/W2737240233	https://escholarship.org/content/qt6dw824z4/qt6dw824z4.pdf?t=ozen4y	English	null	Prevalence of Intracranial Hemorrhage after Blunt Head Trauma in Patients on Pre-injury Dabigatran	The western journal of emergency medicine/Western journal of emergency medicine	2,017	cc-by	4,421	UC Irvine Copyright Information Copyright 2017 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ UC Irvine Western Journal of Emergency Medicine: Integrating Emergenc Care with Population Health Title Prevalence of Intracranial Hemorrhage after Blunt Head Trauma in Patients on Pre-injury Dabigatran Permalink https://escholarship.org/uc/item/6dw824z4 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 18(5) ISSN 1936-900X Authors Chenoweth, James A. Johnson, M. Austin Shook, Laura et al. Publication Date 2017 DOI 10.5811/westjem.2017.5.33092 Copyright Information Copyright 2017 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Western Journal of Emergency Medicine: Integrating Emergenc Care with Population Health Title Prevalence of Intracranial Hemorrhage after Blunt Head Trauma in Patients on Pre-injury Dabigatran Permalink https://escholarship.org/uc/item/6dw824z4 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 18(5) ISSN 1936-900X Authors Chenoweth, James A. Johnson, M. Austin Shook, Laura et al. Publication Date 2017 DOI 10.5811/westjem.2017.5.33092 Copyright Information Copyright 2017 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Western Journal of Emergency Medicine: Integrating Emergen Care with Population Health Title Prevalence of Intracranial Hemorrhage after Blunt Head Trauma in Patients on Pre-injury Dabigatran Permalink https://escholarship.org/uc/item/6dw824z4 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 18(5) ISSN 1936-900X Authors Chenoweth, James A. Johnson, M. Austin Shook, Laura et al. Publication Date 2017 DOI 10.5811/westjem.2017.5.33092 Copyright Information Copyright 2017 by the author(s).This work is made available under the terms of a Creativ Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Powered by the California Digital Library University of California eScholarship.org Original Research Prevalence of Intracranial Hemorrhage after Blunt Head Trauma in Patients on Pre-injury Dabigatran University of California Davis, School of Medicine, Department of Emergency Medicine, Davis, California Veterans Affairs Northern California, Mather Medical Center, Mather, California University of California Davis, School of Medicine, Davis, California * † ‡ University of California Davis, School of Medicine, Department of Emergency Medicine, Davis, California Veterans Affairs Northern California, Mather Medical Center, Mather, California University of California Davis, School of Medicine, Davis, California * † ‡ James A. Chenoweth, MD, MAS† M. Austin Johnson, MD, PHD Laura Shook, BA‡ Mark E. Sutter, MD† Daniel K. Nishijima, MD, MAS James F. Holmes, MD, MPH* Introduction: Dabigatran etexilate was the first direct-acting oral anticoagulant approved in the United States. The prevalence of intracranial hemorrhage after blunt head trauma in patients on dabigatran is currently unknown, complicating adequate ability to accurately compare the risks and benefits of dabigatran to alternative anticoagulants. We aimed to determine the prevalence of intracranial hemorrhage for patients on dabigatran presenting to a Level I trauma center. Methods: This is a retrospective observational study of adult patients on dabigatran who presented to a Level I trauma center and received cranial computed tomography (CT) following blunt head trauma. Patients who met inclusion criteria underwent manual chart abstraction. Our primary outcome was intracranial hemorrhage on initial cranial CT. Results: We included a total of 33 eligible patient visits for analysis. Mean age was 74.8 years (SD 11.2, range 55-91). The most common cause of injury was ground-level fall (n = 22, 66.7%). One patient (3.0%, 95% confidence interval [CI] 0.[1-15.8%]) had intracranial hemorrhage on cranial CT. No patients (0%, 95% CI [0-8.7%]) required neurosurgical intervention. One in-hospital death occurred from infection. Conclusion: To our knowledge, this is the first study to evaluate the prevalence of intracranial hemorrhage after blunt head trauma for patients on dabigatran presenting to the emergency department, including those not admitted. The intracranial hemorrhage prevalence in our study is similar to previous reports for patients on warfarin. Further studies are needed to determine if the prevalence of intracranial hemorrhage seen in our patient population is true for a larger patient population in more diverse clinical settings. [West J Emerg Med. 2017;18(5)794–799.] medications for atrial fibrillation indicated that up to 62% were prescribed a DOAC.3 Western Journal of Emergency Medicine INTRODUCTION Dabigatran etexilate is a direct thrombin inhibitor first approved in October 2010 for primary prevention of stroke in patients with atrial fibrillation.1 It is the first of several direct- acting oral anticoagulants (DOAC) to market. These agents have gained popularity due to the simplicity of dosing and the fact that they do not require routine laboratory testing.2 In fact, a recent cohort study of patients started on anticoagulation Each year traumatic brain injury results in approximately 1.4 million emergency department (ED) visits at an annual cost of U.S. $60 billion.4 An increasing proportion of these patients are elderly and taking anticoagulant medications.5 Patients on pre-injury warfarin or clopidogrel have significant risk of traumatic intracranial hemorrhage, even in Volume 18, no. 5: August 2017 Western Journal of Emergency Medicine 794 Prevalence of Intracranial Hemorrhage after Blunt Head Trauma Chenoweth et al. Population Health Research Capsule What do we already know about this issue? Clinical trial data suggests an improved bleeding profile for dabigatran when compared to warfarin. There are limited data regarding the intracranial hemorrhage risk after head trauma. What was the research question? What is the prevalence of intracranial hemorrhage after blunt head trauma for patients on dabigatran? What was the major finding of the study? Dabigatran appears to have a similar prevalence of intracranial hemorrhage after blunt head trauma as has been reported for warfarin. How does this improve population health? If validated, these findings suggest that previously established guidelines regarding fall- and trauma-risk assessment and warfarin use could be applied to dabigatran. cases with low-impact mechanisms of injury.6 cases with low-impact mechanisms of injury.6 Population Health Research Capsule Population Health Research Capsule In addition to the increased risk of intracranial hemorrhage, pre-injury use of warfarin also significantly increases mortality in elderly patients after head trauma. Early data suggest that this may not be the case for patients on DOACs, although the data for dabigatran is mixed.10-12 Additionally, management of trauma patients on dabigatran is especially challenging due to questions regarding coagulation testing and reversal strategies.13 What do we already know about this issue? Clinical trial data suggests an improved bleeding profile for dabigatran when compared to warfarin. There are limited data regarding the intracranial hemorrhage risk after head trauma. Despite Food and Drug Administration approval six years ago, the true prevalence of intracranial hemorrhage after head trauma for patients on dabigatran is still unknown. Data Collection and Processing The primary outcome of interest was the presence of intracranial hemorrhage on initial cranial CT as interpreted by a board-certified/eligible radiologist. The presence of intracranial hemorrhage as well as the type and extent of injury were directly abstracted from the final radiology report. Specific treatments including attempted reversal (defined as either administration of prothrombin complex concentrates, plasma, recombinant factor VIIa, or dialysis for the specific purpose of reversing the effects of dabigatran), neurosurgical intervention, and hospital length of stay (in days) were abstracted. Final disposition as reported on hospital discharge summary was also recorded. Manual chart abstraction from the electronic medical record was performed by a single abstractor for all patients who met inclusion criteria. Standard chart review methodology was followed for all data abstraction.14 Investigators agreed upon all inclusion/exclusion criteria and definitions prior to chart abstraction. All patient baseline data were abstracted prior to abstraction of CT results. A second abstractor reviewed approximately 20% of charts to measure interrater reliability for the presence of trauma (inclusion/exclusion criteria), intracranial hemorrhage on cranial CT, and the initial Glasgow Coma Scale (GCS) score. Baseline factors including age, sex, and indication for anticoagulation (atrial fibrillation, deep venous thrombosis, pulmonary embolism, other, or unknown) were recorded. Data regarding the mechanism of injury, initial GCS score, international normalized ratio (INR), and activated Selection of Participants All patients who received cranial computed tomography (CT) as part of their ED evaluation and were reported to be on dabigatran during the study period were evaluated for study inclusion. Inclusion criteria were as follows: 1) patient reported head trauma or physical examination findings of head trauma were documented in the ED history and physical examination, and 2) patient was over the age of 18 years at the time of ED presentation. We excluded from the final analysis prisoners, patients who were transferred from an outside hospital, and pregnant patients. thromboplastin time were abstracted from the electronic medical record. For all patients admitted to the hospital we calculated an abbreviated injury severity score (ISS).15 Study Setting and Design This is a retrospective observational study of all patients presenting to a university-based, urban Level 1 trauma center between November 1, 2010 and February 28, 2015. The study was approved by the site’s institutional review board. How does this improve population health? If validated, these findings suggest that previously established guidelines regarding fall- and trauma-risk assessment and warfarin use could be applied to dabigatran. INTRODUCTION Furthermore, there is a paucity of evidence regarding monitoring of the level of anticoagulation in trauma victims on dabigatran. In this study we aimed to determine the prevalence of intracranial hemorrhage for patients on pre-injury dabigatran presenting to a Level I trauma center after blunt head trauma. What was the research question? What is the prevalence of intracranial hemorrhage after blunt head trauma for patients on dabigatran? What was the major finding of the study? Dabigatran appears to have a similar prevalence of intracranial hemorrhage after blunt head trauma as has been reported for warfarin. DISCUSSION In this study we found that the risk of intracranial hemorrhage after blunt head trauma in patients taking dabigatran is similar to the prevalence previously reported for warfarin.16 Furthermore, we have demonstrated that many patients who report taking dabigatran at the time of ED presentation have normal clotting parameters (PT and aPTT), suggesting either non-compliance or a poor correlation between dabigatran effects and currently available anticoagulant tests. There were a significant number of patients who required intensive care unit monitoring. However, a majority of patients were discharged home and the mortality rate was low. Baseline characteristics for the study population are provided in Table 1. Mean age was 74.8 years (SD 11.2, range 55-91). The most common cause of injury was ground-level fall (n = 22, 66.7%). Initial GCS scores were all either 14 (n=4) or 15 (n=29). A total of 19 patients (57.6%, 95% CI 39.2-74.5%) were admitted to the hospital with a median ISS of 6 (IQR 3-9). Treatment and outcomes are reported in Table 2. Initial INR measurements were available in 24 patients, and activated partial thromboplastin time (aPTT) measurements were available in 23 patients. Median INR was 1.2 (IQR 1.1-1.3) and mean aPTT was 38.8 (SD 12.9, range 14.3- 66.1). Thirteen (54%, 95% CI [33-74%]) patients had INR measurements greater than the upper limit of normal (1.18) for the study site, and 13 (57%, 95% CI [34-77%]) had aPTT measurements greater than the upper limit of normal (36.7) for the study site. The relatively low prevalence of intracranial hemorrhage in patients initially presenting to our institution is surprising. Given the large CI, it may be true that a larger sample size would reveal a higher “true” prevalence. It could also partially be explained by the fact that a majority of patients had ground-level falls and low ISS. Previous studies of patients with mild head injury found a prevalence of intracranial hemorrhage of 4.3% if the patient was on pre- injury warfarin.6 i Dabigatran is the first of several DOACs to receive approval by the FDA. It exerts its action by directly binding to thrombin. When it first entered the market it was touted for its benefits of not requiring routine lab testing and very few drug-drug interactions. Characteristics of Study Participants During the study period there were a total of 98 ED visits by 85 patients taking dabigatran during which cranial CT was performed. Of the 98 visits, 33 met inclusion/exclusion criteria and were included in the final study population. We excluded 49 visits because there were no history or physical exam findings of trauma. Eleven patients with traumatic injuries were excluded because they did not sustain head trauma. We excluded an additional five patients were excluded because they were transferred from outside facilities (Figure). Primary Data Analysis We reported normally distributed continuous data as the mean with standard deviations (SD), and we described ordinal or non-normally distributed continuous data as Volume 18, no. 5: August 2017 Western Journal of Emergency Medicine 795 Chenoweth et al. Prevalence of Intracranial Hemorrhage after Blunt Head Trauma medians with interquartile (25%-75%) ranges (IQR). Interrater reliability is reported as Cohen’s kappa. We performed all statistical analyses using STATA 14.1 (STATA Corp., College Station, TX). Interrater reliability To measure interrater reliability of data abstraction, 20 patients had duplicate abstraction for presence of trauma, intracranial hemorrhage on cranial CT, and initial GCS score. There was perfect agreement for presence of trauma and the presence of intracranial hemorrhage on cranial CT (kappa=1.0, 95% CI [0.63-1]). There was excellent agreement for initial GCS (kappa=0.78, 95% CI [0.30-1.0]). Western Journal of Emergency Medicine DISCUSSION 98 ED visits 16 ED visits excluded 5 transfers 11 no head trauma 49 ED visits with history of physical exam findings of trauma 49 ED visits excluded - no trauma 33 head trauma ED visits 98 ED visits 49 ED visits excluded - no trauma 49 ED visits with history of physical exam findings of trauma 16 ED visits excluded 5 transfers 11 no head trauma Figure. Flow of patients in a study of the prevalence of intracranial hemorrhage after blunt head trauma for patients on dabigatran. ED, emergency department. No. % Sex Female 18 54.5 Male 15 45.5 Indication for dabigatran Atrial fibrillation 27 81.8 Deep venous thrombosis 2 6.1 Pulmonary embolism 1 3.0 Unknown 1 3.0 Other 2 6.1 Mechanism of injury Assault 1 3.0 Auto vs pedestrian 1 3.0 Ground-level fall 22 66.7 Motorcycle collision 2 6.1 Motor vehicle collision 3 9.1 Other 4 12.1 ICH 1 3.0 Table 1. Demographics and injury characteristics. No. % Reversal Agent None 30 93.9 aPCC 2 6.1 Transfused with Plasma 1 3.0 Dialysis 2 6.1 PRBC Transfusion 1 3.0 ED disposition Discharged from the ED 14 42.4 Ward 5 15.2 Telemetry 6 18.2 ICU 8 24.2 Final Hospital/ED disposition Died 1 3.0 Skilled Nursing facility 5 15.2 Home 24 72.7 Other 3 9.1 Table 2. Treatment and outcomes of patients with blunt head trauma who were taking dabigatran at time of arrival to Level I trauma center. ICH, intracranial hemorrhage. aPCC, activated 4-factor prothrombin complex concentrate; PRBC, packed red blood cells; ED, emergency department No. % Reversal Agent None 30 93.9 aPCC 2 6.1 Transfused with Plasma 1 3.0 Dialysis 2 6.1 PRBC Transfusion 1 3.0 ED disposition Discharged from the ED 14 42.4 Ward 5 15.2 Telemetry 6 18.2 ICU 8 24.2 Final Hospital/ED disposition Died 1 3.0 Skilled Nursing facility 5 15.2 Home 24 72.7 Other 3 9.1 Table 2. Treatment and outcomes of patients with blunt head trauma who were taking dabigatran at time of arrival to Level I trauma center. aPCC, activated 4-factor prothrombin complex concentrate; PRBC, packed red blood cells; ED, emergency department No. DISCUSSION Initial experience with hemorrhaging patients on dabigatran raised concerns regarding the lack of ability to accurately determine the degree of anticoagulation and unclear method of reversal.12,17 Fortunately, with the recent approval of idarucizumab, a dabigatran-specific antibody fragment, the ability to treat bleeding patients on dabigatran is likely to improve.18 The impact of this agent on patient outcomes or patient need for dialysis, however, is still unknown. One patient was found to have an intracranial hemorrhage on cranial CT (3.0%, 95% CI [0.1%-15.8%]) following a motorcycle collision. This patient had an intra-ventricular hemorrhage on CT and was also noted to have pelvic, cervical spine, and rib fractures. After a hospital stay of 19 days this patient was discharged to a skilled nursing facility. There were no patients that required neurosurgical intervention (0%, 95% CI [0-8.7%]). Patients also suffered the following major injuries: spinal fractures (n=3), long bone fractures (n=2), rib fractures (n=2), and pelvic fracture (n=1). The median hospital length of stay was two days (IQR 1-7 days) for those admitted, and one in-hospital death was identified following infectious complications after a hospital stay of 19 days. Bleeding risk remains the main concern associated with anticoagulant use. The Randomized Evaluation of Long-Term Anticoagulant Therapy (RE-LY) study upon which initial approval of dabigatran was based demonstrated that patients taking dabigatran have a lower risk of major bleeding than patients taking warfarin.2 In this study of 18,113 patients, 46 cases of traumatic intracranial hemorrhage were identified, 24 in patients on warfarin and 22 in the dabigatran groups.19 However, this study did not report the number of patients with Reversal of anticoagulation was attempted in two patients. One patient received both 4-factor activated prothrombin complex concentrate (FEIBA©) and dialysis following a cervical spine fracture. The second patient received a combination of fresh frozen plasma, FEIBA©, and dialysis for intracranial hemorrhage, pelvic fractures, rib fractures, and a cervical spine fracture. Volume 18, no. 5: August 2017 Western Journal of Emergency Medicine 796 Chenoweth et al. Prevalence of Intracranial Hemorrhage after Blunt Head Trauma Figure. Flow of patients in a study of the prevalence of intracranial hemorrhage after blunt head trauma for patients on dabigatran. ED, emergency department. LIMITATIONS This was a retrospective analysis, which has inherent limitations. We attempted to minimize potential bias from the design by adhering to best practice guidelines for retrospective reviews.14 However, because all abstractors were part of the initial study design, they were not blinded to the study hypothesis. The potential bias that this lack of blinding could introduce is mitigated by the fact that the primary study outcome is an objective finding on cranial CT. We also demonstrate excellent interrater reliability enhancing the reliably of the data. Address for Correspondence: Address for Correspondence: James Chenoweth, MD, MAS, University of California Davis, School of Medicine, Department of Emergency Medicine, 4150 V St., PSSB 2100. Sacramento, CA 95817. Email: jachenoweth@ucdavis.edu. Address for Correspondence: Address for Correspondence: James Chenoweth, MD, MAS, University of California Davis, School of Medicine, Department of Emergency Medicine, 4150 V St., PSSB 2100. Sacramento, CA 95817. Email: jachenoweth@ucdavis.edu. Address for Correspondence: Address for Correspondence: James Chenoweth, MD, MAS, University of California Davis, School of Medicine, Department of Emergency Medicine, 4150 V St., PSSB 2100. Sacramento, CA 95817. Email: jachenoweth@ucdavis.edu. Conflicts of Interest: By the WestJEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. The project described was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number UL1 TR000002. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Support for this research was provided by grant number T32HS022236 from the Agency for Healthcare Research and Quality (AHRQ) through the Quality, Safety, and Comparative Effectiveness Research Training (QSCERT) Program. There are no conflicts of interest or sources of funding to declare. We selected only those patients undergoing cranial CT scanning in the ED, resulting in an inability to identify patients with minor trauma treated without cranial imaging. We used this criterion as it gave us the greatest chance of identifying all head trauma patients. It is normal practice in the study site’s ED to image patients with head trauma who are on anticoagulation medications.16 We believe that the bias this decision introduced might have increased the reported prevalence of intracranial hemorrhage due to exclusion of patients at lowest risk. DISCUSSION % Sex Female 18 54.5 Male 15 45.5 Indication for dabigatran Atrial fibrillation 27 81.8 Deep venous thrombosis 2 6.1 Pulmonary embolism 1 3.0 Unknown 1 3.0 Other 2 6.1 Mechanism of injury Assault 1 3.0 Auto vs pedestrian 1 3.0 Ground-level fall 22 66.7 Motorcycle collision 2 6.1 Motor vehicle collision 3 9.1 Other 4 12.1 ICH 1 3.0 Table 1. Demographics and injury characteristics. ICH, intracranial hemorrhage. Western Journal of Emergency Medicine Volume 18, no. 5: August 2017 797 Prevalence of Intracranial Hemorrhage after Blunt Head Trauma Chenoweth et al. blunt head trauma without resultant intracranial hemorrhage. Additional case reports of intracranial hemorrhage have been reported in the literature with some raising concern that pre- injury dabigatran use could result in significant hemorrhage expansion.17 In an animal model, Schaeffer et al. analyzed the size of hemorrhage produced in a standardized fashion in rats given pre-injury dabigatran or warfarin. This study showed smaller hematoma size in rats given dabigatran; however, no differences in neurologic outcomes at day 21 were identified.20 Finally, because we are a tertiary-care referral center, we excluded patients transferred from outside facilities to allow for determination of a “true” prevalence of intracranial hemorrhage in patients presenting to the ED. Two of the five excluded patients were transferred to the study site because they had intracranial hemorrhages. Neither of these patients died during hospitalization at the study site and only one required neurosurgical intervention. Including these patients would have falsely increased the reported prevalence of intracranial hemorrhage on CT. To date, only one human study has evaluated the prevalence of intracranial hemorrhage in patients on dabigatran. This study included elderly patients (>65 years) with ground-level falls who were admitted to a trauma service. The authors compared the intracranial hemorrhage prevalence in the dabigatran group to patients on warfarin and found no difference (13.6% (warfarin) vs. 8.2%(dabigatran)).21 However, this study excluded patients discharged from the ED and included patients transferred to the study site. Both of these factors could result in overestimation of the risk of intracranial hemorrhage after trauma. CONCLUSION In our study, intracranial hemorrhage after blunt head trauma in patients on pre-injury dabigatran was rare. The incidence in our study is similar to previous reports for patients on warfarin, although the wide confidence interval and different methodology make direct comparison difficult. Further studies are needed to determine if the prevalence of intracranial hemorrhage seen in our patient population is true for a larger patient cohort in more diverse clinical settings. LIMITATIONS However, there is the possibility that there were patients who did not receive initial imaging and subsequently presented to other centers with traumatic intracranial hemorrhage. Copyright: © 2017 Chenoweth et al. This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) License. See: http://creativecommons.org/ licenses/by/4.0/ Due to the fact that this was a single-center study, we were unable to evaluate for later presentations for delayed bleeding. Our methods would have discovered any patients that re-presented to the ED during the study period, but could not evaluate for patients who later presented to alternate sites. The single-center nature of this study also resulted in a small sample size, which limits the generalizability of our results. Western Journal of Emergency Medicine REFERENCES 1. US Food and Drug Administration. FDA approves Pradaxa to prevent stroke in people with atrial fibrillation. 2010; Available at: https:// wayback.archive-it.org/7993/20170111150606/http://www.fda.gov/ NewsEvents/Newsroom/PressAnnouncements/2010/ucm230241. Volume 18, no. 5: August 2017 Western Journal of Emergency Medicine 798 Prevalence of Intracranial Hemorrhage after Blunt Head Trauma Chenoweth et al. risk with closed head injuries: are we prepared? J Neurosurg. 2013;119(3):760-5. htm. Accessed November 29, 2015. htm. Accessed November 29, 2015. htm. Accessed November 29, 2015. 2. Connolly SJ, Ezekowitz MD, Yusuf S, et al. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med. 2009;361(12):1139-51. 13. Pakraftar S, Atencio D, English J, et al. Dabigatran etixilate and traumatic brain injury: Evolving anticoagulants require evolving care plans. World J Clin Cases. 2014;2(8):362-6. 3. Desai NR, Krumme AA, Schneeweiss S, et al. Patterns of initiation of oral anticoagulants in patients with atrial fibrillation- quality and cost implications. Am J Med. 2014;127(11):1075-82 e1. 14. Kaji AH, Schriger D, Green S. Looking through the retrospectoscope: reducing bias in emergency medicine chart review studies. Ann Emerg Med. 2014;64(3):292-8. 4. Pearson WS, Sugerman DE, McGuire LC, et al. Emergency department visits for traumatic brain injury in older adults in the United States: 2006-08. West J Emerg Med. 2012;13(3):289-93. 15. Civil ID, Schwab CW. The Abbreviated Injury Scale, 1985 revision: a condensed chart for clinical use. J Trauma. 1988;28(1):87-90. 5. McMillian WD, Rogers FB. Management of prehospital antiplatelet and anticoagulant therapy in traumatic head injury: a review. J Trauma. 2009;66(3):942-50. 16. Nishijima DK, Offerman SR, Ballard DW, et al. Immediate and delayed traumatic intracranial hemorrhage in patients with head trauma and preinjury warfarin or clopidogrel use. Ann Emerg Med. 2012;59(6):460-8 e1-7. 6. Nishijima DK, Offerman SR, Ballard DW, et al. Risk of traumatic intracranial hemorrhage in patients with head injury and preinjury warfarin or clopidogrel use. Acad Emerg Med. 2013;20(2):140-5. 17. Wassef SN, Abel TJ, Grossbach A, et al. Traumatic intracranial hemorrhage in patients taking dabigatran: report of 3 cases and review of the literature. Neurosurgery. 2013;73(2):E368-73; discussion E373-4. 7. Lavoie A, Ratte S, Clas D, et al. Preinjury warfarin use among elderly patients with closed head injuries in a trauma center. J Trauma. 2004;56(4):802-7. 18. US Food and Drug Administration. FDA approves Praxbind, the first reversal agent for the anticoagulant Pradaxa. 2015; Available at: http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ ucm467300.htm. Accessed November 30, 2015. 8. Mina AA, Knipfer JF, Park DY, et al . Western Journal of Emergency Medicine REFERENCES Intracranial complications of preinjury anticoagulation in trauma patients with head injury. J Trauma. 2002;53(4):668-72. 9. Li J, Brown J, Levine M. Mild head injury, anticoagulants, and risk of intracranial injury. Lancet. 2001;357(9258):771-2. 19. Hart RG, Diener HC, Yang S, et al. Intracranial hemorrhage in atrial fibrillation patients during anticoagulation with warfarin or dabigatran: the RE-LY trial. Stroke. 2012;43(6):1511-7. 10. Dezman ZD, Comer AC, Smith GS, Narayan M, Hess JR, Hirshon JM. The severity of bleeding and mortality in trauma patients taking dabigatran. J Emerg Med. 2016;51(3):238-45. 20. Schaefer JH, Leung W, Wu L, et al. Translational insights into traumatic brain injury occurring during dabigatran or warfarin anticoagulation. J Cereb Blood Flow Metab. 2014;34(5):870-875. 11. Maung AA, Bhattacharya B, Schuster KM, et al. Trauma patients on new oral anticoagulation agents have lower mortality than those on warfarin. J Trauma Acute Care Surg. 2016;81(4):652-7. 21. Pozzessere A, Grotts J, Kaminski S. Dabigatran Use Does Not Increase Intracranial Hemorrhage in Traumatic Geriatric Falls When Compared with Warfarin. Am Surg. 2015;81(10):1039-42. 12. Parra MW, Zucker L, Johnson ES, et al. Dabigatran bleed Volume 18, no. 5: August 2017 Volume 18, no. 5: August 2017 Volume 18, no. 5: August 2017 Western Journal of Emergency Medicine 799
https://openalex.org/W4295222700	https://www.nature.com/articles/s41746-022-00670-1.pdf	English	null	Internet- and mobile-based psychological interventions for sexual dysfunctions: a systematic review and meta-analysis	npj digital medicine	2,022	cc-by	10,351	INTRODUCTION sexual dysfunctions via the internet may be a particularly promising treatment avenue. IMIs also meet the preference of many individuals for self-help rather than on-site treatment18. Further, increased access to evidence-based treatments could help reduce the high number of reported unsuccessful treatment attempts for sexual dysfunctions19,20. Sexual dysfunctions are a heterogeneous group of conditions that are characterized by a clinically signiﬁcant impairment in a person’s ability to experience sexual pleasure or to respond sexually1. Sexual dysfunctions include female orgasmic disorder, female sexual interest/arousal disorder, genito-pelvic pain/pene- tration disorder, male hypoactive sexual desire disorder, prema- ture ejaculation, delayed ejaculation, erectile disorder, substance/ medication-induced sexual dysfunction, and other speciﬁc and unspeciﬁed sexual dysfunction1. Sexual dysfunctions are highly prevalent conditions2,3 associated with reduced quality of life4, impaired relationship satisfaction5,6, increased comorbidity of affective and anxiety disorders7, and substantial economic costs8,9. Yet, less than half of women and men with a sexual condition seek help from a healthcare professional10. Despite progressive sexual education and reduced stigma, sexual conditions continue to be a taboo subject in many societies globally11. Fear of stigmatization, feelings of shame, and discomfort surrounding sexuality are major barriers to the utilization of available treatments12–15. Additional treatment barriers include low avail- ability of specialized diagnostics and evidence-based treatments for sexual dysfunctions in routine care and extensive waiting lists for specialists8. Sexual dysfunctions are a heterogeneous group of conditions that are characterized by a clinically signiﬁcant impairment in a person’s ability to experience sexual pleasure or to respond sexually1. Sexual dysfunctions include female orgasmic disorder, female sexual interest/arousal disorder, genito-pelvic pain/pene- tration disorder, male hypoactive sexual desire disorder, prema- ture ejaculation, delayed ejaculation, erectile disorder, substance/ medication-induced sexual dysfunction, and other speciﬁc and unspeciﬁed sexual dysfunction1. Sexual dysfunctions are highly prevalent conditions2,3 associated with reduced quality of life4, impaired relationship satisfaction5,6, increased comorbidity of affective and anxiety disorders7, and substantial economic costs8,9. p y Although IMIs offer an exciting option to address reported barriers to treatment, research and implementation of IMIs for sexual dysfunctions is still in its infancy when compared to other mental disorders21. Despite research on an intelligent computer- based therapy system to diagnose and treat sexual dysfunctions in couples in the 1980s22, the ﬁeld has not received an upsurge in further developments of IMIs as a result. The evidence to date on IMIs for sexual dysfunctions is summarized in one comprehensive meta-analysis for various mental disorders and two reviews. www.nature.com/npjdigitalmed 1Department of Clinical Psychology and Psychotherapy, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. 2Professorship Psychology and Digital Mental Health Care, Department of Sport and Health Sciences, Technical University Munich, Munich, Germany. 3Mental Health Research and Treatment Center, Department of Clinical Psychology and Psychotherapy, Faculty of Psychology, Ruhr University Bochum, Bochum, Germany. 4Department of Clinical Psychology and Psychotherapy, Institute of Psychology and Education, Ulm University, Ulm, Germany. ✉email: anna-carlotta.zarski@fau.de REVIEW ARTICLE OPEN Internet- and mobile-based psychological interventions for sexual dysfunctions: a systematic review and meta-analysis Anna-Carlotta Zarski1,2✉, Julia Velten3, Johannes Knauer4, Matthias Berking1 and David Daniel Ebert Sexual dysfunctions are highly prevalent and undertreated. Internet- and mobile-based psychological interventions (IMIs) could be a promising addition to close this treatment gap, given their accessibility, anonymity, and scalability. This systematic review and meta-analysis investigated the efﬁcacy of IMIs for sexual dysfunctions. A comprehensive literature search was conducted in August 2021 on randomized controlled trials investigating the effects of IMIs on sexual functioning and satisfaction compared to a control condition. Twelve RCTs with 14 comparisons were reviewed with six IMIs targeting female and six IMIs targeting male sexual dysfunctions and n = 952 participants were evaluated in the meta-analysis. IMIs were signiﬁcantly more effective than control conditions (k = 11 waitlist control group, k = 3 online discussion board) at post-treatment for female sexual functioning (g = 0.59, CI: 0.28–0.90, I2 = 0%) and satisfaction (g = 0.90, CI: 0.02–1.79, I2= 82%), and male sexual functioning (g = 0.18, CI: 0.02–0.34, I2= 0%). No signiﬁcant effect was found for male sexual satisfaction (g = 0.69, CI: −0.13–1.51, I2= 88%) with substantial heterogeneity in studies. Most studies showed high dropout, with ten studies indicating some concern of risk of bias, and two studies showing high risk of bias. The results suggest that IMIs can be an effective treatment for sexual dysfunctions, although additional high-quality research is needed. Given the limited availability of specialized treatment for sexual dysfunctions and individual preferences for discrete treatment options, IMIs seem to be a valuable addition to routine care, empowering individuals to promote their sexual health on a guided self-help basis. npj Digital Medicine (2022) 5:139 ; https://doi.org/10.1038/s41746-022-00670-1 INTRODUCTION The meta-analytic ﬁndings from 2012 by Hedman and colleagues comprised four randomized controlled trials (RCTs) with ﬁve comparisons on solely female (k = 1), male (k = 3), or both sexual dysfunctions (k = 1)23. The within-group pre-post effect sizes varied widely between the studies (d = 0.21 to 1.52) with an average treatment effect of d = 0.67 (95%-CI: −0.25 to 1.59), showing moderate success. The IMI for female sexual dysfunctions achieved a large pre-post treatment effect of d = 1.5224, while IMIs for male sexual dysfunctions achieved small to medium effects between d = 0.21 and d = 0.5225–27. Based on these pre-post treatment results, IMIs were classiﬁed as a likely efﬁcacious treatment option. Notably, to date, between-group effects have not been analyzed23. A meta-analysis from 2015 indicated that IMIs can improve sexual function and relationship satisfaction, but y Yet, less than half of women and men with a sexual condition seek help from a healthcare professional10. Despite progressive sexual education and reduced stigma, sexual conditions continue to be a taboo subject in many societies globally11. Fear of stigmatization, feelings of shame, and discomfort surrounding sexuality are major barriers to the utilization of available treatments12–15. Additional treatment barriers include low avail- ability of specialized diagnostics and evidence-based treatments for sexual dysfunctions in routine care and extensive waiting lists for specialists8. It is therefore unsurprising that individuals have been found to increasingly search the web anonymously for treatment options regarding disorders commonly perceived as sensitive and shame- ful, such as sexual dysfunctions10,16. As internet- and mobile-based interventions (IMIs) offer the advantage of scalable, anonymous, and easily accessible evidence-based treatment17, addressing Published in partnership with Seoul National University Bundang Hospital A.-C. Zarski et al. Fig. 1 PRISMA ﬂowchart. A. C. Zarski et al. 2 2 Fig. 1 PRISMA ﬂowchart. Fig. 1 PRISMA ﬂowchart. in their sexual functioning and sexual satisfaction when receiving an IMI compared to a control condition. These outcomes reﬂect the primary goals of psychological treatment for sexual dysfunc- tion of establishing sexual functioning and increasing sexual satisfaction beyond reducing symptom severity37. not distress levels in women. In men, there was a non-signiﬁcant effect of IMIs on sexual function, but insufﬁcient data to be able to draw any conclusions about the effects of IMIs on other outcomes28. npj Digital Medicine (2022) 139 Published in partnership with Seoul National University Bundang Hospital INTRODUCTION A review exclusively on IMIs for female sexual dysfunctions from 201629 showed an increase in research activity, with four completed RCTs24,30–32 of which one RCT included qualitative analyses only31 as well as one ongoing RCT33. Another review from 2018 focusing on IMIs for sexual health among individuals with cancer34 identiﬁed four relevant studies, of which three were RCTs26,30,35,36. No meta-analysis to date has analyzed the between-group effects of IMIs, so no comprehensive statement about its efﬁcacy can be made to date. Study selection A total of 13,089 articles were identiﬁed after removing duplicates. After abstract screening, 75 articles were identiﬁed for full-text review. Twelve articles reporting outcomes for 12 RCTs with 14 comparisons met the inclusion criteria of the study (see Fig. 1). A search update did not reveal any new eligible studies for inclusion in the study. The interrater reliability of the total selection between the two researchers was excellent (Cohen’s Kappa = 0.83)38,39. The study selection process and reasons for exclusion are displayed in Fig. 1. Hence, there is a pressing need to synthesize the existing research on IMIs for female and male sexual dysfunctions to provide a comprehensive overview of the current research, to estimate the overall efﬁcacy of this treatment approach in women and men, and to guide future research efforts. Thus, we conducted a systematic review and meta-analysis to examine whether women and men with sexual dysfunctions improved, on average, Published in partnership with Seoul National University Bundang Hospital A.-C. Zarski et al. 3 Table 1. Study descriptives. Study selection Study Gender Target condition Inclusion and exclusion criteria Medical comor- bidities Conditions n; dropout post Age (SD) Primary outcome (measures), post- treatment measurement time Secondary outcomes (measures) Follow-up (duration) Risk of bias Country Andersson et al., 2011 Male Erectile dysfunction IIEF-5: <21, partner (≥3 m, hetero-/ homosexual); Excl.: medical cause, mental disorder, substance abuse No IMI (ICBT) Active condition (forum) n = 39; 15% n = 39; 10% 57.62 (10.06), 55.50 (9.94) Erectile function (IIEF-5), 7 weeks Erectile function, orgasmic function, sexual desire, intercourse satisfaction, overall satisfaction (IIEF-15); relationship satisfaction (RAS); anxiety (BAI); depression (BDI-II); quality of life (WHOQOL-BREF); Yes (IG, 6 m) Some concerns SW McCabe et al., 2008 Male Erectile dysfunction Partner (heterosexual) Excl.: medical cause, mental disorder, substance abuse No IMI (Rekindle) waitlist control group n = 24; 50% n = 20; 5% NI Erectile function, orgasmic function, sexual desire, overall satisfaction (IIEF-15), 10 weeks marital satisfaction (KMSS); sexual satisfaction (ISS); self- esteem + relationship satisfaction (SEAR) Yes (IG, 3 m) Some concerns AU Schover et al., 2012 Male Erectile dysfunction Partner (heterosexual) (≥1 year) Yes (prostate cancer) IMI (CAREss) waitlist control group n = 55; 51% n = 48; 10% 64 (7), NI Erectile function (IIEF- 15), female sexual functioning (FSFI), 12 weeks distress (BSI-18); dyadic adjustment (A-DAS) Yes (3, IG: 6, 12 m) Some concerns US Van Lankveld et al., 2009 Male Erectile dysfunction, premature ejaculation Excl.: medical cause, mental disorder, substance abuse, severe relationship problems No IMI erectile dysfunction IMI premature ejaculation waitlist control group - erectile dysfunction waitlist control group - premature ejaculation n = 30; 7% n = 22; 5% n = 28; 11% n = 18; 11% Erectile dysfunction: 45.5 (13.0) Premature ejaculation: 40.1 (10.5) Erectile function, sexual desire, overall satisfaction (IIEF-15); premature ejaculation (GRISS- PE), 12 weeks sexual self-esteem + relationship satisfaction (SEAR- CONF), marital functioning (MMQ), impairment (GEQ) Yes (IG, 3, 6 m) Some concerns NL Wootten et al., 2017 Male Erectile dysfunction NI Yes (prostate cancer) IMI (My Road Ahead) IMI + forum Active condition (forum) n = 47, 33 n = 48, 35 n = 47, 18 total: 27% mean: 61 (7) Erectile function, orgasmic function, sexual desire, intercourse satisfaction, overall satisfaction (IIEF-15), 10 weeks depression + anxiety (DASS-21), quality of life (PC-QOL) Yes (3, 6 m) Some concerns AU Zarei et al., 2020 Male Erectile dysfunction Partner (heterosexual) Excl.: mental disorder, substance abuse, sex. dys. Study selection of partner Yes (SCI) App (SAMAR) waitlist control group n = 35; 0% n = 35; 0% 36.7 (39.5) 35.3 (37.7) Sexual adjustment (SAQ), sexual satisfaction (LSSS), marital adjustment (SMAQ), marital satisfaction (EMS), 8 weeks NI Yes (1, 2 m) Some concerns IRN Classen et al., 2012 Female Sexual distress FSDS-R ≥24 Yes (gynecologic cancer) IMI (GyneGals) waitlist control group n = 13; 23% n = 14; 0% 39.9 (29–58) 44.6 (28–59) Sexual distress (FSDS-R), 16 weeks Anxiety + depression (HADS), chronic disease (IIRS) No Some concerns CAN Hucker et al., 2015 Female Multiple sexual dysfunction Partner (heterosexual), Excl.: mental disorder, severe relationship problems No iCBT (Pursuing Pleasure) waitlist control group n = 52; 58% n = 39; 46% 33.31 (7.4) 31.94 (5.17) Female sexual functioning (FSFI), 11 weeks Sexual distress (FSDR-R), erectile function (IIEF-15), premature ejaculation (PEDT) Yes (IG, 3 m) High AU Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 139 Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 139 A.-C. Zarski et al. Study selection 4 Table 1 continued Study Gender Target condition Inclusion and exclusion criteria Medical comor- bidities Conditions n; dropout post Age (SD) Primary outcome (measures), post- treatment measurement time Secondary outcomes (measures) Follow-up (duration) Risk of bias Country Hummel et al., 2017 Female Multiple sexual dysfunction Sexual dysfunction DSM-IV diagnosis, Excl.: mental disorder, substance abuse, severe relationship problems Yes (breast cancer) IMI; waitlist control group n = 84; 19% n = 85; 12% 51.6 (7.7) 50.5 (6.8) Female sexual functioning (FSFI; SAQ), sexual distress (FSDS-R), relationship intimacy (PAIR), 24 weeks Body image (QLQ- BR23), marital functioning (MMQ), menopausal symptoms (FACT-ES), anxiety + depression (HADS), HRQOL (SF- 36) Yes (IG, 3, 9 m) Some concerns NL Jones et al., 2011 Female Multiple sexual dysfunction Partner (heterosexual) No IMI (Revive) waitlist control group n = 26; 58% n = 27; 37% 34.91 (10.27) 33.30 (9.34) Female sexual functioning (FSFI), 10 weeks Sexual functioning (SFS), intimacy (PAIR), anxiety + depression (DASS-21) Yes (IG, 3 m) High AU Zarski et al., 2017 Female Genito-pelvic pain- penetration disorder Partner (>3 m, heterosexual) Excl.: medical cause, mental disorder, substance abuse No IMI, (Vaginismus- Free) waitlist control group n = 40; 10% n = 37; 16% 25.83 (6.46) 28.95 (8.92) Intercourse penetration ability (PEQ), 10 weeks Non-intercourse penetration ability (PEQ), sexual fear (FSQ), female sexual functioning (FSFI), dyadic coping (DCI), treatment satisfaction (CSQ-I) Yes (6 m) Some concerns G Zarski et al., 2021 Female Genito-pelvic pain- penetration disorder Partner (heterosex.) Excl.: medical cause, mental disorder, substance abuse No IMI (Paivina- Care) waitlist control group n = 100; 22% n = 100; 8% 29.46 (9.82) 28.04 (7.84) Intercourse penetration ability (PEQ), 12 weeks Non-intercourse penetration ability (PEQ), sexual fear (FSQ), female sexual functioning (FSFI), dyadic coping (DCI), relationship satisfaction (PFB-K), GAD (GAD-7), anxiety (STAI-T), quality of life (WHO- 5), treatment satisfaction (CSQ-I), neg. ef. (INEP) Yes (6 m) Some concerns G Note: A-DAS dyadic adjustment scale – Abbreviated Form, AU Australia, BAI Beck anxiety inventory, BDI-II Beck depression inventory II, BSI-18 brief symptom inventory-18, CAN Canada, CGI-I clinical global impression – Improvement Scale, CSQ-I client satisfaction questionnaire – internet-based interventions, DASS-21 depression anxiety and stress scales – short version, DCI dyadic coping inventory, EMS ENRICH (evaluation and nurturing relationship issues, communication, and happiness) marital satisfaction scale, Excl. Study selection exclusion criteria, FACT-ES functional assessment of cancer treatment–endocrine symptoms, FSDS-R female sexual distress scale-revised, FSFI female sexual function index, FSQ fear of sexuality questionnaire, G Germany, GAD-7 generalized anxiety disorder questionnaire, GEQ global endpoint question, GRISS Golombok rust inventory of sexual satisfaction, GRISS-PE Golombok rust inventory of sexual satisfaction - premature ejaculation subscale, HADS hospital anxiety and depression scale, HRQOL health-related quality of life, IG intervention group, IIEF-15 international index of erectile functioning – 15 items version, IIEF-5 international index of erectile functioning – 5 items version, IIRS illness intrusiveness ratings scale, INEP inventory for the assessment of negative effects of psychotherapy, IRN Iran, ISS index of sexual satisfaction, KMSS Kansas marital satisfaction scale, LSSS Larson’s sexual-satisfaction scale, m months, MMQ Maudsley marital questionnaire, multiple sexual dysfunctions = desire, arousal, orgasm, pain, n = sample size per group, NI no information, NL the Netherlands, PAIR personal assessment of intimacy in relationships inventory, PC-QOL prostate cancer-related quality of life scales, PE premature ejaculation, PEDT premature ejaculation diagnostic tool, PEQ primary endpoint questionnaire, PFB-K partnership questionnaire – short form, QLQ-BR23 quality of life questionnaire – breast cancer module, RAS relationship assessment scale, SAQ sexual adjustment questionnaire, SCI spinal cord injury, SD standard deviation, SEAR self-esteem and relationship questionnaire, SF-36 36-item short form survey, SFS sexual function scale, SMAQ Spinner’s marital adjustment scale, STAI-T state-trait anxiety inventory – trait scale, SW Sweden, US United States, WHO-5 World Health Organization Well-Being Index, WHOQOL-BREF World Health Organization Quality of Life – Brief Version. Published in partnership with Seoul National University Bundang Hospital Sensitivity and subgroup analyses Sensitivity analyses identiﬁed one outlier for male sexual satisfaction (Zarei et al., 2020). After omitting the outlier study, heterogeneity was reduced (I2 = 31%, 95%-CI: 0–72%), and the effect remained non-signiﬁcant (g = 0.34; 95%-CI: −0.04–0.72, p = 0.07; NNT = 5.26). No statistical outlier was detected for the other outcomes. We did not conduct any subgroup analyses as the recommended number of studies per analysis was not reached. All psychological IMIs were based on the principle of Cognitive Behavioral Therapy (CBT) together with elements of sexual therapy including treatment components regarding: (1) psycho- and sexual education (k = 10), (2) cognitive restructuring (k = 8), (3) mind- fulness (k = 1), (4) emotion regulation (k = 2), (5) relaxation (k = 4), (6) sensate focus (k = 9), (7) communication (k = 9), (8) relapse prevention (k = 6), (9) homework (k = 6) (10), and disorder-speciﬁc interventions (k = 4) such as exposure exercises, pain management for pain-penetration disorders, sexual exploration, and a decision- making algorithm to select suitable additional treatment for erectile dysfunction). IMIs for sexual dysfunctions associated with a medical disorder were also included (e.g., cancer-related topics). IMIs consisted of 3 to 10 modules (median = 5.5) that took between 8 and 24 weeks (median = 10.5) to be completed. Eleven interventions consisted of web-based modules delivered through a website or eHealth platform, and one intervention was smartphone-based. Three IMIs additionally included a moderated online discussion board (k = 2) or online chat groups (k = 1); one study included a smartphone-based diary. Ten studies involved some form of human guidance such as providing written feedback Characteristics of included studies Characteristics of included studies on completed modules (k = 5), moderation in an online discussion board or chat (k = 2), or on demand e-mail-based support (k = 3). The smartphone-based study did not report on any personalized guidance. Adherence reminders for module completion were included in nine studies, two of which also offered additional booster telephone calls. Ten studies provided at least one adherence measure, of which eight studies reported the percen- tage of participants who completed the IMI, which ranged between 8.11% and 87.00% (median: 53.26%). The characteristics of the 14 comparisons from 12 RCTs are described in Table 1 (references of included studies Supplemen- tary References). The RCTs were conducted in Europe (k = 5, one from Sweden, two each from Germany and the Netherlands), Australia (k = 4), Canada (k = 1), Iran (k = 1), and the United States (k = 1). All studies were published between 2008 and 2021. One study received funding by a pharmaceutical company27. The 12 RCTs included a total of 1152 participants. Eleven studies included target outcomes for the meta-analysis evaluating n = 952 participants at post-treatment, 488 in the intervention and 464 in the control groups. Sample sizes at post-treatment ranged from n = 28 to n = 200 (median = 68). In six studies, the individuals affected by a sexual dysfunction were male, and in the other six studies, the affected individuals were female. The treatments were mainly focused on the affected person, but active participation of a sexual partner via couple exercises was mandatory in eight studies. In the other four studies, participation of a sexual partner was optional. Eight interventions were addressed at heterosexual patients only. All IMIs for male sexual dysfunctions targeted erectile dysfunctions, and in one case, additionally premature ejaculation. Three IMIs for female sexual dysfunctions were aimed at multiple, simultaneous sexual dysfunctions, including problems with desire, arousal, orgasm, and pain, whereas two IMIs speciﬁcally treated Genito-Pelvic Pain-Penetration Disorder, and one IMI targeted sexual distress associated with sexual difﬁculties. Of the 12 RCTs, ﬁve studies included samples in which sexual dysfunctions were related to a history of a medical disorder (prostate, gynecological, and breast cancer, and spinal cord injury). The average of mean age reported in 10 of the 12 RCTs was 41.55 years (SD = 10.77). Risk of bias Ten RCTs had an overall risk of bias with some concern, and two RCTs had a high risk of bias. Studies’ risk of bias was classiﬁed based on (1) the randomization process (k = 5 low RoB, k = 7 some concerns), (2) deviations from the intended interven- tions (k = 4 low RoB, k = 8 some concerns), (3) missing outcome data (k = 6 low RoB, k = 4 some concerns, k = 2 high RoB), (4) the measurement of the outcome (k = 12 some concerns), and (5) the selection of the reported results (k = 3 low RoB, k = 9 some concerns). RoB for each study is reported in Table 2 (risk of bias plot per study Supplementary Fig. 7). Main analyses Female sexual functioning and sexual satisfaction were both investigated in ﬁve comparisons (n = 482). The overall signiﬁcant effect size for female sexual functioning was medium with g = 0.59 (95%-CI: 0.28–0.90, k = 5, p < 0.01, NNT = 3.09). Hetero- geneity was low (I2 = 0%; 95%-CI: 0–79%) (forest plot Fig. 2). For female sexual satisfaction, the overall signiﬁcant effect size was large with g = 0.90 (95%-CI: 0.02–1.79, k = 5, p = 0.0475; NNT = 2.10). Heterogeneity was considerable (I2 = 82%; 95%-CI: 59–92%) (forest plot Fig. 3). Male sexual functioning and male sexual satisfaction were investigated in seven comparisons (n = 363) and seven comparisons (n = 400), respectively. The overall signiﬁcant effect size for male sexual functioning was small (g = 0.18, CI: 0.02–0.34, k = 4, p = 0.04; NNT = 7.46) with low heterogeneity (I2 = 0%; 95%-CI: 0–79%) (forest plot Fig. 4). The overall effect size for male sexual satisfaction was medium (g = 0.69; 95%-CI: −0.13–1.51, k = 5, p = 0.09; NNT = 2.67) and non-signiﬁcant with considerable heterogeneity (I2 = 88%; 95%-CI: 78–94%) (forest plot Fig. 5). Eleven comparisons used an inactive control condition (waitlist control condition) while three comparisons used an active control condition (online discussion board). Five studies including male patients assessed sexual functioning with the International Index of Erectile Function (IIEF-15, IIEF-540,41), and ﬁve studies assessed sexual satisfaction with the International Index of Erectile Function in four cases and the Larson’s sexual-satisfaction scale in one case42,43. Of those studies assessing sexual satisfaction, four considered it a primary outcome. Five studies including female patients assessed sexual functioning and sexual satisfaction using the Female Sexual Function Index (FSFI44,45), of which, three considered them as primary outcomes. Six studies included follow-up periods in both the intervention and the control condition, ranging from two to six months (median = 4.5 months); six studies conducted follow-ups in the intervention group only, ranging from 3 to 12 months (median = 6 months). Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital satisfaction (CSQ-I neg. ef. (INEP) A.-C. Zarski et al. 5 Publication bias Egger’s test was signiﬁcant for female sexual satisfaction (p = 0.01) and male sexual functioning (p = 0.04). Two studies were imputed in the trim-and-ﬁll procedure for female sexual satisfaction resulting in a lower non-signiﬁcant effect of g = 0.43 (95%-CI: −0.59–1.44, p = 0.35; NNT = 4.19). No study was imputed for male sexual functioning. Calculating p-curves was only possible for female sexual functioning and sexual satisfaction as two or less signiﬁcant effects emerged in the main analysis for the other two outcomes in men. Signiﬁcant right-skewed p-curves for female sexual functioning and female sexual satisfaction indicated that evidential value was present in both outcomes. The estimate of the true effect size in female sexual functioning resulted in a smaller effect size of d = 0.56 compared to the main outcome Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 139 A.-C. Zarski et al. 6 Table 2. Intervention Content. DISCUSSION Results indicated that IMIs are effective with medium to large effect sizes related to improved sexual functioning (g = 0.59) and satisfaction (g = 90) in women, and with small effect sizes related to improved sexual functioning in men (g = 0.18), compared to a control condition. Effects of IMIs on male sexual satisfaction were medium in size but not signiﬁcant (g = 0.69). The ﬁndings were derived from 11 RCTs which included 952 participants with post- treatment data from seven countries. Heterogeneity of effect sizes varied from low for female and male sexual functioning to substantial for female and male sexual satisfaction. The median percentage of individuals who completed the IMI was 53%. The majority of studies showed some concern regarding risk of bias. There was evidence of publication bias, suggesting that unpub- lished studies with opposing effects may exist. All IMIs were based on the principles of CBT-based sexual therapy. p p py The medium to large effect sizes found in IMIs for female sexual functioning (d = 0.59) and satisfaction (d = 0.90) were comparable to the medium effect sizes found in face-to-face psychological interventions for female and male sexual dysfunc- tions on symptom severity (d = 0.58) and sexual satisfaction (d = 0.47) compared to waitlist conditions37. In contrast to face- to-face treatment, the effects of IMIs on male sexual functioning were smaller (g = 0.18) and non-signiﬁcant for male sexual satisfaction (g = 0.69)37. One explanation for these divergent ﬁndings may be the fact that three comparisons in studies for men compared the IMI with an active control group in the form of an online discussion board. In an online discussion board, important common factors in sexual dysfunction such as mutual understanding and acceptance as well as destigmatization can take place through the exchange of patients among themselves even without speciﬁc therapeutic strategies being presented46. The overall lower effects and high heterogeneity in sexual satisfaction could be explained by differences among the IMIs in the nature and extent of the incorporation of dyadic aspects because sexual satisfaction has been previously found to be related to relational functioning47. g There are several limitations to be considered when interpreting the present ﬁndings. First, the number of identiﬁed RCTs was small. Despite an extensive systematic literature search, we cannot exclude the possibility that additional studies fulﬁlling the eligibility criteria exist. Moreover, publication bias could not be ruled out48. Publication bias 7 Number of intervention modules Duration of intervention Adherence Delivery modality Guidance Psycho- and sexual education Cognitive restructuring Mindfulness Emotion regulation Relaxation Sensate Focus Communication Disorder- speciﬁc intervention Relapse prevention Homework 2017 10 10 w 13/40; 32.50% Web-based modules Written feedback on completed modules; email- based support on demand, adherence reminder Y Y N N Y Y N Y N Y 2021 8 12 w 43/100; 43.00% Web-based modules + diary on smartphone Written feedback on completed modules; email- based support on demand, adherence reminder Y Y N N Y Y Y Y Y Y Disorder-speciﬁc intervention = speciﬁc sexual therapy interventions such as vaginal training, w = weeks, adherence = deﬁned as number and percentage of participants who completed treatment. Y yes, NI no information analysis. No adjusted estimate was calculated for the female sexual satisfaction outcome due to substantial heterogeneity (I2 = 87%) (Funnel plots and p-curves Supplementary Fig. 1–6). analysis. No adjusted estimate was calculated for the female sexual satisfaction outcome due to substantial heterogeneity (I2 = 87%) (Funnel plots and p-curves Supplementary Fig. 1–6). Study Number of intervention modules Duration of intervention Adherence Delivery modality Guidance Psycho- and sexual education Cognitive restructuring Mindfulness Emotion regulation Relaxation Sensate Focus Communication Disorder- speciﬁc intervention Relapse prevention Homework Zarski et al., 2017 10 10 w 13/40; 32.50% Web-based modules Written feedback on completed modules; email- based support on demand, adherence reminder Y Y N N Y Y N Y N Y Zarski et al., 2021 8 12 w 43/100; 43.00% Web-based modules + diary on smartphone Written feedback on completed modules; email- based support on demand, adherence reminder Y Y N N Y Y Y Y Y Y Note: Disorder-speciﬁc intervention = speciﬁc sexual therapy interventions such as vaginal training, w = weeks, adherence = deﬁned as number and percentage of participants who completed treatment. Y yes, N NI i f ti ished in partnership with Seoul National Univ Publication bias Study Number of intervention modules Duration of intervention Adherence Delivery modality Guidance Psycho- and sexual education Cognitive restructuring Mindfulness Emotion regulation Relaxation Sensate Focus Communication Disorder- speciﬁc intervention Relapse prevention Homework Andersson et al., 2011 7 7 w 3/37, 8.11% Web-based modules Written feedback on completed modules; email- based support on demand Y Y N N Y Y Y N Y Y McCabe et al., 2008 5 10 w 12/24; 50.00% Web-based modules Email-based support on demand; adherence reminder N N N N N Y Y N N N Schover et al., 2012 3 12 w 48/55, 87.27% Web-based modules Written feedback on completed modules; email-based support on demand; adherence reminder, 2 booster telephone calls Y Y N N N Y Y Y Y Y Van Lankveld et al., 2009 NI 12 w - Web-based modules Email-based support on demand Y Y N N N Y N N N N Wootten et al., 2017 6 10 w - Web-based modules + online forum Adherence reminder, automated feedback, moderation in online forum Y Y N Y N N Y N Y N Zarei et al., 2020 NI 8 w - Mobile application - Y NI NI NI NI NI NI NI NI NI Classen et al., 2012 1 12 w - Web-based modules + online forum 90-min text-based chat session, moderation in online forum, adherence reminder Y N N Y N N Y N N N Hucker et al., 2015 6 11 w 26/46, 56.52% Web-based modules + online chat groups Email-based support on demand; adherence reminder, moderation in online chat groups Y Y Y N N Y Y N Y Y Hummel et al., 2017 4–5 max. 24 w 52/84, 61.90% Web-based modules Written feedback on completed modules; Email- based support on demand; adherence reminder, 2 telephone calls Y Y N N Y Y Y Y Y Y Jones et al., 2011 5 10 w 17/26, 65.38% Web-based modules Email-based support on demand; adherence reminder N N N N N Y Y N N N Digital Medicine (2022) 139 Published in partnership with Seoul National University Bundang npj Digital Medicine (2022) 139 Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital A.-C. Zarski et al. DISCUSSION Dismantling and additive-design studies, as well as studies compar- ing different treatment approaches, are needed to investigate different treatment components and approaches such as cognitive or mindfulness-based therapy60. The extent to which the efﬁcacy of IMIs could be fostered by tailoring the number of treatment components and modules, individualizing support, and adapting the content more precisely to the respective situation or constellation of symptoms of the affected individuals, still remains an open research question61. We also need a better understanding of the speciﬁc mechanisms between guidance, adherence and outcomes as half of all individuals in the studies reviewed did not complete the IMI62,63. To increase the limited treatment effects, theory-based interventions are needed in order to conﬁrm and further develop model assumptions64. While stepped treatment approaches starting with psychoeducation and sexual counselling are explicitly recom- mended, to the best of our knowledge, preventive approaches for sexual dysfunction have not yet been evaluated, and despite their accessibility, IMIs are often used after prolonged symptom duration. Moreover, many interventions have limited scope in terms of their target population and should be adapted and studied for different target populations regardless of sexual orientation or existing partnerships. Lastly, most of the existing IMIs are currently not available in routine care outside of research projects. Therefore, studies on the implementation of IMIs for sexual dysfunctions in routine care should be conducted to create tangible and accessible treatment options for the large number of affected individuals64. sexual dysfunctions. Fifth, we were not able to pool the long-term follow-up outcome data because only six studies included a long- term follow-up assessment in both the intervention and the control condition, and follow-up periods varied strongly among studies. Thus, no conclusions about the long-term efﬁcacy could be drawn from the present study. It must, however, also be noted that the post-treatment measurement times also varied between the studies (range: 7 to 24 weeks). ( g ) More adequately powered studies are needed to investigate the efﬁcacy of IMIs for sexual dysfunctions in women and men, people with sex variation (intersex), and gender-diverse communities, e.g., transgender and non-binary individuals, and individuals in same- gender and opposite-gender partnerships. Moreover, comprehensive diagnostics of sexual dysfunctions and associated conditions according to current guidelines should be included to determine the efﬁcacy of IMIs for speciﬁc sexual dysfunctions and populations. Published in partnership with Seoul National University Bundang Hospital DISCUSSION Most of the identiﬁed studies included small sample sizes and appeared to be pilot investigations, which limited the identiﬁcation of small effects and restricted the generalizability of the ﬁndings. Thus, a number of the meta-analyses included only a small number of studies and were not adequately powered to detect clinically relevant differences between the IMI and the control conditions for all outcome measures. In addition, the small number of studies with small sample sizes meant that no further subgroup analyses could be performed49. Moreover, heterogeneity was substantial in most analyses but should be interpreted with caution due to the limited number of studies available50. Third, the majority of studies reviewed displayed at least an overall risk of bias with some concern (e.g., high study dropout rates), or no use of intention-to- treat analyses. Due to missing intention-to-treat data, calculations in most of the studies were performed with observed data, which might have led to an overestimation of the effect sizes. Thus, the results should be interpreted with caution. Fourth, most studies did not use diagnostic questionnaires with predeﬁned symptom cut- offs or clinical interviews to establish a diagnosis of sexual dysfunction. Thus, variation in applied diagnostic criteria might limit the generalizability of the results to other populations with Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 139 A.-C. Zarski et al. 8 Fig. 2 Forest plot for female sexual functioning outcomes. Note: All comparison groups were waiting control groups. Fig. 2 Forest plot for female sexual functioning outcomes. Note: All comparison groups were waiting control groups. Fig. 2 Forest plot for female sexual functioning outcomes. Note: All comparison groups w Fig. 3 Forest plot for female sexual satisfaction outcomes. Note: All comparison groups were waiting control groups. Fig. 3 Forest plot for female sexual satisfaction outcomes. Note: All comparison groups were waiting control groups. ot for female sexual satisfaction outcomes. Note: All comparison groups were waiting control groups. Fig. 3 Forest plot for female sexual satisfaction outcomes. Note: All comparison groups were waiting co studied in detail which intervention components are responsible for positive treatment outcomes55–58. Due to their structured and standardized presentation of treatment components, IMIs are particularly well suited to investigate mechanisms of action and the signiﬁcance of different components for treatment success59. npj Digital Medicine (2022) 139 DISCUSSION Little is also known regarding which individuals are most likely to beneﬁt from an IMI versus an in-person form of sexual therapy51. Investigating moderators of treatment efﬁcacy for digital self-help and for other formats of sexual therapy can help to match affected individuals with the approach best suited to their needs. Most studies developed their program for heterosexual couples and examined their program in heterosexual samples. For this reason, the general- izability to sexual minorities and gender-diverse communities has been limited to date and should be the subject of further research. In this context, transcultural adaptation of IMIs for sexual dysfunction should also be explored52,53. Critically, a scientiﬁc investigation of potential negative outcomes and contraindications of treatment for sexual dysfunctions, irrespective of treatment modality, should also be conducted, which to date, has rarely been investigated. It is also unclear to what extent comorbid conditions such as anxiety or depression might complicate the treatment of sexual dysfunctions and which disorder should be addressed ﬁrst54. Beyond clarifying who beneﬁts from IMIs for sexual dysfunction, it has not yet been Despite the stated limitations, especially concerning number and sample size of the RCTS identiﬁed, our ﬁndings indicated that sexual dysfunctions can be effectively treated with IMIs targeted npj Digital Medicine (2022) 139 A.-C. Zarski et al. 9 Fig. 4 Forest plot for male sexual functioning outcomes. Note: ED erectile dysfunction, PE premature ejaculation; two comparisons included an active control condition (Andersson et al., 2011, Wootten et al., 2016). Fig. 4 Forest plot for male sexual functioning outcomes. Note: ED erectile dysfunction, PE premature ejaculation; two comparisons included an active control condition (Andersson et al., 2011, Wootten et al., 2016). Fig. 5 Forest plot for male sexual satisfaction outcomes. Note: ED erectile dysfunction, PE premature ejaculation, IMI IMI alone, IMI + ODF = IMI plus online discussion board; two comparisons included an active control condition (Andersson et al., 2011, Wootten et al., 2016). Fig. 5 Forest plot for male sexual satisfaction outcomes. Note: ED erectile dysfunction, PE premature ejaculation, IMI IMI alone, IMI + ODF = IMI plus online discussion board; two comparisons included an active control condition (Andersson et al., 2011, Wootten et al., 2016). exclude studies based on participants’ age, sex, or any other demographic characteristic; comorbid general medical disorders were also not used as an exclusion criterion. Search strategy A systematic literature search was completed (until September 5th, 2020 with a search update covering the period up to August 3rd, 2021) in three major bibliographical databases (Cochrane Central Register of Controlled Trials, PsycInfo, PubMed). Key search terms included a combination of terms indicative of sexual dysfunctions and internet- and mobile-based psycholo- gical treatment (Search String Supplementary Methods). Addi- tionally, references in identiﬁed studies were checked for earlier publications, and authors were contacted in case further information was necessary to clarify study eligibility. The interrater reliability was assessed with Cohen’s Kappa for the initial agreement on full text eligibility with values <0 indicating no agreement, 0.00–0.20 none to slight, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, and 0.81–1.00 indicating excellent agreement38,39. METHODS This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta- Analyses (PRISMA) guidelines and the Cochrane Handbook for Systematic Reviews of Interventions65–67. The study was registered in the Open Science Framework (OSF; https://osf.io/m8kng). DISCUSSION Studies evaluating blended interventions (i.e., combined IMIs and face-to-face treatment), telemedicine, treatments in which the internet was only used for communication between therapist and patient, or those equivalent to face-to-face therapy (e.g., video therapy) were excluded. RCT arms including other treatments such as face-to- face psychotherapy, pharmacological, or physical therapies were excluded. No language restrictions were applied apart from formulating the search string in English. towards female sexual functioning, female sexual satisfaction, and male sexual functioning. More high-quality research is needed to strengthen the evidence base for IMIs in the treatment of sexual dysfunctions and to generate knowledge to reach individuals who are most likely to beneﬁt from an internet-based treatment. Nevertheless, the results indicate that IMIs for sexual dysfunction are ready to be implemented in routine care and more practice research is needed under routine conditions to accompany and advance implementation in health care systems. Published in partnership with Seoul National University Bundang Hospital Statistical analyses Meta-analytic procedure. The meta-analysis was performed using the meta68, metafor69, and dmetar70 packages in R version 3.6.2 (R Core Team, 2017). A random-effects pooling model was applied as considerable heterogeneity among studies was expected. For each comparison between an IMI and a control condition at post- treatment, we calculated the between-group effect size Hedges’ g (g) to adjust for small sample bias71, the 95% conﬁdence interval (95%-CI), and the p-value (p) for each target outcome. In line with recommendations from Cohen (1988), Hedges’ g was interpreted with an effect size of 0.2 being considered small, 0.5 moderate, and 0.8 being considered a large effect72. Prediction intervals (95%) were calculated around the pooled effect sizes, indicating the interval within which the effect size of a similar future study would fall if selected at random from the same population of studies presently investigated73. Additionally, the effects were transformed into numbers-needed-to-treat (NNT) using the formula by Kraemer and Kupfer to indicate the number of patients that need to be treated to gain one additional positive outcome in comparison to a control condition74. Heterogeneity was evaluated using the I² statistic and its 95% conﬁdence interval (Higgins & Thompson, 2002). I² heterogeneity of 25% can be regarded as low, 50% as moderate, and 75% as substantial heterogeneity75. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Study selection After removing duplicates, titles and abstracts of identiﬁed studies were ﬁrst screened for potential eligibility. Second, full texts of potentially eligible studies were reviewed for inclusion criteria. Study selection, subsequent full text screening, rating for eligibility, and data extraction were performed indepen- dently by two researchers (J.K. & A.-C.Z.). All discrepancies were resolved by consensus with an additional third and fourth researcher (D.D.E. & J.V.). Sensitivity and subgroup analyses. To assess the inﬂuence of individual studies and potential outliers on the overall effect, we conducted “leave-one-out” analyses. We ﬁrst identiﬁed signiﬁcant outlying studies exerting the greatest inﬂuence on the results. In case of signiﬁcant outliers, we recalculated the analyses with the outlier removed77. Subgroup analyses to investigate potential sources of heterogeneity were only performed if the meta-analysis contained at least k = 10 studies per research question following the recommendation by Schwarzer and colleagues78. DATA AVAILABILITY Data collected and used in this meta-analysis can be requested from the corresponding author. Received: 11 February 2022; Accepted: 2 August 2022; Quality assessment We evaluated the validity of included studies using the ﬁve domains of the risk of bias 2.0 tool by the Cochrane Collabora- tion, based on ﬁve criteria: (1) the randomization process, (2) deviations from the intended interventions, (3) missing outcome data, (4) outcome measurement, and (5) selection of the reported results (Sterne et al., 2019). Following the guidelines and related decision trees, the overall risk of bias was judged with either “low”, “some concerns”, or “high”. Given the difﬁculty of blinding participants and study staff in psychological intervention RCTs with non-interventional control conditions, the judgement guidelines for domain speciﬁc risk of bias were adjusted. Risk of bias assessment was conducted independently by two researchers (J.K. & A.-C.Z.). Publication bias Indications of publication bias were evaluated via two approaches based on different assumptions of the origin of the bias. Assuming that publication bias operates through effect sizes, we investigated the funnel plot visually79 and tested for asymmetry via Egger’s test80. If the test for asymmetry was signiﬁcant, indicating publication bias, Duval and Tweedie trim- and-ﬁll procedure was used to adjust for possible bias by obtaining an estimation of the pooled effect when accounting for missing studies81. Alternatively, p-curve analyses were applied as a second approach, assuming that the publication bias was mainly driven by statistical signiﬁcance82,83. A signiﬁcant test of right‐skewness of the p-curve indicates the presence of evidential value in an analysis. To calculate p-curves, three or more signiﬁcant effects were needed70. We only reported the adjusted effect size estimate for analyses with I2 < 50%, as a lack of robustness has been noted for analyses with substantial heterogeneity84. Data extraction Extracted data included (1) bibliographical data (ﬁrst author, year), (2) study design features (target condition, inclusion and exclusion criteria, study conditions, intervention name, type of control group (inactive control condition such as waitlist control conditions and active control condition such as attention control conditions), sample size, primary and secondary outcomes, outcome mea- sures, follow-up, duration of follow-up, country of origin), (3) intervention characteristics (number of treatment modules, duration, treatment adherence, delivery modality, guidance, type of guidance, treatment components), (4) sample characteristics (gender, age, medical comorbidity), and (5) data for calculation of effect sizes (means and dispersion measures, preferably intention- to-treat post-treatment data, study dropout rates, handling of missing data). If data was not retrievable from the publication, study authors were contacted for clariﬁcation. Eligibility criteria Our eligibility criteria included a broad assessment of psycholo- gical IMIs for sexual dysfunctions among diverse populations. We included (1) peer-reviewed (2) randomized controlled trials using (3) a computer, internet- or mobile-based (4) psychological intervention as the primary treatment, (5) aiming to reduce symptoms of sexual dysfunctions in adult female or male individuals or couples (6) compared to an active or inactive control condition (7) at post treatment, with (8) symptom severity, sexual functioning, or sexual satisfaction as outcomes. We did not npj Digital Medicine (2022) 139 Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital A.-C. Zarski et al. 10 Main analyses. In the main outcome analyses, we pooled studies with the same target outcome of female or male sexual functioning or sexual satisfaction to generate a mean effect size for each investigated outcome. We pooled all target outcome data of eligible studies for the respective analysis, irrespective of whether the measure was deﬁned as a primary or secondary outcome. If trials were multi-armed, reporting two or more intervention groups to the same control comparison, we divided the sample size of the comparison to avoid inﬂating power76. REFERENCES The effectiveness of an internet-based psychological treatment program for female sexual dysfunction. J. Sex. Med. 8, 2781–2792 (2011). 50. Higgins, J. P. & Thompson, S. G. Quantifying heterogeneity in a meta-analysis. Stat. Med. 21, 1539–1558 (2002). 51. Brotto, L. A., Zdaniuk, B., Rietchel, L., Basson, R. & Bergeron, S. Moderators of improvement from mindfulness-based vs traditional cognitive behavioral therapy for the treatment of provoked vestibulodynia. J. Sex. Med. 17, 2247–2259 (2020). 25. Andersson, E. et al. A randomized controlled trial of guided internet-delivered cognitive behavioral therapy for erectile dysfunction. J. Sex. Med. 8, 2800–9 (2011). 26. Schover, L. R. et al. A randomized trial of internet-based versus traditional sexual counseling for couples after localized prostate cancer treatment. Cancer 118, 500–509 (2012). 52. Heim, E. & Kohrt, B. A. Cultural adaptation of scalable psychological interventions: a new conceptual framework. Clin. Psychol. Eur. 1, (2019). 53. Bhavsar, V. & Bhugra, D. Cultural factors and sexual dysfunction in clinical prac- tice. Adv. Psychiatr. Treat. 19, 144–152 (2013). 27. Van Lankveld, J., Leusink, P., Van Diest, S., Gijs, L. & Slob, A. K. Internet-based brief sex therapy for heterosexual men with sexual dysfunctions: a randomized Ccontrolled pilot trial. J. Sex. Med. 6, 2224–2236 (2009). 54. Hoyer, J., Uhmann, S., Rambow, J. & Jacobi, F. Reduction of sexual dysfunction: by-product of cognitive-behavioural therapy for psychological disorders? Sex. Relatsh. Ther. 24, 64–73 (2009). 28. Hobbs, L. J. Psychotherapeutic interventions for sexual difﬁculties delivered via the Internet: Effectiveness, acceptability and reach. (University College London, 2015). 55. Godbout, N., Bakhos, G., Dussault, É. & Hébert, M. Childhood interpersonal trauma and sexual satisfaction in patients seeing sex therapy: examining mindfulness and psychological distress as mediators. J. Sex. Marital Ther. 46, 43–56 (2020). 29. van Lankveld, J. J. Internet-based interventions for women’s sexual dysfunction. Curr. Sex. Health Rep. 8, 136–143 (2016). 56. Carvalheira, A., Godinho, L. & Costa, P. The impact of body dissatisfaction on distressing sexual difﬁculties among men and women: the mediator role of cognitive distraction. J. Sex. Res. 54, 331–340 (2017). 30. Schover, L. R. et al. Efﬁcacy Trial of An Internet-Based Intervention for Cancer- Related Female Sexual Dysfunction. J. Natl Compr. Cancer Netw. 11, 1389–1397 (2013). 57. ter Kuile, M. M., Melles, R. J., Tuijnman-Raasveld, C. C., de Groot, H. E. & van Lankveld, J. J. D. M. Therapist-Aided exposure for women with lifelong vagi- nismus: mediators of treatment outcome: a randomized waiting list control trial. REFERENCES & Najafabadi, G. M. The study of sexual satisfaction in Iranian women applying for divorce. Int. J. Fertil. Steril. 8, 281–288 (2014). 18. Ebert, D. D. et al. Internet and mobile-based psychological interventions: Appli- cations, efﬁcacy and potential for improving mental health. A report of the EFPA E-Health Taskforce. Eur. Psychologist 23, 167–187 (2018). 44. Rosen, R. et al. The female sexual function index (FSFI): a multidimensional self- report instrument for the assessment of female sexual function. J. Sex. Marital Ther. 26, 191–208 (2000). 19. Bond, K., Mpofu, E. & Millington, M. Treating women with genito-pelvic pain/ penetration disorder: Inﬂuences of patient agendas on help-seeking. J. Fam. Med. 2, 1–8 (2015). 45. Berner, M. M., Kriston, L., Zahradnik, H.-P., Härter, M. & Rohde, A. Überprüfung der Gültigkeit und Zuverlässigkeit des Deutschen Female Sexual Function Index (FSFI-d). Geburtshilfe Frauenheilkd. 64, 293–303 (2004). 20. Reissing, E. D. Consultation and treatment history and causal attributions in an online sample of women with lifelong and acquired vaginismus. J. Sex. Med. 9, 251–258 (2012). 46. Rains, S. A. & Young, V. A meta-analysis of research on formal computer-mediated support groups: examining group characteristics and health outcomes. Hum. Commun. Res. 35, 309–336 (2009). 21. Carlbring, P., Andersson, G., Cuijpers, P., Riper, H. & Hedman-Lagerlöf, E. Internet- based vs. face-to-face cognitive behavior therapy for psychiatric and somatic disorders: an updated systematic review and meta-analysis. Cogn. Behav. Ther. 47, 1–18 (2018). 47. Stephenson, K. R. & Meston, C. M. Differentiating components of sexual well- being in women: are sexual satisfaction and sexual distress independent con- structs? J. Sex. Med. 7, 2458–2468 (2010). 22. Binik, Y. M., Servan-Schreiber, D., Freiwald, S. & Hall, K. S. Intelligent computer- based assessment and psychotherapy: an expert system for sexual dysfunction. J. Nerv. Ment. Dis. 176, 387–400 (1988). 48. van Aert, R. C. M., Wicherts, J. M. & van Assen, M. A. L. M. Publication bias examined in meta-analyses from psychology and medicine: a meta-meta- analysis. PLoS ONE 14, (2019). 23. Hedman, E., Ljótsson, B. & Lindefors, N. Cognitive behavior therapy via the Internet: a systematic review of applications, clinical efﬁcacy and cost- effectiveness. Expert Rev. Pharmacoecon Outcomes Res. 12, 745–764 (2012). 49. Brookes, S. T. et al. Subgroup analyses in randomized trials: Risks of subgroup- speciﬁc analyses; power and sample size for the interaction test. J. Clin. Epidemiol. 57, 229–236 (2004). 24. Jones, L. M. & McCabe, M. P. REFERENCES J. et al. Help-seeking for sexual difﬁculties and the potential role of interactive digital interventions: ﬁndings from the third british national survey of sexual attitudes and lifestyles. J. Sex. Res. 56, 937–946 (2019). 37. Frühauf, S., Gerger, H., Schmidt, H. M., Munder, T. & Barth, J. Efﬁcacy of psycho- logical interventions for sexual dysfunction: a systematic review and meta- analysis. Arch. Sex. Behav. 42, 915–933 (2013). sexual attitudes and lifestyles. J. Sex. Res. 56, 937–946 (2019). 11. Waling, A., Fraser, S. & Fisher, C. Young People and Sources of Sexual Health Information. https://doi.org/10.26181/5efa95dd0e5ec. (2020). 38. Cohen, J. A coefﬁcient of agreement for nominal scales. Educ. Psychological Meas. XX, 37–46 (1960). 12. Hinchliff, S. & Gott, M. Seeking medical help for sexual concerns in mid- and later life: a review of the literature. J. Sex. Res. 48, 106–117 (2011). 39. Landis, J. R. & Koch, G. G. The measurement of observer agreement for catego- rical data. Biometrics 33, 159–174 (1977). 13. Bergvall, L. & Himelein, M. J. Attitudes toward seeking help for sexual dysfunc- tions among US and Swedish college students. Sex. Relatsh. Ther. 29, 215–228 (2014). 40. Rosen, R. et al. The international index of erectile function (IIEF): a multi- dimensional scale for assessment of erectile dysfunction. Urology 49, 822–830 (1997). 14. Shifren, J. L. et al. Help-seeking behavior of women with self-reported distressing sexual problems. J. Women’s Health 18, 461–468 (2009). 41. Wiltink, J., Hauck, E., Phädayanon, M., Weidner, W. & Beutel, M. Validation of the german version of the international index of erectile function (IIEF) in patients with erectile dysfunction, peyronie’s disease and controls. Int. J. Impot. Res. 15, 192–197 (2003). 15. Gott, M. & Hinchliff, S. Barriers to seeking treatment for sexual problems in primary care: a qualitative study with older people. Fam. Pract. 20, 690–695 (2003). 42. Larson, J. H., Anderson, S. M., Holman, T. B. & Niemann, B. K. A longitudinal study of the effects of premarital communication, relationship stability, and self-esteem on sexual satisfaction in the ﬁrst year of marriage. J. Sex. Marital Ther. 24, 193–206 (1998). 16. Russo, G. I. et al. Consulting “Dr Google” for sexual dysfunction: a contemporary worldwide trend analysis. Int. J. Impot. Res. 32, 455–461 (2020). 17. Andersson, G. & Titov, N. Advantages and limitations of Internet-based inter- ventions for common mental disorders. World Psychiatry 13, 4–11 (2014). 43. Gheshlaghi, F., Dorvashi, G., Aran, F., Shaﬁei, F. REFERENCES 1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. (American Psychiatric Association, 2013). 1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. (American Psychiatric Association, 2013). 2. Mitchell, K. R. et al. Sexual function in Britain: ﬁndings from the third National Survey of Sexual Attitudes and Lifestyles (Natsal-3). Lancet 382, 1817–1829 (2013). 3. McCool, M. E. et al. Prevalence of female sexual dysfunction among pre- menopausal women: a systematic review and meta-analysis of observational studies. Sex. Med. Rev. 4, 197–212 (2016). 4. Nappi, R. E. et al. Female sexual dysfunction (FSD): Prevalence and impact on quality of life (QoL). Maturitas 94, 87–91 (2016). 4. Nappi, R. E. et al. Female sexual dysfunction (FSD): Prevalence and impact on quality of life (QoL). Maturitas 94, 87–91 (2016). 5. McCabe, M. P. & Connaughton, C. Sexual dysfunction and relationship stress: how does this association vary for men and women? Curr. Opin. Psychol. 13, 81–84 (2017). Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 139 A.-C. Zarski et al. 11 6. Velten, J. & Margraf, J. Satisfaction guaranteed? How individual, partner, and relationship factors impact sexual satisfaction within partnerships. PLoS ONE 23, 1–17 (2017). 33. Hummel, S. B. et al. Internet-based cognitive behavioral therapy for sexual dys- functions in women treated for breast cancer: design of a multicenter, rando- mized controlled trial. BMC Cancer 15, 321 (2015). 34. Kang, H. S., Kim, H. K., Park, S. M. & Kim, J. H. Online-based interventions for sexual health among individuals with cancer: a systematic review. BMC Health Serv. Res. 18, 1–10 (2018). 7. Van Lankveld, J. J. & Grotjohann, Y. Psychiatric comorbidity in heterosexual couples with sexual dysfunction assessed with the Composite International Diagnostic Interview. Arch. Sex. Behav. 29, 479–498 (2000). 8. Goldmeier, D., Malik, F., Phillips, R. & Green, J. Cost implications of sexual dys- function: The female picture. Int. J. Impot. Res. 16, 130–134 (2004). 35. Classen, C. C. et al. Psychosexual distress in women with gynecologic cancer: a feasibility study of an online support group. Psycho-Oncol. 22, 930–935 (2012). 9. Xie, Y. et al. Economic burden and quality of life of vulvodynia in the united states. Curr. Med. Res. Opin. 28, 601–608 (2012). 36. Wootten, A. C. et al. Development, feasibility and usability of an online psycho- logical intervention for men with prostate cancer: My Road Ahead. INVENT 1, 188–195 (2014). 10. Hobbs, L. Published in partnership with Seoul National University Bundang Hospital ADDITIONAL INFORMATION 73. Borenstein, M., Higgins, J. P. T., Hedges, L. V. & Rothstein, H. R. Basics of meta- analysis: I2 is not an absolute measure of heterogeneity. Res. Synth. Methods 8, 5–18 (2017). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41746-022-00670-1. 74. Kraemer, H. C. & Kupfer, D. J. Size of treatment effects and their importance to clinical research and practice. Biol. Psychiatry 59, 990–6 (2006). Correspondence and requests for materials should be addressed to Anna-Carlotta Zarski. 75. Higgins, J. P., Thompson, S. G., Deeks, J. J. & Altman, D. G. Measuring incon- sistency in meta-analyses. Br. Med. J. 327, 557–560 (2003). Reprints and permission information is available at http://www.nature.com/ reprints ACKNOWLEDGEMENTS 59. Domhardt, M., Cuijpers, P., Ebert, D. D. & Baumeister, H. More Light? Opportu- nities and pitfalls in digitalized psychotherapy process research. Front. Psychol. 12, 1–5 (2021). The ﬁrst author received funding for personnel costs as part of a grant from the Emerging Talents Initiative of the Friedrich-Alexander-Universität Erlangen-Nürnberg. The authors wish to thank all investigators who have provided additional information regarding their studies upon our request. This study was registered in The Open Science Framework (https://osf.io/m8kng). 60. Meyers, M., Margraf, J. & Velten, J. Psychological treatment of low sexual desire in women: Protocol for a randomized, waitlist-controlled trial of internet-based cognitive behavioral and mindfulness-based treatments. JMIR Res. Protocols 9, (2020). 61. Furukawa, T. A. et al. Dismantling, optimising, and personalising internet cogni- tive behavioural therapy for depression: a systematic review and component network meta-analysis using individual participant data. Lancet 03, 1–12 (2021). FUNDING 65. Higgins, J. P. et al. Cochrane Handbook for Systematic Reviews of Interventions. (Cochrane, 2020). Open Access funding enabled and organized by Projekt DEAL. 66. Moher, D., Liberati, A., Tetzlaff, J. & Altman, D. G. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. J. Clin. Epidemiol. 62, 1006–1012 (2009). AUTHOR CONTRIBUTIONS 62. Schueller, S. M., Tomasino, K. N. & Mohr, D. C. Integrating human support into behavioral intervention technologies: The efﬁciency model of support. Clin. Psychol.: Sci. Pract. 24, 27–45 (2016). A.C. and J.K. conducted the searches, screened the search records and full-text papers against the eligibility criteria and extracted the study characteristics and effect size data. J.V. and D.D.E. advised on data-extraction disagreements, and D.D.E. also advised on data-analysis. A.C.Z. and J.K. conducted the analyses, and A.C.Z., J.K. and J.V. wrote the manuscript. All authors provided feedback on and approved the ﬁnal manuscript. 63. Musiat, P., Johnson, C., Atkinson, M., Wilksch, S. & Wade, T. Impact of guidance on intervention adherence in computerised interventions for mental health pro- blems: a meta-analysis. Psychological Med. 52, 229–240 (2022). 64. Gurney, K., Hobbs, L. J., Adams, N. J. & Bailey, J. V. The Sextherapylondon inter- active website for sexual difﬁculties: content, design and rationale. Sex. Relatsh. Ther. 35, 210–226 (2020). REFERENCES J. Sex. Med. 12, 1807–1819 (2015). 31. Wiljer, D. et al. A Qualitative Study of an Internet-Based Support Group for Women with Sexual Distress Due to Gynecologic Cancer. J. Canc Educ. 26, 451–458 (2011). 32. Hucker, A. & McCabe, M. P. Incorporating mindfulness and chat groups into an online cognitive behavioral therapy for mixed female sexual problems. J. Sex. Res. 52, 1–13 (2015). 58. Brotto, L. A., Bergeron, S., Zdaniuk, B. & Basson, R. Mindfulness and cognitive behavior therapy for provoked vestibulodynia: mediators of treatment outcome and long-term effects. J. Consulting Clin. Psychol. 88, 48–64 (2020). Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 139 npj Digital Medicine (2022) 139 A.-C. Zarski et al. 12 COMPETING INTERESTS A.C.Z. uses an e-health platform for online health trainings (HelloBetter/Get.On) for intervention development and research purposes as part of a free cooperation. D.D.E. has served as a consultant to/on the scientiﬁc advisory boards of Sanoﬁ, Novartis, Minddistrict, Lantern, Schoen Kliniken, Ideamed and German health insurance companies (BARMER, Techniker Krankenkasse), and a number of federal chambers for psychotherapy. He is also a stakeholder of the Institute for health training online (GET.ON), which aims to implement scientiﬁc ﬁndings related to digital health interventions into routine care. The remaining authors declare no competing interests. 67. Liberati, A. et al. The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: Explanation and elaboration. PLoS Med. 6, (2009). 68. Schwarzer, G. meta: An R Package for Meta-Analysis. R News vol. 7 (2007). 69. Viechtbauer, W. Conducting meta-analyses in R with the metafor package. J. Stat. Softw. 36, 1–48 (2010). 70. Harrer, M., Cuijpers, P. & Ebert, D. Doing meta-analysis in R: A hands-on guide. (2019). 71. Hedges, L. V. & Olkin, I. Statistical methods for meta-analysis. (Academic Press, 2014). 72. Cohen, J. Statistical power analysis for the behavioral sciences. (Routledge Member of the Taylor and Francis Group, 1988). Reprints and permission information is available at http://www.nature.com/ reprints 76. Higgins, J. P. & Green, S. Cochrane Handbook for Systematic Reviews of Interven- tions. (The Cochrane Collaboration, 2011). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 77. Viechtbauer, W. & Cheung, M. W.-L. Outlier and inﬂuence diagnostics for meta- analysis. Res. Synth. Methods 1, 112–125 (2010). 78. Schwarzer, G., Carpenter, J. R. & Rücker, G. Meta-Analysis with R. (Springer Inter- national Publishing, 2015). 79. Peters, J. L., Sutton, A. J., Jones, D. R., Abrams, K. R. & Rushton, L. Contour- enhanced meta-analysis funnel plots help distinguish publication bias from other causes of asymmetry. J. Clin. Epidemiol. 61, 991–996 (2008). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/. 80. Egger, M., Smith, G. D., Schneider, M. & Minder, C. Bias in meta-analysis detected by a simple, graphical test. Br. Med. J. 13, 629–34 (1997). 81. Duval, S. & Tweedie, R. Trim and ﬁll: a simple funnel-plot-based method of testing and adjusting for publication bias in meta-analysis. Biometrics 56, 455–463 (2000). 82. Simonsohn, U., Nelson, L. D. & Simmons, J. P. P-curve: a key to the ﬁle-drawer. J. Exp. Psychol.: Gen. 143, 534–547 (2014). 83. Simonsohn, U., Nelson, L. D. & Simmons, J. P. p-Curve and effect size: correcting for publication bias using only signiﬁcant results. Perspect. Psychological Sci. 9, 666–681 (2014). 84. van Aert, R. C. M., Wicherts, J. M. & van Assen, M. A. L. M. Conducting meta- analyses based on p values: reservations and recommendations for applying p-uniform and p-curve. Perspect. Psychological Sci. 11, 713–729 (2016). Reprints and permission information is available at http://www.nature.com/ reprints © The Author(s) 2022 © The Author(s) 2022 Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 139
https://openalex.org/W2783760658	https://link.springer.com/content/pdf/10.1007%2Fs00339-018-1585-6.pdf	English	null	An analytical model to predict and minimize the residual stress of laser cladding process	Applied physics. A, Materials science & processing	2,018	cc-by	3,553	Applied Physics A (2018) 124:202 https://doi.org/10.1007/s00339-018-1585-6 Abstract Laser cladding is one of the advanced thermal techniques used to repair or modify the surface properties of high-value compo- nents such as tools, military and aerospace parts. Unfortunately, tensile residual stresses are generated in the thermally treated area of this process. This work focuses on to investigate the key factors for the formation of tensile residual stress and how to minimize it in the clad when using dissimilar substrate and clad materials. To predict the tensile residual stress, a one- dimensional analytical model has been adopted. Four cladding materials (Al2O3, TiC, TiO2, ZrO2) on the H13 tool steel substrate and a range of preheating temperatures of the substrate, from 300 to 1200 K, have been investigated. Thermal strain and Young’s modulus are found to be the key factors of formation of tensile residual stresses. Additionally, it is found that using a preheating temperature of the substrate immediately before laser cladding showed the reduction of residual stress. An analytical model to predict and minimize the residual stress of laser cladding process N. Tamanna1 · R. Crouch1 · I. R. Kabir1 · S. Naher1 Received: 31 October 2017 / Accepted: 14 January 2018 / Published online: 2 February 2018 © The Author(s) 2018. This article is an open access publication 1 City, University of London, Nothampton Square, London EC1V HB, UK 1 Introduction to the style guide simplified analytical model was established based on the elastic mechanics for dissimilar materials by Wang et al. [3]. Numerical methods are applied to calculate three-dimen- sional state of residual stresses in the treated object. Mainly, tensile residual stresses were predicted on the surface while the quantity of these stresses was different in different direc- tions, along with experimental validation [4]. The funda- mental understanding of the formation of residual stress in dissimilar material is not well established because of the lack of knowledge about materials’ behaviour in the process. Models are focused on the same cladding and the substrate material [11]. In some cases, dissimilar cladding and sub- strate materials have been examined [4]. However, these models and experimental works do not explain the effect of different properties of cladding materials on residual stresses when the same substrate material was used. Doping is a popular method to improve materials properties by inten- tionally introducing impurities in the substrate. It is found from doping mechanism that residual stresses varied when dissimilar doping materials were used [12]. Other thermal cladding process such as thermal spray [13] and plasma spray [14] showed the differences of residual stress for dif- ferent cladding materials on the same substrate. Recently, to minimize residual stresses, deposition of functionally graded material are used. This process can minimize the differences of mismatch of thermal strain between materials [15]. So, it is required to develop an understanding of the influence of materials properties on the formation of residual stresses. Laser cladding (LC) is one of the advantageous thermal techniques over thermal spraying, plasma spraying and arc welding [1]. In this process, a laser heat source is used to deposit a thin layer, mostly at micro-scale, of a desired mate- rial on a substrate material [2]. One of the challenges of this process is the formation of tensile residual stresses in the thermally treated area [3–5]. Residual stresses are the locked in stress in any engineering components after thermal or mechanical treatment in the absence of any external load [6]. These residual stresses deteriorate the performance of engineering components such as fatigue life [7], corrosion resistance [8] and dimensional accuracy [9] in the finished products. A one-dimensional analytical model was proposed to predict the stress evolution considering elastic, plastic and thermal strain [10]. To predict the tensile residual stress, a * N. Tamanna Nusrat.Tamanna@city.ac.uk R. Crouch Roger.Crouch.1@city.ac.uk I. R. 2 Methodology residual stress of clad and the substrate. However, the coating and the substrate are connected with a metallurgical bonding. So the actual contraction at the interface of the clad and the substrate is H13 tool steel was used as a substrate material and Al2O3, TiC, TiO2 and ZrO2 were used for cladding materials. The preheating temperature of substrate was varied from 300 to 1200 K. The material properties are given in Table 1.i (1) 훼cl (Tmc −Tr ) −휎cl∕Ec = 훼sl (T0 −Tr ) + 휎sl∕Es. (1) A simplified model was adopted to calculate the residual stress as shown in Fig. 1 considering the elastic and the ther- mal strain. 휎 is the normal tensile residual stress [3, 22]. 훼, E and h are defined as the thermal expansion coefficient, Young’s modulus and thickness, respectively. The subscripts c and s represent the clad and the substrate, respectively. Tr, To and Tmc are room temperature, preheating temperature of the substrate and melting temperature or operating temperature of cladding materials, respectively. In the laser cladding process, the oper- ating temperature should be equal or more than the melting temperature of cladding materials. If the clad and the substrate contract freely, the thermal contractions are 훼cl (Tmc −Tr ) and 훼sl (T0 −Tr ) correspondingly. The higher thermal strain of cladding material was assumed than the substrate material, see Fig. 1. The induced elastic strains are σc/Ec and σs/Es due to The uniform stress distribution is assumed in the clad and the substrate while the height of the clad was lower than the height of the substrate. From the internal force balance, The uniform stress distribution is assumed in the clad and the substrate while the height of the clad was lower than the height of the substrate. From the internal force balance, (2) hs휎s = hc휎c, hs휎s = hc휎c, (2) where hs and hc are the heights of the substrate and the clad, respectively. 휎c and 휎s are the tensile residual stresses of the clad and the substrate. From Eq. 1 and Eq. 2, the generated stress in the clad can be derived, (3) 휎c = hsEsEc [훼c (Tmc −Tr ) −훼s (T0 −Tr )] hsEs + hcEc . Table 1 Material properties used to calculate the residual stress 1 Introduction to the style guide Kabir Israt.Kabir.1@city.ac.uk S. Naher Sumsun.Naher.1@city.ac.uk (0121 3456789) 3 3456789) 3 202 Page 2 of 5 202 Page 2 of 5 N. Tamanna et al. Substrate Clad , , , , , , , , ( ) ( ) Fig. 1 The geometry of residual stress model based on thermo-elastic mechanics [3] The high temperature gradient from the clad to the sub- strate material is one of the main reasons for the formation of cracks. The higher cooling rate generates higher misfit of strain, resulting higher residual stress [13]. Preheating of the substrate reduces the thermal gradient which signifi- cantly minimizes residual stresses and prevents cracks on the surface [16]. In this work, an analytical model is presented to calcu- late the tensile residual stress in dissimilar materials. Four widely used cladding materials have been used to observe the change of residual stresses on the H13 tool steel substrate which is a popular material for tooling application [17]. This technique provides a unique method to compare residual stresses for different cladding materials, with capabilities to integrate material properties. Furthermore, preheating temperature of the substrate was applied to check the effect on residual stresses. Fig. 1 The geometry of residual stress model based on thermo-elastic mechanics [3] 2 Methodology (3) a Substrate material b Cladding materials Height of the substrate () and height of the clad () Materials Thermal expansion coef- ficient × 10−6(K−1) Young’s modulus (GPa) Melting tempera- ture (K) Height (m) H13 tool steela 10.90 [4] 210 1752 0.005 Al2O3 b 5.40 [18] 380 2338 0.001 TiCb 7.70 [19] 449 3363 0.001 TiO2 b 11.80 [20] 288 2123 0.001 ZrO2 b 12.20 [21] 250 2973 0.001 1 3 Page 3 of 5 202 Page 3 of 5 202 An analytical model to predict and minimize the residual stress of laser cladding process The effect of different materials and preheating tem- perature of the substrate on the generation of residual stresses were theoretically investigated using this model. The results of residual stresses are normalized with the residual stress of TiC clad. Thermal strain of the mate- rial is related with the thermal expansion coefficient of materials and temperature difference of the operating temperature and the room temperature. The mismatch of the thermal strain between the clad and the substrate ( 훼c (Tmc −Tr ) −훼s (T0 −Tr ) in Eq. 3) was found one of the reasons of formation of residual stresses in the system. Another material property, Young’s modulus ( Ec in Eq. 3), was considered with the mismatch of thermal strain to understand its effect on the formation of residual stress. The effect of different materials and preheating tem- perature of the substrate on the generation of residual stresses were theoretically investigated using this model. 3 Results and discussion Figure 2 shows the distribution of predicted tensile residual stresses for different cladding materials (Al2O3, TiC, TiO2, ZrO2) on the H13 tool steel substrate at a range of preheat- ing temperature (300 to 1200 K) of the substrate. TiC clad- ding material formed higher residual stress than TiO2 clad- ding material. The minimum residual stress was formed for Al2O3 cladding material. TiC cladding material formed the maximum tensile residual stress below 900 K preheating temperature of the substrate, while ZrO2 cladding material formed the maximum tensile residual stress above 900 K preheating temperature of the substrate, see Fig. 2. The pre- dicted residual stress of four cladding materials at 300 K and at 1200 K preheating temperature of the substrate is shown in Fig. 3a, b, respectively. To understand the variation of residual stresses for different material system, thermal strain and Young’s modulus have been analysed. T h e m i s m a t c h o f t h e r m a l s t r a i n , 훼c (Tmc −Tr ) −훼s (T0 −Tr ) in Eq. 3, between the clad and the substrate material is one of the reasons of the forma- tion of residual stress. The differences of the thermal strain of four cladding materials from the substrate material are shown in Fig. 4a, b at the 300 and 1200 K preheating tem- perature of the substrate, respectively. T h e m i s m a t c h o f t h e r m a l s t r a 훼c (Tmc −Tr ) −훼s (T0 −Tr ) in Eq. 3, between the clad the substrate material is one of the reasons of the for tion of residual stress. The differences of the thermal st of four cladding materials from the substrate material shown in Fig. 4a, b at the 300 and 1200 K preheating t perature of the substrate, respectively. ZrO2 cladding material showed higher mismatch of th mal strain with the substrate at any preheating tempera than other clad materials, see Fig. 4a, b. However, be 900 K preheating temperature of the substrate, TiC cladd material generated higher tensile residual stress than Z cladding material. To clarify this issue, another mate property, Young’s modulus, was considered. The Youn modulus of TiC and ZrO2 cladding materials is 449 G and 380 GPa, respectively. From Eq. 3 Results and discussion 3, we have taken 300 400 500 600 700 800 900 100011001200 0.0 0.2 0.4 0.6 0.8 1.0 Residual stress Temperature (K) Al2O3 ZrO2 TiO2 TiC Fig. 2 Distribution of the normalized residual stress with preheating temperature of the substrate for four cladding materials 0.0 0.2 0.4 0.6 0.8 1.0 Residual stress Al2O3 ZrO2 TiC TiO2 (b) 0.0 0.2 0.4 0.6 0.8 1.0 TiC TiO2 ZrO2 Al2O3 Residual stress (a) Fig. 3 The normalized residual stress a at 300 K and b at 1200 K preheating temperature of the substrate for four cladding materials 300 400 500 600 700 800 900 100011001200 0.0 0.2 0.4 0.6 0.8 1.0 Residual stress Temperature (K) Al2O3 ZrO2 TiO2 TiC Fig. 2 Distribution of the normalized residual stress with preheating temperature of the substrate for four cladding materials 300 400 500 600 700 800 900 100011001200 0.0 0.2 0.4 0.6 0.8 1.0 Residual stress Temperature (K) Al2O3 ZrO2 TiO2 TiC ZrO2 cladding material showed higher mismatch of ther- mal strain with the substrate at any preheating temperature than other clad materials, see Fig. 4a, b. However, below 900 K preheating temperature of the substrate, TiC cladding material generated higher tensile residual stress than ZrO2 cladding material. To clarify this issue, another material property, Young’s modulus, was considered. The Young’s modulus of TiC and ZrO2 cladding materials is 449 GPa and 380 GPa, respectively. From Eq. 3, we have taken the Temperature (K) Fig. 2 Distribution of the normalized residual stress with preheating temperature of the substrate for four cladding materials Fig. 2 Distribution of the normalized residual stress with preheating temperature of the substrate for four cladding materials 0.0 0.2 0.4 0.6 0.8 1.0 Residual stress Al2O3 ZrO2 TiC TiO2 (b) 0.0 0.2 0.4 0.6 0.8 1.0 TiC TiO2 ZrO2 Al2O3 Residual stress (a) Fig. 3 The normalized residual stress a at 300 K and b at 1200 K preheating temperature of the substrate for four cladding materials 0.0 0.2 0.4 0.6 0.8 1.0 Residual stress Al2O3 ZrO2 TiC TiO2 (b) 0.0 0.2 0.4 0.6 0.8 1.0 TiC TiO2 ZrO2 Al2O3 Residual stress (a) (b) (a) Fig. 3 The normalized residual stress a at 300 K and b at 1200 K preheating temperature of the substrate for four cladding materials 1 1 3 202 Page 4 of 5 202 Page 4 of 5 N. Tamanna et al. 3 Results and discussion 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 Mismatch of thermal strain at 300 K Al2O3 ZrO2 TiC TiO2 (a) 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 Mismatch of thermal strain at 1200 K Al2O3 ZrO2 TiC TiO2 (b) Fig. 4 Mismatch of thermal strain between the cladding materials and the substrate a at 300 K and b at 1200 K preheating temperature of the substrate 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 Mismatch of thermal strain at 1200 K Al2O3 ZrO2 TiC TiO2 (b) (a) Fig. 4 Mismatch of thermal strain between the cladding materials and the substrate a at 300 K and b at 1200 K preheating temperature of the substrate material is higher than ZrO2 (250 GPa) cladding material. However, ZrO2 cladding material produced higher residual stress. Then, it is important to focus on the mismatch of thermal strain. The mismatch of thermal strain of Al2O3 is much lower than ZrO2 which generating lower residual stress in Al2O3 cladding material. part related with mismatch of thermal strain and Young’s modulus of clad material, Ec [훼c (Tmc −Tr ) −훼s (T0 −Tr ) , to explain this trend. The product of mismatch of thermal strain and Young’s modulus was shown in Fig. 5a, b. At 300 K, TiC cladding material showed maximum value shown in Fig. 5a. Moreover, TiC has higher Young’s modu- lus and very small difference between mismatch of thermal strain with ZrO2. However, mismatch of thermal strain was decreased with increasing the preheating temperature of the substrate. The degree of decrement of the mismatch of ther- mal strain between TiC cladding material and the substrate was higher than ZrO2 cladding material above 900 K gen- erating lower residual stress, though the Young’s modulus of TiC was higher. The residual stress decreased with the rise of the pre- heating temperature of the substrate for all types of clad- ding materials. The reason behind the descendant trend of the residual stress is the lower thermal gradient between the clad and the substrate material and relatively slower cooling rate [16, 23]. The slower the cooling rate, the material got longer time to come back to the original dimension. As it takes longer time to come back to room temperature, the mismatch of thermal strain also decreased between two materials. 4 Conclusions 40, 5007 (2014) 13. T.W. Clyne, S.C. Gill, J. Therm. Spray Technol. 5, 401 (1996) 13. T.W. Clyne, S.C. Gill, J. Therm. Spray Technol. 5, 401 (1996) 14. J. Matejicek, S. Sampath, P.C. Brand, H.J. Prask, Acta Mater. 47, 607 (1999) 15. Z. He, J. Ma, G.E.B. Tan, J. Alloys Compd. 486, 815 (2009) 16. Y.-F. Tao, J. Li, Y.-H. Lv, L.-F. Hu, Opt. Laser Technol. 97, 379 (2017) 17. F. Klocke, K. Arntz, M. Teli, K. Winands, M. Wegener, S. Oliari, Procedia CIRP. 63, 58 (2017) 18. P. Auerkari, Mechanical and Physical Properties of Engineering Alumina Ceramics, Technical Research Centre of Finland, VTT Manufacturing Technology, Research Notes 1792, 3–36 (1996) Acknowledgements Sincere thanks to EU funded Erasmus Mundus project, EM LEADERS reference: 551411-EM-1-2014-1-UK-ERA MUNDUS-EMA21_EM, and School of Mathematics, Computer Sci- ence and Engineering of City, University of London for funding the Project. 19. D.Y. Dang, J.L. Fan, H.R. Gong, J. Appl. Phys. 116, 0 (2014) 20. AZoM (2002), https://www.azom.com/article.aspx?Artic leID=1179. Accessed 31 Oct 2017 21. AZoM (2002), https://www.azom.com/properties.aspx?Artic leID=133. Acessed 31 Oct 2017 22. F.P. Incropera, T.L. Bergman, A.S. Lavine, D.P. DeWitt, Funda- mentals of Heat and Mass Transfer, 7th edn. (Don Fowley, Jef- ferson, 2011) Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creativeco mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu- tion, 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. ) 23. S. Zhou, X. Dai, H. Zheng, Opt. Laser Technol. 43, 613 (2011) 3 Results and discussion If we compare between Al2O3 and ZrO2 cladding mate- rials, the Young’s modulus of Al2O3 (380 GPa) cladding 0 2 4 6 8 10 Product of mismatch of thermal strain and Young's modulus (GPa) at 300 K Al2O3 ZrO2 TiC TiO2 (a) 0 2 4 6 8 10 Product of mismatch of thermal strain and Young's modulus (GPa) at 1200 K Al2O3 ZrO2 TiC TiO2 (b) Fig. 5 Product of mismatch of thermal strain and Young’s modulus a at 300 K and b at 1200 K preheating temperature of the substrate for four cladding materials 0 2 4 6 8 10 Product of mismatch of thermal strain and Young's modulus (GPa) at 1200 K Al2O3 ZrO2 TiC TiO2 (b) 0 2 4 6 8 10 Product of mismatch of thermal strain and Young's modulus (GPa) at 300 K Al2O3 ZrO2 TiC TiO2 (a) (b) (a) Fig. 5 Product of mismatch of thermal strain and Young’s modulus a at 300 K and b at 1200 K preheating temperature of the substrate for four cladding materials 1 3 3 An analytical model to predict and minimize the residual stress of laser cladding process Page 5 of 5 Page 5 of 5 202 202 1. E. Toyserkani, A. Khajepour, S.F. Corbin, Laser Cladding, 1st edn. (CRC Press, New York, 2004) 4 Conclusions 2. C.S. Chien, C.W. Liu, T.Y. Kuo, C.C. Wu, T.F. Hong, Appl. Phys. 2. C.S. Chien, C.W. Liu, T.Y. Kuo, C.C. Wu, T.F. Hong, Appl. Phys. A Mater. Sci. Process. 122, 1 (2016) A Mater. Sci. Process. 122, 1 (2016) g pp y A Mater. Sci. Process. 122, 1 (2016) 3. D. Wang, Q. Hu, X. Zeng, Surf. Coatings Technol. 274, 51 (2015) An analytical model has been used to calculate and compare the stress generation in dissimilar clad and substrate material system with different preheating temperatures of the sub- strate. Four cladding materials, TiO2, TiC, ZrO2 and Al2O3 were used on the H13 tool steel substrate. Below 900 K preheating temperature of the substrate, maximum residual stress was generated in the TiC clad. The maximum residual stress was formed in ZrO2 clad above 900 K. The minimum residual stress was found for Al2O3 clad. Thermal strain and Young’s modulus were found as key factors of the genera- tion of residual stress. The minimum mismatch of thermal strain was found in Al2O3 clad. The reduction of mismatch of thermal strain can reduce the residual stress in the clad. This analysis can be used to find a combination of materials that can generate minimum residual stress. This model is capable to include preheating of the substrate and predicts its effect on the distribution of the residual stress. The incre- ment of preheating temperature of substrate decreased the residual stress. 3. D. Wang, Q. Hu, X. Zeng, Surf. Coatings Technol. 274, 51 (2015) 4. P. Farahmand, R. Kovacevic, Opt. Laser Technol. 63, 154 (2014) 4. P. Farahmand, R. Kovacevic, Opt. Laser Technol. 63, 154 (2014) 5. N. Tamanna, R. Crouch, S. Naher, 20th Int. ESAFORM Conf. Mater. Form. 40015, (2017) 6. M. Eslami, R. Hetnarski, J. Ignaczak, N. Noda, N. Sumi, Theory of Elasticity and Thermal Stresses, 1st edn. (Springer, New York, 2013) 7. Y. Chew, J.H.L. Pang, Int. J. Fatigue. 87, 235 (2016) 8. A. Martinez Hurtado, J.A. Francis, N.P.C. Stevens, Mater. Sci. Technol. 32, 1484 (2016) 9. J. Liu, L. Li, Opt. Laser Technol. 36, 477 (2004) 10. F. Brückner, D. Lepski, E. Beyer, J. Therm. Spray Technol. 16, 355 (2007) 11. M. Hao, Y. Sun, Int. J. Heat Mass Transf. 64, 352 (20 12. P. Han, F.R. Xiao, W.J. Zou, B. Liao, Ceram. Int. 40, 5007 (2014) 12. P. Han, F.R. Xiao, W.J. Zou, B. Liao, Ceram. Int. References 1 3 1 3
https://openalex.org/W2103707810	https://ccsenet.org/journal/index.php/ijef/article/download/40095/23353	English	null	The Dilemma of Graduate Unemployment within a Context of Poverty, Scarcity and Fragile Economy: Are there Lessons for the University?	International journal of economics and finance	2,014	cc-by	7,622	International Journal of Economics and Finance; Vol. 6, No. 12; 2014 ISSN 1916-971X E-ISSN 1916-9728 Published by Canadian Center of Science and Education International Journal of Economics and Finance; Vol. 6, No. 12; 2014 ISSN 1916-971X E-ISSN 1916-9728 Published by Canadian Center of Science and Education The Dilemma of Graduate Unemployment within a Context of Poverty, Scarcity and Fragile Economy: Are there Lessons for the University? Marcellus Forh Mbah1 1 Faculty of Education, Canterbury Christ Church University, Canterbury, United Kingdom Correspondence: Marcellus Forh Mbah, Research Centre for Children, Families and Communities, Canterbury Christ Church University, North Holmes Campus, CT1 1QU, United Kingdom. E-mail: Marcellus.mbah@canterbury.ac.uk Accepted: October 7, 2014 Online Published: November 25, 2014 URL: http://dx.doi.org/10.5539/ijef.v6n12p27 Received: September 5, 2014 doi:10.5539/ijef.v6n12p27 Abstract The context of the work of universities in Sub-Saharan Africa is one of high levels of poverty, scarcities and fragile economies. Even though historically, African universities have been very useful in providing the human resources needed to serve in public and private sectors, the rising trend in graduate unemployment is a call for concern. Whilst graduate unemployment is also a phenomenon across the globe, the situation is particularly severe in Africa where many inhabitants see university education as a live wire to gain economic freedom, acquire prosperity and step into a better living condition. Whilst many Africans still harbour hope in tertiary education (and this can be evident by the increasing demand for university education and enrolment), the resulting effect of higher education is often an acquisition of prestige for having completed that level of education than the economic benefits they aspired for. Given this context, this article draws on a single interpretive case study involving a State-owned university, as well as semi-structured interviews and focus group discussions carried out with participants from disparate backgrounds to uncover voices on graduate unemployment, including the disturbing impact this phenomenon is having on families and the future of many youths. It concludes with recommendations of ways the university and its educational system can be fashioned to foster graduate employability and improvement in the living condition of those living in uncertain and fragile economies. Keywords: university education, graduate employability, capacity building, entrepreneurship, Cameroon Keywords: university education, graduate employability, capacity building, entrepreneurship, Cameroon 1. Introduction 1. Introduction ployment, Ca rticle therefor ameroon univ re sets out to e versities still explore the sub face the ch bject of gradu hallenge of h uate unemploy high rate of yment within a f graduate a confined rise in y unemploy context in youth unemp yment. This ar n Cameroon. ployment, Ca rticle therefor ameroon univ re sets out to e versities still explore the sub face the ch bject of gradu hallenge of h uate unemploy high rate of yment within a f graduate a confined 2. Methoodology and Contextual Baackground The resea a qualitat Employin participan adopted. 2006; Yin 25th of M arch underpinn tive approach ng convention nts the latitud Given that a n, 2009), the March to the 1st ning this artic was informed nal quantitativ de to articula case study w research emp t of June 2013 cle can best be d by the need ve methods t ate their ideas will typically ployed semi-st 3 to capture th e described as d to give parti to the researc s. Thus, a sin utilise multip tructured inte he voices of 31 a qualitative cipants an op ch would hav ngle interpret ple ways of co rviews and tw 1 participants study. The de portunity to h ve inhibited t tive case-stud ollecting data wo focus grou drawn from d eliberate choic have their voi the intention dy research d a (Mackenzie up discussions disparate back ce to adopt ces heard. of giving esign was & Knipe, s from the grounds. Figuure 1. Illustrattion of the muunicipality of BBuea Figuure 1. Illustrattion of the muunicipality of BBuea The site Cameroo municipa is made u above 20 residing i at the foo capital of British co West Reg English a The mun over 12,0 of the researc n carved out ality is situated up of eighty-fi 00,000 inhabit in the villages ot of Mount Ca f German Kam olonial rule, th gion. The maj and French ar nicipality also 000, and abou ch was the mu administrative d in the South five villages sp tants. The prin s. The urban b ameroon, whi merun during he capital of t ority of the in re two official consists of a t 300 permane unicipality of e units into vi h West Region pread across a ncipal ethnic but cosmopoli ich is the high German colo he Federated nhabitants rely l languages u State univers ent and 200 p f Buea in Cam illages, towns n which is one a surface area group in the itan setting w hest mountain onial rule. It la State of West y on agricultu sed for gener sity created in part-time staff meroon. It is w s, cities, munic e of two Engli of 870 Sq.km municipality within the mun in West and C ater became c t Cameroon, a ure (small scal ral interaction n 1993. The u f (UB, 2014). 1. Introduction Despite the timid and restricted start, several universities sprung up in Africa following independence and were faced with the mounting task of providing a labour force that would serve the Independent States (Eisemon & Salmi, 1993). Apart from having a mission to provide a labour force to serve the respective Independent States, newly established African universities were also charged to play a leading role in addressing the continent’s outstanding problems of poverty, low production, hunger, unemployment, diseases and illiteracy (Mosha, 1986; Sawyerr, 2004). Taking the case of Cameroon for instance, the country had no university and many Cameroonians went abroad for university studies prior to independence in 1960, though such studies were poorly adapted to meet the needs of the Africa continent in general and Cameroon in particular (Njeuma et al., 1999). After independence, Cameroon faced an immediate need for trained senior civil servants to fill some of the positions that had hitherto been occupied by expatriates. The Federal University of Cameroon which evolved from the National Institute for Universities Studies established in 1961 and later renamed the University of Yaoundé had as urgent goal to meet the needs of the nation (Ngu & Kwankam, 1992). Given the escalating need to decongest and decentralise the University of Yaoundé, Cameroon’s Higher Education underwent a series of reforms which accounts for the eight State universities the nation currently has. Although the rising demand in university education and increase in enrolment meant that Cameroonian universities have seen a surge in the number of graduates being released to seek for jobs, there has not been a corresponding rise in the rate of graduate employment (Neneh, 2014). This is worrying, given that the current dilemma of graduate unemployment falls within the shadow of the university reforms of 1993 which also had as goal the professionalisation of higher education with an intention to produce graduates with the capacity needed by the private sector and the public service (Njeuma et al., 1999). In spite of several attempts by the State to curb the 27 Innternational Jouurnal of Econommics and Finance www.ccsenet.org/ijef Vol. 6, No. 12; 2014 rise in y unemploy context in youth unemp yment. This ar n Cameroon. 3.1 Graduate Employability for Self-Reliance In a deliberate move to convey a message when asked the purpose of university education, a community participant who was divorced and had two dependents stated: “If it is not to have a job then what is the essence of a child going to the university?” (Agnes, female, 51–60, Bokwai, 06-04-2013). Given this quotation and the desire expressed by many other participants to see university graduates employed or find jobs, it was evidently clear that one of the underlining reasons points to the notion of “self-reliance”. But what is “self-reliance”? The following quotation from a community participant who relied on small scale farming to take care of her twenty-six dependents illustrated this notion: “When we the parents send our children to the university, it is that they should study and have something to do; If I, as a struggling farmer spend money and my child goes to the university and comes out, and there is no job, then the child turns to me and says ‘mummy I want a pair of shoes or bathing lotion’; it does not sound well. The weight of financial burden you thought had been uplifted from your shoulder by sending that child to the university to study and pick up a job returns to you again. This is because the child cannot find a job, thus continuing to rely on you for provision of his/her basic needs” (Docas, female, 31–40, Bokuva, 03-05-2013). The key message communicated by this participant can be construed to be the need for university graduates to be employed in order for them to be self-sufficient or demonstrate an ability to provide for their personal needs. It can be argued that rather than graduates exercising continuous reliance on their “struggling” parents or sponsors, these sponsors will be relieved of the financial burden of not only sponsoring a student at the university but also meeting his or her daily exigencies due to the opportunities graduate employment brings. 2. Methoodology and Contextual Baackground A worth noting cipalities, divi ish Speaking r m with a total e is Bakweri w nicipality is th Central Africa capital of Sout and now regio le farming) as while “pidgin university has A different mu that the gove visions and reg regions of Cam estimated pop with a majorit he town of Bu a. Historically, thern Camero onal capital of s a source of l in” is the ling a student enr municipality an ernment of gions. The meroon. It pulation of ty of them uea located it was the oons under f the South livelihood. gua franca. rolment of nd perhaps 28 International Journal of Economics and Finance Vol. 6, No. 12; 2014 www.ccsenet.org/ijef in the French speaking part of Cameroon could have been chosen for the research but was decided against due to the language barrier and the potential additional costs of securing translations. Figure 1 illustrates the position of the municipality of Buea in the world. in the French speaking part of Cameroon could have been chosen for the research but was decided against due to the language barrier and the potential additional costs of securing translations. Figure 1 illustrates the position of the municipality of Buea in the world. During the enquiry process, participants were invited to explore and describe their own understandings and definitions of the university and its role within their local community. Participants’ contributions were transcribed and thematically analysed (Ryan, 2003). Direct quotations from participants have been inserted in the article to justify the different themes, with each quotation bearing the pseudonym of the participant, the gender, the age range, his/her village of residence and the date the interview or focus group discussion was conducted. From the responses gathered, the university was construed as a) an educational and teaching centre b) a place to acquire prestige and status c) a venue for capacity building in order to gain employment and d) a place for intellectual and personal development amongst others. Whilst the subject of the university being seen as a venue for students to gain capacity in order to secure employment was a recurrent theme, it is also in the interest of this article to probe this subject further. 3. University for Capacity Building and Employment Universities have been very useful in providing the human resources needed to serve in public and private sectors worldwide (Elliots et al., 1996; Eisemon & Salmi, 1993). Even though the first university established in Cameroon had the mandate to provide a labour force to serve the demands of the newly independent nation– Cameroon (Njeuma et al., 1999); yet, many decades after independence, participants’ responses revealed that university education in Cameroon is still being overwhelmingly regarded as a gateway to employment. Respondents uncovered how many sponsors of university students in Cameroon rely on very small scale farming and other menial tasks to generate the funds needed to support a child or loved one undergo university education with the intention that upon completion of studies, the graduate would be gainfully employed and begin to assist other loved ones economically. In this regard, many participants emphatically stated that the university should demonstrate responsibility in ensuring that graduates are job or employment ready in order to guarantee their ability for self-reliance and readiness to assume family responsibilities. This may not be considered surprising given the revealed challenging circumstances sponsors endure in order to see a loved one through university education due to widespread poverty and the fragile economy of the nation. 3.2 Graduate Employability to Assume Family Responsibility In Africa in general and in Cameroon in particular, there is a strong sense of the family in almost every sector of social life. Family members tend to stick together and assist one another economically, morally and spiritually underpinned by a communal spirit of solidarity. This solidarity also aligns with the Ubuntu ideology expressed in many African communities. According to Makgoba (1996), Ubuntu’s uniqueness is underpinned by its emphasis on the respect for the non-material order that exists in and amongst people. It fosters respect within humankind and for the environment; it is spiritually inclined; it is non-racial; it accommodates other cultures, and it is an invisible uniting force amongst Africans (Enslin & Horsthemke, 2004). It can be seen as a value system which provides the source of many actions and attitudes in African communities. As an African way of viewing the world, Ubuntu represents an alternative to individualism and competiveness in western communities by underlining the need for care, hospitality, respect and responsibility towards one another. Given this context, many community participants associated graduate employment with family solidarity. A family member who succeeds in a venture is often considered the success of the family and families can be seen relying on a member’s economic fortune. In many instances, the employed is the sole bread winner of the entire family, one who assumes most of the financial responsibilities associated with it. The following excerpt from a participant’s response captures this understanding: “The mother thinks the child should finish studying at the university and gets a good job and starts to provide his or her needs and the needs of other family members” (Helen, female, 51–60, Bokwai New Layout, 05-05-2013). This participant suggested that graduates should find jobs so that they can provide the needs of the family. Being an African, it is no hidden secret that the burden of economic responsibility that rest on an employed graduate goes beyond his or her immediate family to also include extended family members. The nature of family economic responsibilities associated with graduate employability as captured by participants’ narratives can be illustrated thus: Figure 2. Family economic responsibilities associated with graduate employability Parents/sponso 1 University education 2 Graduate 3 Other family members (brothers, sisters, …) 4 2 University education 1 Graduate Parents/sponso Figure 2. 3.1 Graduate Employability for Self-Reliance To corroborate this notion of self-reliance, another respondent who was involved in small scale farming in one of the remote communities underscored: “The mother thinks the child should have finished studying at the university and got a good job and started to provide his or her needs and the needs of other family members but he or she comes out of the university and continues to be a burden and the both of you are just looking at each other. It does not make sense if he is unable to find a job when you know that he is educated. If he is educated and is not using his degree to earn a living, it does not mean anything” (Helen, female, 51–60, Bokwai New Layout, 05-05-2013). From the narrative of this participant and many others captured within the enquiry process, it can also be 29 International Journal of Economics and Finance Vol. 6, No. 12; 2014 www.ccsenet.org/ijef underscored that the reason articulated for graduate employment is not limited to self-reliance but also included the need for employed graduates to assume family responsibilities. A strong link can therefore be uncovered between participants’ desire for graduate employment and the concept of the family. underscored that the reason articulated for graduate employment is not limited to self-reliance but also included the need for employed graduates to assume family responsibilities. A strong link can therefore be uncovered between participants’ desire for graduate employment and the concept of the family. 3.2 Graduate Employability to Assume Family Responsibility Family economic responsibilities associated with graduate employability Figure 2 can be explained: Starting from the position of a sponsor/parent “1”, he/she may engage in small scale farming or small business to sponsor a child at the university at position “2”. The sponsor or parent struggles in the process to secure the required funds to enable his or her child enrol and go through university education with great anticipation that after graduation, the child would find a job or be employed at position “3” and eventually assist his/her siblings and other family members at position “4” to gain university education or meet any other 30 International Journal of Economics and Finance Vol. 6, No. 12; 2014 www.ccsenet.org/ijef socio-economic demands, which can in turn be of economic help to the parents or sponsors at position “1” who had hitherto gone through tough times to see her/him through university education and need to recuperate, or be able to assist other loved ones go through university education. In addition to economic responsibilities associated with graduate employability, a closer look at Figure 2 also reveals that university education can be positioned at the centre of its own sustainability, as well as the economic sustainability of families. This sustainability is depicted by the cyclic nature of the figure. From this perspective, it can be argued that the rate of graduate employability may spur university enrolment, and hence the sustainability of university education in communities. Not only would graduate employment provide a convincing reason why parents should send their children to the university but also, it provides an economic opportunity to afford the costs of seeing someone through it. In spite of participants’ acknowledgment that the university carry the potential to build human capacities for employment, their responses also raised the concern of rise in graduate unemployment. 4. The Dilemma of Graduate Unemployment Most of the participants interviewed on the nature of university education, pointed fingers to the lack of jobs for graduates. They overwhelmingly considered the university to be specialising in releasing graduates without prior preparation for employment or job creation and consequently contributing to the rising trend of youth unemployment in the community. The following excerpts from participants’ responses revealed the nature of graduate unemployment in their communities: “A lot of students graduate from the university and they do not have any employment. We the parents are taken aback because we expected that as soon as a child gets to that level, he or she should automatically obtain a job. But now we are realising that we spend more and more money each time in education and there is no solution” (Sherley, male, 51–60, Bonakanda, 10-05-2013). “Many of them that leave from the university just come back to stay at home jobless. A mother goes to the farm and is harvesting cassava and the son who has graduated from the university is also fighting to harvest that same cassava. The child who has completed from the university is requesting money from the mother to buy soap. The mother is stressed up all the time” (Helen, female, 51–60, Bokwai New Layout, 05-05-2013). “My daughter has just graduated with a degree in economics and she has a good GPA but what can she do? Is all theory and there is no job” (Margaret, female, 41–50, Bolifamba, 15-04-2013). Whilst the strategic plan of the university at the centre of this article covering the period 2007–2015 underscores that one of the university’s objective is to “make university programmes more professional and more responsive to market forces” (Mbuntum et al., 2008), the views of participants strongly suggested graduate unemployment is still a significant problem in the community. Given the state of this crisis and in an attempt to begin to seek ways to address it, it is essential to explore its underlining causes. In this light and from participants’ narratives, the rise in graduate unemployment can be considered to have an endogenous root within the university and exogenous root outside the university. Endogenous factors responsible for graduate unemployment are factors the university can bring under its control whereas exogenous factors are factors largely beyond the reach of the university’s means and capacity. 5. Endogenous Factors Responsible for Graduate Unemployment Lack of student orientation, absence of course relevance and lack of practical skills are some of the factors raised by participants to be responsible for the undesirable situation of graduate unemployment. 5.2 Absence of Course Relevance It was noted that some students enrolled at the university due to the extrinsic benefit of securing the employment opportunities a particular academic degree presents. One female student on an agricultural course maintained: “I wanted to do Agriculture because of the employment prospects it offers. Self-employment, employment with the private sector, and employment with the government are possible options after my studies. If I apply the skills I have acquired here as a self-employer, that is, doing my own farming, I will still make a lot of money and earn a living” (Sophie, female, 21–30, Molyko, 19-04-2013). This was the scenario of a student who saw the need to enrol on an academic programme which provides a prospect for employment after completion. Enrolling on an academic programme like Agriculture may guarantee employment, even as the economy of Cameroon largely relies on the agricultural sector. An analysis of the contribution of the various economic sectors to Cameroon’s 2009 real economic growth of 1.8% reveals that food crop production made the most contribution with a 0.9% (Fouda, 2012). Not all programmes at the university were seen in the same light. One male student participant noted: “About one quarter of academic programmes at the university are not useful in the contemporary world, they are not profitable and as such, it makes it difficult to get a job, whereas, if half of the population at the university is orientated towards subjects like the sciences, technology and agriculture, it will help completing students to at least be self-employed. Looking at the Cameroonian society, I think our main source of livelihood is agriculture and the majority of the students of the university should be orientated towards it and even technology because globalisation has made technology to gain ground in the developing world” (Lucas, male, 21–30, Molyko, 12– 04–2013). This assertion suggested the need for the university to run academic programmes which are relevant to the local community but also globally significant. The university may therefore need to keep pace with changing local and global market demands and trends and train/educate students suited for these changes. To corroborate the need for academic programmes at the university to also demonstrate global relevance, one female participant maintained that the university should “enlarge its scope and diversify its programmes. They should open the students not only to the Cameroonian community but to communities abroad” (Susan, female, 21–30, Molyko, 12–04–2013). 5.1 Lack of Proper Orientation One participant suggested that the lack of proper orientation have led many students to embark on the wrong academic programmes at the university which resulted in the difficulty of finding a job, basically because there are no jobs in that academic field. This female student participant elaborated: “You would notice that some students before being enrolled into the university are not orientated. When they are not orientated, they come in and they study something. But when you study something that the community is already saturated with or may not have immediate need of, you will automatically have some graduates jobless” (Susan, female, 21–30, Molyko, 12–04–2013). Whilst students can benefit from parental counselling or the advice of a loved one before university enrolment, it can be suggested that the university can also set up a unit to present opportunities for counselling to students caught up at the crossroad of confusion or doubt about which academic path to pursue, which have accompanying employment prospects. However, when many of the participants spoke about graduate employment, it was noticed that the idea of employment they conveyed was one that centres on being recruited by the private or public sector rather than on being self-employed. Accordingly, having a relevant orientation could lead to a change of mind set and prospective graduates can acquire skills for job creation or 31 International Journal of Economics and Finance Vol. 6, No. 12; 2014 www.ccsenet.org/ijef self-employment. One student participant sustained this argument by maintaining that the youths of Cameroon should depart from the idealistic position of seeking for jobs that would enable them to sit in offices. He elaborated: “There are other forms of employment; you must not only sit in an office as a lawyer or a doctor” (Lucas, male, 21–30, Molyko, 12–04–2013). Although the university at the centre of this research has staff members who were recruited to provide guidance and counselling to students in need, how effectively the service is running is worth exploring. 5.2 Absence of Course Relevance This underscores a desire for the university to position prospective graduates such that they are able to maximise the advantages of globalisation by being able to also compete for jobs or demands out of the country. Whilst the number of graduates from Cameroonian universities exceeds the demand for new recruits, it would be beneficial if graduates are able to trade their skills and abilities abroad. Furthermore, where training programmes at the university are not relevant to the needs of both local and global markets, there is need for reorientation of such programmes for suitability. 6. Exogenous Factors Responsible for Graduate Unemployment Inadequate facilities to conduct training, as well as public corruption, heavy taxes and cumbersome governmental processes are some of the exogenous factors raised by participants which are responsible for graduate unemployment. 5.3 Lack of Practical Skills In addition to revealing their desire to see university education as relevant to local and global markets, some participants suggested that such relevance can be enhanced when practical components are associated with each academic programme. One female university student suggested: “The university needs to bring in more practical elements into their academic programmes. It has been theory, theory, and you discover how boring it is the more you remain in class. We do not even know what we are going to face outside. They need to widen the scope a little bit” (Pauline, female, 21–30, Molyko, 12-04-2013). The view of this participant can be said to also fall in line with the notion of the engaged scholarship propounded by Ernest Boyer (1996) where he argued that scholarship should not be confined to theory but should also find expression on the field through engagement. Engaging students in practical activities, tend to give them the opportunity to apply classroom knowledge in a real world or practical situation and this has the potential to prepare them for life after studies. Furthermore, experience enhances learning (Dewey, 1938) by promoting reflective and critical thinking. Whilst these endogenous factors responsible for graduate unemployment can be addressed by the university’s capacity, there are other factors termed exogenous factors which the university could find challenging to address as they do not fit so much within its remit. 32 International Journal of Economics and Finance Vol. 6, No. 12; 2014 www.ccsenet.org/ijef 6.2 Public Corruption, Heavy Taxes and Cumbersome Governmental Processes Although employment may be regarded as work under contractual arrangements involving economic benefits (Jahoda, 1982), it is not restricted to being recruited to work in a public or private sector, but also involves being self-employed. Self-employment can be expressed through the setting up of businesses. Entrepreneurs and sole proprietors setting up businesses, would normally seek a relevant environment and climate that would enable their businesses to flourish and to maximise profit. According to one student participant, this is not the situation in Cameroon where he maintained the political and economic climate is hostile to sole proprietorship. He posited: “The government of the country is corrupt which makes it difficult to even start a business. The procedure to start a new business is cumbersome, coupled with heavy taxes. The system should facilitate private investments” (Paul, male, 21–30, Molyko, 02-04-2013). This narrative suggests that the university can impart students with entrepreneurial skills for self-employment but State policies and corruption can make it difficult for them to engage in profitable ventures like starting up a new business. Corruption in Cameroon is a widespread concern and the country was ranked 144th out of 177 countries in the 2013 Corruption Perceptions Index (CPI, 2013). Whilst many participants expected the university to equip students with what it takes to seek or create jobs, they also want the machinery of the State to be less corrupt and the process of registering a new business be rendered less burdensome and bureaucratic. Furthermore, as a means to curb unemployment, this article maintains the need for new businesses not to be allowed to crumble under the weight of heavy taxes. Rather, the State and its policies can provide incentives to enable new businesses to emerge and grow to the point whereby they can begin to make realistic tax contributions to the system which will not be counterproductive to their long term profitability and survival. 7. Repositioning the University as a Gateway to Employment It suffices to highlight that the State-owned university at the hub of this article has a mandate to enhance graduate employability. However, there is mounting evidence that depicts the need for the university to be repositioned to act accordingly. A number of factors emerged from participants’ narratives on how the university can help students to be employed upon finishing their studies. Some of these factors which have been mentioned before in the previous section will be reiterated in addition to new insights. 6.1 Inadequate Facilities The university at the nucleus of this article is State-owned and relies heavily on State subsidies for its recurrent and investment expenditures. The current economic situation of the country has led to dwindling subsidies to State universities, irrespective of an increasing need for investment in facilities that would enhance the capacity building of young Cameroonians for employment and societal transformation. This situation has affected the quantity and quality of infrastructures at State universities. One student participants noted: “We do not have the necessary facility to foster our learning so that we can come out with skills to serve the society. I think that if the university has a good science department with equipment, it would be of help to us” (Lucas, male, 21–30, Molyko, 12–04–2013). Whilst it can be argued that the university’s ability to secure relevant facilities to enhance the training of young Cameroonians with transferable skills needed for gainful employment after their studies and effective contribution to the country’s GDP is dependent on the quality of financial or budget management at the level of the university, it can be argued that it is also largely dependent on State allocations. This article therefore suggests that the State may need to revisit its budgetary allocations by prioritising university education and investing significantly in its infrastructures, facilities and capacity building of the students, as well as the staff. ublic Corruption, Heavy Taxes and Cumbersome Governmental Processes 7.1 Customising Education and Training to Serve the Needs of the Job Market As an English teacher, I will say that the English examination at the Ordinary Level which I set and which I mark and which is an artificial test of language cannot be given priority over the language which he used to pass his Advance Level which is applied English. This is education we are talking about. We are talking about development to bring out skills from people not because the English man qualifies us for it but because we need those skills to develop our environment” (Gregory, male, 51–60, Wokoko, 28-04-2013). To clarify, the State-owned university in the municipality was established to emulate the standards of a traditional British university in terms of governance, structure and operations. As the participant above posited, having the required result in an English Language test is a prerequisite for admission into the university irrespective of the fact that one may have passed other subjects conducted in English at the General Certificate of Examination (GCE) Ordinary and Advance Levels organised by the GCE Board. It is worth to note that Cameroon is multilingual, and English is one of official languages, spoken by 30% of the population (PRC, 2014). Irrespective of the fact that the English Language is an official language and Cameroon has historical ties with Britain, the participant above argued that the label of “Anglo-Saxon” over the State university has become a barrier to the community’s development and the potential for graduates to integrate into the society and be agents of transformation. On this basis, the concept of Anglo-Saxonism over the local university can be argued to have alienated many prospective students from university education because of failure to meet the English Language requirement and given that Higher Education has the potential to enhance one’s capacity for participation in the nation, as well as community building, this “language entry” barrier could be removed or simplified. By simplification, one participant underscored the need for the university to examine its operations and modify it in order to enhance access to Higher Education and to enable its educational system to serve local market needs instead of adopting a foreign system of education which is deficient in contributing to local community development and the needs of the citizenry. 7.1 Customising Education and Training to Serve the Needs of the Job Market 7.1 Customising Education and Training to Serve the Needs of the Job Mark A significant number of participants emphasised the need for education and training at the university to be tailored to address the needs of the job market. They suggested that the university can connect with the civil society, the industrial world or other services in the wider community so that academic programmes can be customised to address their concerns. Another benefit of customising academic programmes could be that if a student, for example, reads English Literature at the university, something can be added to it such as teaching of English Literature, editing or creative writing to provide an opportunity to demonstrate the relevance of the subject to the society. Furthermore, the university could also work with community partners to negotiate 33 International Journal of Economics and Finance Vol. 6, No. 12; 2014 www.ccsenet.org/ijef academic pathways, modules and endorsement consistent with areas of community/market needs. However, these measures should also take into account the need for quality and standard in conformity with regulations for institutional conferment of awards. academic pathways, modules and endorsement consistent with areas of community/market needs. However, these measures should also take into account the need for quality and standard in conformity with regulations for institutional conferment of awards. In addition, as many participants picked up issues with the educational system at the State-owned university in the municipality, one respondent suggested that foreign models of education which are obsolete and no longer relevant in serving the developmental or market needs of the community should be replaced with an updated educational system which is relevant to national and local contexts. This participant went on to interrogate the relevance of the Anglo Saxon system of education adopted by the university at the centre of this project. He probed: “Has the Anglo-Saxon system helped our development, in terms of meeting our needs or hindering it? If you tell a boy with Mathematics, Chemistry and Physics at the Advanced Level who can read electronics normally, that he would not enter the university because of failure in Ordinary Level English, does that not hinder his development? 2 Demonstrating Consciousness and Providing Students with Adequate Counselling Participants’ narratives also highlighted the need for the university to demonstrate awareness and desire for affecting the community and its ecological landscape through the quality of its graduates. The university can be a service oriented agent of education and that consciousness could take a level of emphasis. One participant posited that “to be awarded a PhD by the university does not mean that one is useful because a PhD needs an HPD which means ‘Help People Develop’. If one’s PhD does not have a corresponding HPD, it simply implied one had but a PHD which means ‘Pull Humans Down’” (Gregory, male, 51–60, Wokoko, 28-04-2013). From this insight, the need for university education to have an ethical and moral dimension which seeks to contribute to improving the livelihood of community residents can be suggested. Furthermore, the consciousness of contributing to societal transformation can be fostered through a strong counselling unit at the university to assist students with choice of career path as earlier noted. Although it was observed that the university has professional counsellors recruited by the State to serve this purpose, they are often redeployed into other units of the university which tend to invalidate their initial role. Therefore, it can be suggested that there is a need for the university to recognise the role of these counsellors and let the students maximise their services for proper orientation and guidance towards profitable careers. 7.3 Providing Entrepreneurial Skills for Job Creation 7.1 Customising Education and Training to Serve the Needs of the Job Market In accordance with this view, this article suggests that the university can revise the Anglo-Saxon model of education it has adopted to suit relevant markets and graduate employability within a context of sustainability. 7.2 Demonstrating Consciousness and Providing Students with Adequate Counselling 7.3 Providing Entrepreneurial Skills for Job Creation In order to address the need for graduate employment, some participants advocated that students should be given entrepreneurial skills to not only look for jobs after completion of studies but also to create one and be 34 International Journal of Economics and Finance Vol. 6, No. 12; 2014 www.ccsenet.org/ijef self-employed. The following interview transcript with a tomato farmer (Martin, male, 31–40, Molyko, 26-04-2013) emphasises this: Q: what do you want the university to do in the future? Martin: They should go more vocational, if they go more vocational then it would be so wonderful Martin: Somebody being trained will not come out and seek employment; they will create employment for others Q: So indirectly you want the university to give people entrepreneurial skills? Martin: Somebody being trained will not come out and seek employment; they will create employment for others Although the above mentioned factors can boost graduate employment, it can also be argued that the purpose of university education should not be limited to employment. There is therefore a need to strike a balance between university education for personal/intellectual development or some other reason and university education for local/global market consumption. Whilst liberal education may be concerned with the cultivation of the mind as the business of the university, Newman asserts that some great minds would be very slow to understand this. According to Newman (1955), “they insist that Education should be confined to some particular and narrow end, and should issue in some definite work, which can be weighed and measured. They argue as if everything, as well as every person, had its price; and that where there has been a great outlay, they have a right to expect a return in kind. This they call making Education and Instruction ‘useful’, and ‘utility’ becomes their watchword” (1955, p. 77). This assertion is consistent with the views of many participants who called for university education to guarantee a foothold in the job market for graduates. Moreover, the subject of graduate employability should not be limited to graduates finding jobs in the local community but should also incorporate the global community. The limited size of a local job market means that it can easily become saturated with too many applicants competing for few job vacancies, hence the need for the university to train students who can also their skills and knowledge abroad or be competitive in the global market. 8. Conclusion Even though the role of the university is increasingly being subjected to scrutiny, a university can be perceived to have the potential which enables it operate as a gateway to employment within a context of poverty, scarcity and fragile economy. On the basis of findings from research data, this article argues that graduate employment can 1) foster self-reliance as graduates would no longer depend on sponsors or parents as a source of livelihood, and 2) contribute to the economic sustainability of families as employed graduates would be able to earn income and economically assist both immediate and extended family members. Given that the family can be construed as the basic unit of a society or community, graduate employment also has the potential to enhance the economic prosperity of a community and a nation. Although a university can be perceived to have the capacity to enhance the employability of its graduates, it could take appropriate measures to enforce and operationalise this capacity. By connecting the university with relevant segments of the local and global communities so that academic programmes at the university can be customised to serve local and global interests; adopting and operationalising an appropriate counselling unit to provide career guidance to students and fostering entrepreneurial capabilities for self-employment, the university can make effective contributions towards graduate employability and also maintain its ethos of fostering academic rigour and quality. In addition, the university’s efforts toward enhancing graduate employability can be complemented with relevant inputs from State and the community within a framework of collaborative engagement and shared interests. Elliot, J., Francis, H., Humphreys, R., & Istance, D. (1996). Communities and their Universities: The Challenge of Lifelong Learning. London: Lawrence & Wishart Limited. Enslin, P., & Horsthemke, K. (2004). Can Ubuntu provide a model for citizenship education in African Eisemon, T. O., & Salmi, J. (1993). African Universities and the State: Prospects for Reform in Senegal and Uganda. Higher Education, 25(2), 151–168. http://dx.doi.org/10.1007/BF01384746 Dewey, J. (1938). Democracy and Education: An Introduction to the Philosophy of Education. New York: Macmillan. Boyer, E. L. (1996). The Scholarship of Engagement. Journal of Public Service and Outreach, 1(1), 11 20. CPI. (2013). Corruption Perceptions Index 2013. Retrieved October 07, 2014 from http://cpi.transparency.org/cpi2013/results/ Dewey, J. (1938). Democracy and Education: An Introduction to the Philosophy of Education. New York: Macmillan. References Case study research: Design and methods (4th ed.). Thousand Oaks, CA: Sage. References CPI. (2013). Corruption Perceptions Index 2013. Retrieved October 07, 2014 from http://cpi.transparency.org/cpi2013/results/ Dewey, J. (1938). Democracy and Education: An Introduction to the Philosophy of Education. New York: Macmillan. Eisemon, T. O., & Salmi, J. (1993). African Universities and the State: Prospects for Reform in Senegal and Uganda. Higher Education, 25(2), 151–168. http://dx.doi.org/10.1007/BF01384746 Elliot, J., Francis, H., Humphreys, R., & Istance, D. (1996). Communities and their Universities: The Challenge of Lifelong Learning. London: Lawrence & Wishart Limited. 35 International Journal of Economics and Finance Vol. 6, No. 12; 2014 www.ccsenet.org/ijef democracies? Comparative Education, 40(4), 545–558. http://dx.doi.org/10.1080/0305006042000284538 Fouda, M. S. (2012). A Pragmatic Approach to Policy for Sustainable Growth. In E. Aryeetey, S. Devarajan, R. Kanbur, & L. Kasekende (Eds.), The Oxford Companion to the Economics of Africa (pp. 457–459). Oxford: Oxford University Press. Jahoda, M. (1982). Employment and Unemployment: A Social Psychological Analysis. Cambridge: The Press Syndicate of the University of Cambridge. Mackenzie, N., & Knipe, S. (2006). Research delimmas: Paradigms, methods and methodology. I Educational Research, 16(2), 193–205. Makgoba, M. W. (1996). In search of the ideal democratic model for SA. Sunday Times, 23. Mbuntum, F., Tiayong, C., Tendo, J., Foba, F. N., Talla, R., Tita, C., & Mbake, M. (2008). The University of Buea Strategic Plan 2007–2015. Buea: Vincent P.K. Titanji. Mosha, J. H. (1986). The role of African universities in national development: a critical analysis. Comparative Education, 22(2), 93–109. http://dx.doi.org/10.1080/0305006860220202 Neneh, B. N. (2014). An Assessment of Entrepreneurial Intention among University Students in Cameroon. Mediterranean Journal of Social Sciences, 5(20), 542. , J. H. (1955). The Idea of a University. London: Cambridge University Press. Newman, J. H. (1955). The Idea of a University. London: Cambridge University Press. Ngu, L. J., & Kwankam, Y. S. (1992). At what price higher education in Africa? A case study of higher education systems in Cameroon. Ottawa: International Development Research Centre. Njeuma, D. L., Endeley, H. N., Mbuntum, F. F., Lyonga, N., Nkweteyim, D. L., Musenja, S. & Elizabeth, E. (1999). Reforming a National System of Higher Education: The Case of Cameroon. Washington: ADEA Working Group on Higher Education. PRC. (2014). Presentation of Cameroon. Retrieved October 06, 2014 from https://www.prc.cm/en/cameroon/presentation Ryan, W. G. (2003). Techniques to Identify Themes. Field Methods, 15(1), 85–109. http://dx.doi.org/10.1177/1525822X02239569 Sawyerr, A. (2004). Challenges Facing African Universities: Selected Issues. African Studies Review, 47(1), 1–59. UB. (2014). About UB. Retrieved October 06, 2014, from http://ubuea.cm/about/ Yin, R. K. (2009). Copyrights py g Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 36

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.